WRNA 12 E1658

Received: 30 January 2021 Revised: 26 March 2021 Accepted: 26 March 2021
DOI: 10.1002/wrna.1658
ADVANCED REVIEW
Ribosome dynamics and mRNA turnover, a complex

relationship under constant cellular scrutiny
Christelle Morris | David Cluet | Emiliano P. Ricci
Laboratory of Biology and Modeling of the

Cell, Université de Lyon, ENS de Lyon,
Abstract
Université Claude Bernard, CNRS UMR Eukaryotic gene expression is closely regulated by translation and turnover of
5239, Inserm U1293, Lyon, France
mRNAs. Recent advances highlight the importance of translation in the con-
Correspondence trol of mRNA degradation, both for aberrant and apparently normal mRNAs.
Emiliano P. Ricci, Laboratory of Biology During translation, the information contained in mRNAs is decoded by ribo-
and Modeling of the Cell, Université de
somes, one codon at a time, and tRNAs, by specifically recognizing codons,
Lyon, ENS de Lyon, Université Claude
Bernard, CNRS UMR 5239, Inserm translate the nucleotide code into amino acids. Such a decoding step does not
U1293, Lyon, France. process regularly, with various obstacles that can hinder ribosome progression,
Email: emiliano.ricci@ens-lyon.org
then leading to ribosome stalling or collisions. The progression of ribosomes is
Funding information constantly monitored by the cell which has evolved several translation-
Agence Nationale de la Recherche, Grant/ dependent mRNA surveillance pathways, including nonsense-mediated decay
Award Number: ANR-20-CE15-0025;
Agence Nationale de Recherches sur le (NMD), no-go decay (NGD), and non-stop decay (NSD), to degrade certain
Sida et les Hepatites Virales, Grant/Award problematic mRNAs and the incomplete protein products. Recent progress in
Number: ANRS-ECTZ3306; Fondation sequencing and ribosome profiling has made it possible to discover new mech-
Innovations en Infectiologie; H2020
European Research Council, Grant/Award anisms controlling ribosome dynamics, with numerous crosstalks between
Number: ERC-StG-LS6-805500 translation and mRNA decay. We discuss here various translation features crit-
ical for mRNA decay, with particular focus on current insights from the com-
Edited by: Jeff Wilusz, Editor-in-Chief plexity of the genetic code and also the emerging role for the ribosome as a
regulatory hub orchestrating mRNA decay, quality control, and stress signal-
ing. Even if the interplay between mRNA translation and degradation is no
longer to be demonstrated, a better understanding of their precise coordination
is worthy of further investigation.
This article is categorized under:

RNA Turnover and Surveillance > Regulation of RNA Stability
Translation > Translation Regulation
RNA Interactions with Proteins and Other Molecules > RNA-Protein
Complexes
KEYWORDS
decay, degradation, mRNA, ribosome, translation
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2021 The Authors. WIREs RNA published by Wiley Periodicals LLC.
WIREs RNA. 2021;12:e1658. wires.wiley.com/rna 1 of 34

https://doi.org/10.1002/wrna.1658
2 of 34 MORRIS ET AL.
1 | INTRODUCTION
Messenger RNAs (mRNAs) are ephemeral intermediates between DNA, the carrier of genetic information, and proteins
that display a functional role. Their primary sequence contains not only the information ribosomes need to synthesize
proteins but also additional sequence and structural elements that regulate the translation process as well as other steps
including transcription, splicing, polyadenylation, nuclear export, localization, and degradation. These elements can be
located within untranslated regions (UTR) or in the coding region, owing to the degeneracy of the genetic code that
allows some sequence flexibility. An intricate set of regulatory elements is therefore embedded within genes, under
selective pressure, to play a specific role during the expression, function, or turnover of mRNAs.
mRNA translation is a sophisticated process that involves a large number of protein and RNA actors assisting ribo-
somal subunits in binding and assembling onto the mRNA. Thereafter, the ribosome translocates over the coding region
and dissociates once the stop codon is reached, consequently releasing the newly synthesized protein. As such, translation
is tightly regulated through a multitude of pathways involving cis-acting features and trans-acting factors that can affect
the process in a transcript-specific or global manner. Although the initiation of translation is generally considered as the
rate-limiting step, it has long been known that elongation is not a uniform process and termination is also tightly regu-
lated, although these studies were technically challenging. The advent of high-throughput sequencing and the ribosome
profiling protocol (Ingolia et al., 2009), together with structural biology approaches, have greatly contributed to the char-
acterization of ribosome dynamics from the recruitment of the 40S to the elongation of 80S ribosomes across the open
reading frame and the recycling of post-termination ribosomes. Collectively, these works have led to the discovery of new
mechanisms involved in the regulation of translation and highlighted the numerous crosstalks between translation and
other processes such as co-translational protein folding and mRNA turnover. This review summarizes recent findings that
contribute to improve our understanding of the intricate relationship between mRNA decay and the translation process,
with particular emphasis on how the translating ribosome has emerged as a central regulatory hub not only for the quality
control pathways that degrade faulty mRNAs but also for canonical mRNA degradation.
2 | TR ANSLATION- DEPENDENT m RNA S URVEILLANCE P ATHWAYS
To ensure high fidelity of gene expression, eukaryotic cells have evolved three major cytosolic mRNA surveillance path-
ways that rely on translation to recognize and trigger degradation of aberrant transcripts. These pathways, known as
nonsense-mediated decay, non-stop decay, and no-go decay, respectively, signal RNAs with a premature termination
codon (PTC), RNAs lacking a termination codon and RNAs containing diverse ribosome-stalling sequences (Powers
et al., 2020; Shoemaker & Green, 2012). Despite their several mechanistic differences, the three mRNA surveillance
pathways survey mRNA quality through careful inspection of the translation process, alerting when ribosomes are not
progressing normally, in order to degrade aberrant mRNAs and their nascent polypeptides and to recycle ribosomes.
2.1 | Nonsense-mediated decay
This surveillance pathway causes decay of transcripts harboring a PTC that can be brought in by genetic mutations or
introduced into an mRNA through stochastic errors in transcription or splicing. In addition to its role in eliminating
aberrant mRNAs with a PTC, nonsense-mediated decay (NMD) also degrades a large number of normal transcripts that
contain an upstream open reading frame (uORF) or a long 30 -untranslated region (UTR; Kurosaki et al., 2019; Nasif
et al., 2018). In all cases, it is assumed that RNA features downstream of a termination codon must interfere with trans-
lating ribosomes and, depending on the NMD trigger, the termination codon is defined as premature or genuine. The
core NMD factors comprise a set of up-frameshift (UPF) proteins (UPF1, 2, and 3B) and suppressors with morphoge-
netic effects on genitalia (SMG) proteins (SMG1, 5, 6, and 7). The NMD machinery is thought to be recruited to target
mRNAs because translation termination at a PTC is aberrant. In normal translation, ribosomes decode transcripts until
they encounter a stop codon, usually present in the most 30 -exon in close proximity to the poly(A)-binding protein
(PABPC1 in mammals, Pab1 in yeast) bound to the poly(A) tail. PABPC1 promotes translation termination by stimulat-
ing the recruitment of eukaryotic release factors eRF1 and eRF3a to the ribosomal A-site, which releases the nascent
peptidic chain (Ivanov et al., 2016). Afterwards, ribosomes are dissociated into 40S and 60S ribosomal subunits that are
recycled by the factor ABCE1 (Rli1 in yeast) with help of several initiation factors (Figure 1(a)).
MORRIS ET AL. 3 of 34
In the case of a PTC, two predominant models of NMD activation have been put forward. The first, referred to as
the exon junction complex (EJC) model, postulates that, during the pioneer round of translation, when a ribosome
encounters a stop codon located sufficiently upstream of an EJC, a warning signal is sent to the NMD pathway to
degrade the mRNA carrying a PTC (Nagy & Maquat, 1998; Thermann et al., 1998; Zhang et al., 1998). In this scenario,
(a) (b)
F I G U R E 1 Translation termination at a normal stop codon and at a premature termination codon. (a) Normal translation termination.
Panel 1: The translating 80S ribosome, composed of 60S and 40S subunits, encounters the STOP codon (red STOP sign) located near the
poly(a) tail bound by PABPC1 in mammals. Ribosomal A-site, P-site, and E-site are indicated. tRNAs are bound in the P-site and E-site.
PABPC1 promotes the recruitment of the eRF1 and eRF3a release factors to the ribosomal A-site. Panel 2: After GTP hydrolysis by eRF3a,
eRF1 triggers hydrolysis of the peptidyl-tRNA, releasing the completed nascent protein. This leaves a ribosome still bound to the mRNA,
which has to be disassembled and recycled. Panel 3: The ribosome recycling factor ABCE1 is recruited and it supports, with help of other
initiation factors, the dissociation of the 80S ribosome into 40S and 60S subunits, which can then be recycled for extra rounds of translation.
(b) Translation termination at a premature termination codon (PTC) and induction of nonsense-mediated mRNA decay (NMD). Panel 1:
The ribosome stops at a PTC located upstream of an EJC and distant from the normal stop codon. The presence of the NMD factor UPF3B at
the EJC allows the association of eRF1 and eRF3a in the ribosomal A-site. Panel 2: A truncated protein product is released through the
action of eRF3a and eRF1. Panel 3: Ribosome dissociation is mediated by ABCE1 together with UPF3B. Subsequently, UPF2 and UPF3B
are thought to activate the SMG1 kinase for UPF1 phosphorylation. Panel 4: Phosphorylated UPF1 mainly associates with the
SMG5-SMG7 complex which recruits decapping and deadenylation activities (1), followed by the exoribonucleases with 50 -to-30 (XRN1) and
30 -to-50 (exosome or DIS3L2) activities (2). In addition, phospho-UPF1 can recruit the endonuclease SMG6 that cleaves RNA in the vicinity
of the PTC
PABPC1 cannot interact with eRF3a due to their distance and, instead, the EJC allows the recruitment of NMD factors
which, in turn, associate with eRF3a. Importantly, it has been reported that eRF3a interacts directly with UPF3B, but
not UPF1 as previously thought, and UPF3B acts to dissociate post-termination ribosomes (Neu-Yilik et al., 2017). In
addition, the ribosome recycling factor ABCE1, required for ribosome dissociation at a normal termination codon, is
also essential for termination at a PTC, contrary to what was previously believed (Zhu et al., 2020). Indeed, loss of
ABCE1 was found to induce an accumulation of unrecycled ribosomes in the 30 -UTR of NMD targets, leading to dis-
placement of downstream EJC, followed by an impaired recruitment of the central NMD factor UPF1. Following proper
ribosome dissociation, UPF1, activated by SMG1-mediated phosphorylation and aided by its partners UPF2 and UPF3B,
can remodel the 30 -UTR and recruit the enzymes involved in mRNA decay (Figure 1(b)). The second model for NMD,
called the faux UTR model, has been proposed in yeast. This model postulates that the inherently aberrant nature of
premature translation termination allows the binding of UPF factors to mRNAs, thus triggering NMD. Supporting this
model, early results showed that translation termination at a PTC is inefficient, likely because the PTC is distant from
the poly(A)-binding protein Pab1 located at the 30 -end of the ORF (Amrani et al., 2004). In vivo experiments in yeast
now substantiate these previous findings by demonstrating that the efficiency of translation termination increases as
PTCs are closer to ORF 30 -ends, and furthermore this positional effect depends on Pab1 (W. Chan et al., 2020). The
authors proposed from their results that NMD is activated as soon as the efficiency of termination is below a certain
threshold.
By developing a method that allows real-time imaging of both translation and NMD of single mRNA molecules
in live human cells, a recent work provided a unifying model for NMD activation by recapitulating most aspects of
NMD, but this study also unveils that the mechanism of NMD activation is more complicated than previously
believed (Hoek et al., 2019). This powerful method to study NMD kinetics and heterogeneity reveals that each ribo-
some (the first or one of the following ribosomes) that terminates translation at the PTC has an equal probability
of triggering NMD. In addition, NMD occurs with the same probability during the pioneer round of translation
(CBC-bound mRNAs) or during a later round (eIF4E-bound mRNAs). Surprisingly, for a given NMD substrate, a
fraction ranging from 5 to 30% of mRNA molecules is resistant to NMD. This could be explained by some variabil-
ity in splicing or EJC deposition. Contrariwise, NMD probability can vary substantially depending on several key
parameters such as the distance between the PTC and the downstream EJC, the number of EJCs, or the mRNA
sequence downstream of the PTC. On the other hand, changing the 30 -UTR length of the mRNA reporters did not
have a major effect on NMD efficiency. This finding agrees with a recent study reporting that NMD in human cells
occurs independently of stable ribosome stalling at PTCs (Karousis et al., 2020). To monitor ribosome density at
stop codons, the authors developed a toeprinting assay based on in vitro translation of NMD reporter mRNAs with
human cell lysates. Their results demonstrated a comparable ribosomal density at stop codons of NMD-sensitive
and NMD-insensitive reporters. In line with this, ribosome profiling analyses also did not show any increase in
ribosome occupancy at termination codon of endogenous NMD-sensitive transcripts, compared with NMD-
insensitive mRNAs. Together, both studies do support that NMD is induced by a mechanism more complicated
than just ribosome stalling at stop codons.
Another work was designed to define NMD targets and decay intermediates on a transcriptome-wide level
(Kurosaki et al., 2018). Because NMD substrates are degraded by nucleases common to other RNA decay pathways
and the UPF1 factor is involved in NMD but also in other decay mechanisms, the authors took advantage of the
fact that, in human embryonic kidney (HEK) 293T cells, UPF1 is activated by phosphorylation exclusively during
NMD and not during other decay pathways. The use of an antibody specific of phosphorylated UPF1 combined
with high-throughput sequencing allowed them to selectively identify direct NMD targets and their decay interme-
diates. Importantly, their findings provided evidence that NMD initiates on mRNAs that are bound by one or more
translating ribosomes. This strongly suggests that NMD does not occur in P-bodies (see Box 1), in agreement with
previous studies (Eulalio, Behm-Ansmant, Schweizer, & Izaurralde, 2007; Stalder & Mühlemann, 2009) but in dis-
agreement with others (Durand et al., 2007; Sheth & Parker, 2006). Their data also support that phospho-UPF1 pre-
dominantly associates with the SMG5-SMG7 complex which recruits decapping and deadenylation activities,
followed by the action of exoribonucleases with 50 -to-30 (XRN1) and 30 -to-50 (exosome or DIS3L2) activities. Alter-
natively, phospho-UPF1 can interact with the endonuclease SMG6, which cleaves in close proximity to the PTC
(Figure 1(b)). Finally, the authors found that NMD decay intermediates were modified by uridylation at their 30 -
ends by the poly(U) polymerases TUT4 and TUT7, which further increases 30 -to-50 decay and also XRN1-mediated
50 -to-30 decay.
BOX 1 Processing-bodies
Processing-bodies (P-bodies) are cytosolic membraneless organelles that form through a process known
as liquid–liquid phase separation (for a comprehensive review see Luo et al., 2018; Standart & Weil, 2018).
P-bodies are composed of aggregates of untranslating mRNAs and proteins related to RNA metabolism. Among
these proteins are factors involved in mRNA degradation (including decapping factors, the 50 -30 exonuclease
XRN1, deadenylation factors, miRNA-mediated silencing factors, and nonsense-mediated decay factors), as well
as proteins involved in translational repression (for an exhaustive list of proteins see Eulalio, Behm-Ansmant, &
Izaurralde, 2007; Parker & Sheth, 2007). The enrichment of such factors together with that of mRNAs targeted
by mRNA degradation pathways such as miRNAs or ARE-elements, led to the hypothesis that P-bodies were
sites of active mRNA degradation. However, this model has been recently challenged by studies showing that
mRNAs found within P-bodies are intact (no specific detection of degradation intermediates), thus suggesting
that P-bodies are sites of regulated mRNA storage instead of degradation (Courel et al., 2019; Standart &
Weil, 2018). Further supporting this hypothesis, mRNAs stored in P-bodies have been shown under specific
conditions to exit P-bodies and reengage in translation (Bhattacharyya et al., 2006; Brengues, 2005). Neverthe-
less, mRNAs enriched in P-bodies appear to follow a distinct pathway for their degradation that involves the
action of the PAT1B protein (Courel et al., 2019).
2.2 | No-go decay
The no-go decay (NGD) pathway acts to recognize and degrade transcripts containing sequence features that induce
ribosome stalling during translation elongation (Doma & Parker, 2006; Tsuboi et al., 2012). The most prevalent stalling
features into mRNAs comprise stable secondary structures as stem-loop motifs or GC-rich sequences, tracts of rare
codons that leave the ribosomal A-site empty, or damaged RNA bases. Such obstacles that hinder the movement of ribo-
somes along mRNAs lead to ribosome stalling and potentially ribosome collision, which implies a collision between a
stalled ribosome and the elongating ribosome behind it. Depending on the context, two different mechanisms evolved
to cope with the event of ribosomal arrest. In the case of a stalled ribosome at the end of a truncated mRNA, which
leaves empty the ribosomal A-site, it was found that the yeast complex Dom34-Hbs1 (or Pelota-Hbs1 in mammals)
binds to the A-site and is thought to recruit an endonuclease for mRNA cleavage in the vicinity of the stalled ribosome
(Doma & Parker, 2006). The ensuing cleavage products are degraded by either the Xrn1 exonuclease or the Ski-exosome
complex. Some mechanistic details were obtained using high resolution ribosome profiling and biochemistry
approaches in yeast (D'Orazio et al., 2019). The authors identified Cue2 as the primary endonuclease that cleaves
mRNA precisely within the A-site of the stalled ribosome and they found that exonucleolytic decay of RNA intermedi-
ates proceeds primarily with Xrn1 rather than the exosome. Furthermore, the NGD process closely couples decay of
faulty mRNAs with ribosome rescue and degradation of the nascent polypeptide. Dom34/Pelota and Hbs1, which are
structural homologs of the canonical termination factors eRF1 and eRF3, recruit the ribosome-recycling factor Rli1/
ABCE1 to promote dissociation of the stalled ribosome (Tsuboi et al., 2012). Because Dom34/Pelota is unable to manage
peptidyl-tRNA hydrolysis, the nascent peptide remains attached to the 60S ribosomal subunit and is then handled by
the ribosome-associated quality control (RQC) pathway for subsequent ubiquitination, extraction, and degradation by
the proteasome (Shao et al., 2013; Verma et al., 2013; Figure 2(a)).
A second major mechanism is employed during NGD to resolve ribosome collision events. This mechanism does
not involve external factors interfacing with ribosomes, but it relies on direct modifications of ribosomes. When the
trailing ribosome collides with the leading stalled ribosome, the complex formed by the two is called a disome. The
structure of the disome was solved by cryo-electron microscopy (cryo-EM) and revealed a unique conformation suitable
for recognition and modification of some ribosomal proteins by the ubiquitin ligase ZNF598 (Hel2 in yeast; Ikeuchi
et al., 2019; Juszkiewicz et al., 2018). In particular, ubiquitination of the ribosomal protein uS10 (RPS20) was found to
be required for elimination of the disome unit by the NGD pathway in yeast (Ikeuchi et al., 2019). Consistent with these
findings, it was demonstrated that ribosome collisions are crucial for robust induction of the NGD process (Simms
et al., 2017; Figure 2(a), Panel 4).
(a) (b)
F I G U R E 2 No-go decay and non-stop decay. (a) No-go decay (NGD). Panel 1: The ribosome stops before reaching the stop codon
because it encounters a stalling feature such as a stem-loop motif, a GC-rich tract, or rare codons. These obstacles lead to ribosome stall and
potentially ribosome collision. Two different mechanisms evolved to cope with this particular event of ribosomal arrest. The first
mechanism, aimed to resolve a stalled ribosome, is illustrated on panels 2 and 3. The mammalian complex Pelota-Hbs1 (Dom34-Hbs1 in
yeast) binds to the empty ribosomal A-site and recruits the ribosome recycling factor ABCE1 (Rli1 in yeast) and also the Cue2 endonuclease
(in yeast), which cleaves the mRNA in the A-site of the stalled ribosome, further allowing exonucleolytic decay mediated primarily by the
XRN1 exonuclease rather than by the exosome assisted with the Ski complex. Panel 3: NGD closely couples decay of faulty mRNA with
ribosome recycling and degradation of the nascent polypeptide. Because Pelota-Hbs1 cannot hydrolyze the peptidyl-tRNA bound to the P-
site, peptide release does not occur. The nascent peptide still attached to the 60S ribosomal subunit is handled by the ribosome-associated
quality control (RQC) pathway and degraded via the proteasome. Panel 4 illustrates the second mechanism aimed to resolve collided
ribosomes. This event induces conformational changes of the ribosome that are recognized by the ubiquitin ligase ZNF598 (in mammals).
ZNF598 ubiquitinates several ribosomal proteins and these modifications are required for robust induction of NGD. (b) Non-stop decay
(NSD). NSD relies on the same factors as NGD, but differs in its mRNA targets. Panel 1: Due to the lack of a stop codon, the ribosome
translates a poly(A) sequence and translation elongation progressively slows down and eventually stops. Activation of NSD results from
interactions between the positively charged lysine residues of the nascent polypeptide and the negatively charged exit channel of the
ribosome. Panel 2: The complex Pelota-Hbs1 in the A-site recruits the endonuclease NONU-1 (in C. elegans, homolog of Cue2 in
S. cerevisiae). The interactions between positively-charged residues with the exit channel result in the retention of the nascent peptide when
the ribosome subunits are dissociated. Panel 3: Ribosome stalling caused by translation of poly(A) sequences is recognized by ZNF598.
Ubiquitination of ribosomal proteins by ZNF598 are important modifications for ribosome rescue during NSD
2.3 | Non-stop decay
Despite its name, the non-stop decay (NSD) pathway is not dedicated to the decay of all transcripts lacking a stop codon
but primarily dedicated to the decay of transcripts inappropriately polyadenylated in their coding region, resulting in
formation of nonstop mRNAs (Frischmeyer et al., 2002). Therefore, although they differ in their substrate specificity
and ribosome stalling mechanism, NSD and NGD both degrade problematic RNAs with stalled ribosomes and collided
ribosomes if severe ribosome slowdown. In line with this, NSD relies on the same factors as NGD to induce mRNA
decay and ribosome rescue (Figure 2(b)). These include Dom34/Pelota-Hbs1 and Rli1/ABCE1 (Tsuboi et al., 2012).
Recently, NONU-1 was identified in C. elegans as a novel endonuclease that induces RNA cleavage in the vicinity of
stalled ribosomes during both NSD and NGD (Glover et al., 2020). Homology searches revealed that one homolog of
NONU-1 in S. cerevisiae is Cue2, which was previously identified as the primary endonuclease functioning in NGD
(D'Orazio et al., 2019). Mechanistically, earlier studies suggested that ribosome stalling during NSD was caused by the
interaction of the nascent polypeptide, positively charged because of consecutive lysine residues encoded by the poly(A)
sequence, with the ribosomal exit channel that is charged negatively (Koutmou et al., 2015; Lu & Deutsch, 2008). Fur-
ther biochemical and structural studies in mammalian systems have elucidated the mechanism by which ribosomes
stall during translation of a poly(A) tail (Chandrasekaran et al., 2019). By cryo-EM, the authors visualized a suboptimal
peptidyl-tRNA conformation for peptide bond formation and a reconfiguration of the decoding center by the poly(A)
mRNA that conflict with incoming aminoacyl-tRNA, thus impeding further elongation. Accordingly, they proposed
that NSD is selectively activated upon coincident detection by the ribosome of poly-lysine chains in the exit tunnel and
poly(A) tracts in the decoding center. Additionally, it was found that ribosome stalling caused by translation of poly(A)
sequences is recognized by ZNF598. This E3 ligase, also involved in NGD, ubiquitinates the 40S ribosomal proteins
eS10 (RPS10), uS10 (RPS20), and eS1 (RPS3A) in mammals (Figure 2(b), Panel 3), and these modifications are crucial
to detect and resolve ribosomes that have stalled during decoding of poly(A) sequences (Garzia et al., 2017;
Juszkiewicz & Hegde, 2017; Sundaramoorthy et al., 2017). In yeast, similar ubiquitinations catalyzed by Hel2 were also
described, yet targeting distinct ribosomal proteins (Matsuo et al., 2017).
3 | TR ANSLATION I NITIATION A ND mRNA DECAY
In eukaryotic cells, coupling between mRNA translation and decay seems to be widespread and extends beyond the
aforementioned mRNA surveillance mechanisms. While individual steps of translation and decay processes are rather
well elucidated, less is known about the upstream determinants that control mRNA fate. Translation initiation is gener-
ally viewed as the most influential step in translation and numerous studies have attempted to address its impact on
mRNA decay. Early observations in S. cerevisiae have shown that active translation initiation protects certain mRNAs
from degradation (Schwartz & Parker, 1999). For those transcripts, inhibition of translation initiation by mutation of
initiation factors led to higher rates of mRNA decay, associated with an increase in both decapping and deadenylation
activities. A transcriptome-wide study in S. cerevisiae revealed that this relationship between mRNA translation initia-
tion and decay is broadly generalizable (P. P. Chan & Lowe, 2016). By combining a minimally invasive metabolic
labeling-based assay to measure mRNA decay rates with selective perturbations of the translation process, these authors
found that slowing translation initiation caused a higher rate of transcript degradation on a transcriptome-wide level.
Consistent with this global destabilization of mRNAs upon inhibition of translation initiation, they observed an
enhanced formation of P-bodies, which are sites of mRNA storage and decay. By contrast, they found that slowing
translation elongation led to an overall stabilization of transcripts and no change on P-body formation. Collectively,
their results provide further evidence that translation initiation is an important determinant of mRNA stability in yeast.
Two mutually non-exclusive models have been proposed to explain how translation may affect mRNA stability. The
first model, referred to as the stalled ribosome-triggered decay model, predicts that a slow rate of translation elongation
at suboptimal codons is perceived as a signal to trigger mRNA decay. The second model, referred to as the translation
factor-protection model, predicts that the translation initiation machinery protects mRNAs from decay by a competition
mechanism for mRNA binding. The data obtained by (P. P. Chan & Lowe, 2016) are consistent with mRNA decay being
regulated by translation initiation but not elongation.
In higher eukaryotes, a study now provides a broad and comprehensive view regarding the impact of the 50 -UTR on
mRNA translatability and stability (L. Jia et al., 2020). By designing a mRNA library of over one million 50 -UTR variants
with a randomized sequence preceding a small uORF and downstream GFP, the authors identified several elements in
the 50 -UTR that contribute to reporter mRNA outcomes in mammalian cells. While translation of the GFP-encoding
main ORF preserves mRNAs from degradation, uORF translation was found to inhibit translation of downstream GFP,
by triggering mRNA decay in a way reminiscent of NMD since this uORF-mediated decay depends on both UPF1 and
translation. Additionally, enrichment of GGC motifs in the 50 -UTR inhibits translation and confers a shorter half-life to
mRNAs. These sequence elements form RNA G-quadruplex (RG4) structures that impair the scanning process and relo-
cate the RG4 reporter mRNAs into P-bodies for DCP2-dependent or XRN1-dependent decay. Finally, a 50 -UTR unstruc-
tured A-rich motif was uncovered as an internal ribosome-entry site to mediate cap-independent translation. This
poly(A) tract element, while stabilizing mRNAs engaged in translation, destabilizes ribosome-free mRNAs likely by rec-
ruiting nucleases. Together, these findings reveal that the 50 -UTR contributes to a tight coupling between mRNA trans-
lation initiation and degradation. Furthermore, this coupling can occur through various mechanisms depending on the
50 -UTR sequence features.
4 | THE RIBOSOME A S A HUB FOR mR NA DECAY, QUALITY CONTROL,

AND STRESS S IGNALING
To ensure protein homeostasis, eukaryotic cells have evolved co-translational quality control mechanisms that respond
to abnormal translation by targeting the associated mRNA and nascent polypeptide chain for degradation and by
recycling ribosomes (Joazeiro, 2019). Emerging evidence highlights crucial roles for the ribosome in quality control
mechanisms, acting as a hub for recruiting factors that determine the fate of mRNAs and polypeptides. In addition,
ubiquitin modifications at the ribosome emerge as part of the quality control pathways. Furthermore, recent work has
revealed that the ribosome is a new central player to trigger various stress responses.
4.1 | The ribosome and mRNA decay
It was long believed that mRNA degradation required the removal of ribosomes first until a pioneering study in yeast
uncovered that mRNA decay could occur while transcripts were still bound by translating ribosomes (Hu et al., 2009).
The prevailing model of cytoplasmic mRNA decay is that it starts with shortening of the poly(A) tail by the Ccr4-Not
deadenylase complex and removal of the 50 -cap structure by the Dcp1–Dcp2 decapping complex. Then, mRNA decay
proceeds through 50 -to-30 degradation by the exoribonuclease Xrn1 and/or 30 -to-50 exonucleolytic degradation by the
exosome assisted with the Ski complex (Figure 3(a)). The study by Hu et al. showed that decapping and 50 -to-30
exonucleolytic degradation take place on certain mRNAs still associated with ribosomes. Their findings indicate that
removal of ribosomes is not a prerequisite for initiation of mRNA decay and, furthermore, suggest that Xrn1-mediated
mRNA degradation from the 50 -end follows the last elongating ribosome. By using high-throughput sequencing of 50 -
phosphorylated mRNA degradation intermediates, another work revealed that up to approximately 35% of transcripts
in S. cerevisiae undergo co-translational decay, which is inferred from the characteristic 3-nucleotide periodicity pattern
observed in the mRNA coding region (Pelechano et al., 2015). Additionally, the use of novel sequencing technologies to
identify RNA decay intermediates has shown that co-translational mRNA degradation is much more widespread than
previously thought (Ibrahim et al., 2018). Ibrahim et al. have developed a new transcriptome-wide method coined
“Akron-seq” to simultaneously capture and sequence the 30 -ends of capped mRNAs (Akron3) or the 50 -ends of
polyadenylated transcripts (Akron5) in human cells (Ibrahim et al., 2018). Their results showed that most 30 and 50
RNA ends are mapped within the mRNA body. Specifically, up to 63% of the 30 -ends of capped mRNAs are mapped
within coding sequences and only 11% of capped transcripts contain a poly(A) sequence at their 30 -end, thus indicating
that fragmented mRNAs are much more represented in cells than full-length RNA molecules. Importantly, relative
positioning analyses of Akron reads with ribosome profiling data revealed that 50 -end and 30 -end reads exhibit a striking
3-nucleotide periodicity for all transcripts, suggesting that canonical mRNA degradation may be coupled with translat-
ing ribosomes. Furthermore, their positioning analyses stipulated that RNA intermediates are not generated by exonu-
clease activities but by a repeated ribosome-associated endonucleolytic activity, which cleaves translated RNAs at the
exit site of mRNA ribosome channel. This cleavage generates a 50 -capped mRNA fragment with potentially active elon-
gating ribosomes. When the leader ribosome on this 50 -capped fragment reaches the 30 -cleavage site, it undergoes a hard
stall (the 30 -end of the mRNA being in the A-site) that induces a new endonucleolytic cleavage at the exit site of mRNA
ribosome channel, generating a final degradation product of 16 nt in length. Ibrahim et al called “ribothrypsis” this
(a) (b)
F I G U R E 3 Conventional mRNA decay pathways in yeast and mammals. Cytoplasmic mRNA decay occurs independently of the
translation process for the most part (a), but it can also proceed co-translationally (b). (a) The translation-independent mRNA decay starts
with deadenylation by the CCR4–Not complex, followed by removal of the 50 -cap by the DCP1–DCP2 complex (Panel 1). Thereafter, mRNA
decay proceeds through 50 -to-30 degradation by the exoribonuclease XRN1 and/or 30 -to-50 degradation by the exosome assisted with the Ski
complex (Panel 2). (b) Co-translational mRNA decay is initiated when, for various reasons, a ribosome is not loaded by a new tRNA in its A-
site. As above, mRNA decay starts with deadenylation by the CCR4–Not complex, then decapping by the DCP1–DCP2 complex, leading to
exonucleolytic cleavage by XRN1 (50 -to-30 ) and the ski-exosome complex (30 -to-50 ). In mammals, the mRNA decay pathways are not
redundant, but instead perform specialized functions. XRN1 predominantly mediates bulk mRNA decay with the aid of normal translation
(Panel 1). The Ski-exosome complex functions in global mRNA surveillance and triggers mRNA decay in the case of aberrant translation
events as stalled ribosomes at a premature stop codon (Panel 2). Alternatively, mRNA decay can occur through a repeated ribosome-
associated endonucleolytic activity, for which the endonuclease remains to be identified (Panel 3)
novel process of ribosome-phased endonucleolysis, for which the endonuclease remains to be identified (Figure 3(b)).
Beyond that, their study provides evidence that translation-dependent mRNA degradation is much more widespread
than previously thought, as it is very widely used in canonical mRNA decay. Final ribothrypsis products are similar to
the small ribosome footprints (15–18 nt) observed at known sites of mRNA truncation in yeast lacking the auxiliary
exosome factor Ski2 (Guydosh & Green, 2014). However, final ribothrypsis products are naturally generated by a
ribosome-associated endonuclease while the 50 -end of the small ribosome footprints observed in yeast are generated by
the RNaseI treatment following cell lysis. It is, therefore, not known yet whether a mechanism similar to ribothrypsis is
conserved in lower eukaryotes.
Recently, Tuck et al. wanted to find out whether decay factors could interact with ribosomes in mammals, and to
what extent mRNA decay was coupled to translation in higher eukaryotes (Tuck et al., 2020). Using mouse embryonic
stem cells, they first performed a global comparison of transcripts bound by XRN1 or by the Ski complex helicase
SKIV2L (homolog of yeast Ski2), and found a different specificity for a large number of transcripts, suggesting that the
two cytoplasmic decay pathways could have specialized functions. Their results showed that XRN1 and SKIV2L are
both recruited on translating ribosomes but their distribution is different, with XRN1 associating uniformly throughout
the mRNA length and SKIV2L accumulating specifically on RNAs occupied with stalled ribosomes. This work, as a
whole, sustains the idea that XRN1 mediates bulk mRNA decay with the aid of normal translation, whereas the Ski
complex functions in global mRNA surveillance and assists the exosome in triggering mRNA decay in the case of aber-
rant translation events as stalled ribosomes (Figure 3(b)).
Consistent with mRNA decay occurring co-translationally, a cryo-EM structure of yeast Xrn1 bound to a ribosome
was solved and the overall architecture of the Xrn1–ribosome complex is compatible with Xrn1 binding at the mRNA
exit site of the ribosome (Tesina et al., 2019). Also, Schmidt et al. determined the structure of a yeast ribosome associ-
ated with the Ski2–Ski3–Ski8 complex and found that the Ski complex is positioned near the ribosomal mRNA entry
tunnel (Schmidt et al., 2016). Additionally, a ribosome tagging method developed in mouse embryonic stem cells rev-
ealed that the mammalian “ribo-interactome” is composed of a repertoire of approximately 400 proteins including the
expected ribosomal proteins and translation factors but also many unexpected proteins controlling diverse cellular pro-
cesses (Simsek et al., 2017). Moreover, this dataset contains several RNA helicases including SKIV2L and UPF1, as well
as certain subunits of the Ccr4-Not deadenylase complex and m6A-binding proteins, all of which are known to regulate
the efficiency of mRNA translation and decay.
4.2 | The ribosome and selective co-translational degradation of functional mRNAs
Besides the role of ribosomes in orchestrating bulk mRNA degradation, other pathways involving co-translational deg-
radation of selected functional transcripts exist. These involve ribosome interacting proteins that can either contact
nascent peptides or specific RNA motifs within translated mRNAs to trigger their degradation.
Staufen-mediated mRNA decay (SMD) is a translation-dependent degradation pathway mediated by the mamma-
lian double-stranded RNA (dsRNA) binding proteins Staufen1 and Staufen2 (Y. K. Kim et al., 2005; E. Park &
Maquat, 2013). Staufen proteins bind dsRNA structures located in the 30 -UTR of specific mRNAs or resulting from
intermolecular interactions between two different RNA species (de Lucas et al., 2014; Gong et al., 2013; Gong &
Maquat, 2011; Ricci et al., 2014; Sugimoto et al., 2015). Staufen proteins interact directly with ribosomes as well as with
UPF1, inducing phosphorylation of the latter and stimulating its helicase activity (Y. K. Kim et al., 2005; E. Park &
Maquat, 2013). Similarly to NMD, SMD requires active translation of the target mRNA to trigger its decay. Interestingly,
Staufen proteins and UPF2 were shown to bind to UPF1 at overlapping regions thus suggesting that their association to
UPF1 is mutually exclusive (Gong et al., 2009). As a consequence, NMD and SMD were shown to be in competition,
with the down-regulation of Staufen proteins activating NMD while UPF2 down-regulation stimulates SMD (Gong
et al., 2009). However, biochemical reconstitution of UPF1 recruitment to Stau1 in vitro indicates a crucial role for
UPF2 in facilitating UPF1 binding to Stau1 and its activation, indicating that the observed competition between NMD
and SMD does not result from a competition of UPF2 and Stau1 to bind UPF1 (Gowravaram et al., 2019). Regarding its
biological roles, SMD has been implicated in the regulation of myogenesis, adipogenesis, and in the local degradation
of neuronal mRNAs upon their transport in axons, among others (Cho et al., 2012; Gong et al., 2009; J. Y. Kim
et al., 2017).
Similarly to Staufen proteins, Regnase-1 is an RNA-binding protein with RNase activity that mediates translation-
dependent mRNA degradation of specific transcripts through UPF1 (Mino et al., 2015). Regnase-1 binds to specific
RNA stem-loops in the 30 -UTRs of inflammation-related mRNAs and plays an essential role in modulating the immune
response (Cui et al., 2017; Kidoya et al., 2019; Mino et al., 2015; Tanaka et al., 2019; Uehata et al., 2013). Regnase-1 can
bind to target mRNAs before they are translated but, in the absence of translation, this interaction does not result in
mRNA degradation (Mino et al., 2019). Following the pioneer round of translation, UPF1 interacts with Regnase-1 and
this association induces UPF1 phosphorylation and stimulates its RNA-helicase activity. As a consequence, UPF1
unwinds the RNA stem-loop structure bound to Regnase-1 and this conformational switch licenses Regnase-1 to cleave
the target mRNA (Mino et al., 2019). Together, SMD-mediated and Regnase-1-mediated mRNA decay depend on spe-
cific RNA structures to recognize their target RNAs and exploit UPF1 to mediate translation-dependent mRNA decay,
although through different mechanisms.
Translation-dependent mRNA degradation can occur independently of a sequence or a structural motif within the
target mRNA but instead rely on the co-translational recognition of nascent peptides. Such a mechanism has been
described for the degradation of mRNAs coding for secretory proteins whenever they fail to be correctly addressed to
the endoplasmic reticulum (ER). Secretory proteins contain an N-terminal signal peptide that mediates co-translational
recruitment of the signal recognition particle (SRP; Walter et al., 1981). Following its recruitment to the nascent pep-
tide, the SRP induces a transient arrest of elongation and interacts with its receptor located in the rough ER to mediate
the correct translocation of the nascent protein into the ER lumen before translation is resumed (Wolin & Walter, 1988,
1989). Mutations of the signal peptide that impair SRP recruitment lead to a decrease in protein expression and degra-
dation of the corresponding mRNA (Karamyshev et al., 2014). Similarly, when SRP levels are down-regulated, the
mRNAs coding for secretory proteins are destabilized within the cell. The absence of the SRP at the ribosome exit tun-
nel was shown to allow the Argonaute2 (Ago2) protein to interact with the nascent peptide and the ribosome to trigger
mRNA degradation through a still uncharacterized mechanism that is independent of its slicing activity (Karamyshev
et al., 2014). However, Ago2 is not required for the degradation of all mRNAs encoding secretory proteins that fail to
recruit SRP, thus suggesting that other proteins could be involved in this degradation pathway (Pinarbasi et al., 2018).
Furthermore, it is not clear how the cell discriminates between mRNAs coding for secretory proteins that fail to recruit
an SRP from mRNAs coding for cytosolic proteins.
Another example of co-translational mRNA degradation through recognition of nascent peptides occurs for the reg-
ulation of tubulin expression. Indeed, the levels of alpha and beta tubulins are tightly controlled within cells, through
an autoregulation mechanism that induces the degradation of tubulin mRNAs when the concentration of soluble tubu-
lin proteins (i.e., non-polymerized) is above a certain threshold (Cleveland, 1988; Cleveland et al., 1981; Gasic
et al., 2019). This regulation occurs co-translationally and depends on the recognition of the first four residues of the
alpha and beta tubulin proteins (Met–Arg–Glu–Ile for β-tubulin and Met–Arg–Glu–Cys for α-tubulin) by the
tetratricopeptide protein 5 (TTC5) (Bachurski & Cleveland, 1994; Gay et al., 1989; Lin et al., 2020; Pachter, 1987; The-
odorakis & Cleveland, 1992; Yen et al., 1988). TTC5 associates with nascent tubulin peptides and contacts the ribosome
near the exit tunnel through interactions with 28S rRNA and ribosomal protein uL24 (Z. Lin et al., 2020). Mutation of
TTC5 residues that interact with ribosomes leads to loss of the regulation. The authors also found that, when soluble
tubulin levels are low, the binding of TTC5 to nascent tubulin peptides is prevented by a still unknown factor. In con-
trast, the activity of this factor is inhibited whenever cells detect an excess of soluble tubulin, thus allowing TTC5 to
bind to nascent tubulin peptides and ribosomes and to induce mRNA degradation. The precise mechanism by which
TTC5 mediates mRNA degradation is yet to be characterized. It could rely on the recruitment of mRNA decay factors
to TTC5-mediated stalled ribosomes or on the recognition of a specific ribosomal conformational change induced
by TTC5.
Altogether, these studies highlight the crucial role of ribosomes in integrating multiple signals originating from the
nascent peptide or from cis-acting elements within mRNAs to mediate co-translational degradation of functional
mRNAs.
4.3 | The ribosome and associated quality control mechanisms
In addition to the emerging role of the ribosome as a platform for a large number of interactors, ribosome
ubiquitination appears to be crucial, particularly in the context of ribosome collisions. Obviously, collided ribosomes
are detrimental to protein homeostasis and thus require mechanisms to cope with them. The vast majority of insights
about ribosome collisions has been obtained from model substrates consisting of aberrant mRNAs with stalling
sequences which, when translated, engender the leading ribosome to stall, causing collisions with trailing ribosomes
(see the Sections 2.2 and 2.3 about the NGD and NSD mRNA surveillance pathways). A few studies have now sought to
systematically identify the proteins associated with collided ribosomes. Widespread ribosomal collisions can be induced
using low-doses of the elongation inhibitor emetine. Using quantitative proteomics on polysome fractions from cells
treated with low-dose emetine, two groups identified the protein Endothelial differentiation-related factor 1 (EDF1) as
being the new first-acting ribosome collision sensor (Juszkiewicz et al., 2020; Sinha et al., 2020). Cryo-EM structure
reveals that EDF1 and its highly conserved yeast homolog Mbf1 bind at the disome interface close to the mRNA entry
channel (Sinha et al., 2020). If collisions persist, EDF1 is needed for stabilization of both the E3 ubiquitin ligase
ZNF598 and the protein Grb10-interacting GYF (glycine-tyrosine-phenylalanine) domain protein 2 (GIGYF2) at col-
lided ribosomes (Juszkiewicz et al., 2020; Sinha et al., 2020). GIGYF2 and ZNF598 were previously found to interact
with the eIF4E-homologous protein (4EHP) (Morita et al., 2012), which inhibits translation by competing with eIF4E
for binding to the 50 -cap structure (Rom et al., 1998). Several findings suggest that both GIGYF2-4EHP and ZNF598
aim to minimize ribosome collisions: the translational repressor GIGYF2-4EHP blocks new rounds of translation onto
problematic mRNAs and marks these transcripts for decay whereas ZNF598 catalyzes 40S ribosomal subunit
ubiquitinations required for ribosome rescue by the ribosome-associated quality control (RQC) pathway (Hickey
et al., 2020; Juszkiewicz et al., 2020; Sinha et al., 2020; Weber et al., 2020; Figure 4). In particular, ZNF598 is able to dis-
criminate between transient or long-lasting collisions and is essential to resolve ribosome queuing that can form on
mRNAs containing ribosome stalling sequences (Goldman et al., 2021). In addition to ZNF598, a second E3 ubiquitin
ligase was reported to function in RQC. The makorin ring finger protein 1 (MKRN1) binds to prematurely
polyadenylated transcripts and ubiquitinates the ribosomal protein eS10 and PABPC1 (Hildebrandt et al., 2019). While
it is clear that ubiquitinations are key for proper rescue of stalled ribosomes, their precise role and temporal sequence
are just beginning to emerge. It has been found that the ZNF598-catalyzed ubiquitinations are reversed by the
deubiquitinating enzymes USP21 and OTUD3 (Garshott et al., 2020). Furthermore, the authors reported that
ubiquitination events are hierarchically organized, suggesting the existence of an ubiquitin code on ribosomes. Alterna-
tively, ubiquitination marks may serve as a scaffold for recruiting downstream quality control factors involved in resolu-
tion of ribosome stalls.
4.4 | The ribosome and associated stress responses
A novel function was ascribed to ribosome collisions, apart from their role in quality control. Two studies have now
shown that general cellular stress conditions induce high levels of ribosome collisions and that the resulting colliding
ribosomes are a key signaling node to coordinately regulate stress responses (C. Wu et al., 2020; Yan & Zaher, 2020).
Specifically, a first study in human cells revealed that cellular stress caused by amino acid starvation or UV irradiation
induce a large number of colliding ribosomes that serve as a platform to recruit the kinase ZAKα (C. Wu et al., 2020).
This protein is a common activator of the p38/JNK-mediated ribotoxic stress response and the integrated stress response
(ISR), the core event of which is the phosphorylation of the initiation factor eIF2α by one of four kinases, including
GCN2. The second study in yeast showed that alkylation or oxidation stresses promote ribosome collisions and trigger
both the Gcn2-mediated ISR pathway and the RQC pathway activated by the Hel2 ubiquitin ligase (ZNF598 in mam-
mals; Yan & Zaher, 2020). Notably, an apparent competition was observed between these two pathways, suggesting that
ISR and RQC must be coordinated somehow. What seems to emerge from these two studies is that the extent of ribo-
some collisions is the major determinant of which signaling pathway should be activated. In unstressed cells, basal
levels of colliding ribosomes, which reflect occasional problematic mRNAs, are suggested to induce the RQC pathway
in order to degrade the aberrant transcript and nascent peptide. In stressed cells, intermediate levels of ribosome colli-
sions could activate the GCN2-mediated ISR pathway that blocks global translation initiation but induces translation of
specific mRNAs to promote cell survival. In contrast, high levels of colliding ribosomes are thought to trigger the
p38/JNK-mediated stress response in order to induce apoptotic cell death. From this perspective, ZAKα would have a
role in monitoring the extent of ribosome collisions.
5 | C O D O N AN D A M I N O AC I D US A G E M O D U L A T E m R N A S T A B I L I T Y
Because the genetic code is degenerate, most amino acids, except methionine and tryptophan, are encoded by multiple
codons known as synonymous codons. In eukaryotes, the number of tRNA genes in the genome can vary from 275 in
yeast (S. cerevisiae) to 416 in humans, and more than 8000 in zebrafish (P. P. Chan & Lowe, 2016). However, in all
known organisms there are fewer tRNA anticodons than the 61 codons encoding the 20 canonical amino acids. The
limited number of available anticodons is compensated by the possibility of non-Watson–Crick base pairings between
the first position of the anticodon and the third position of the codon, known as the wobble decoding (Crick, 1966).
Usage of synonymous codons is not random and differs between species and within genes for a given organism in
what is described as “codon usage bias” (Grantham et al., 1980). Codon usage bias is thought to depend on global geno-
mic GC content, resulting from mutational bias, and on selective pressure acting on coding sequences (Chen
et al., 2004). Codon usage has been shown to affect ribosome decoding speed and co-translational folding of nascent
proteins (Drummond & Wilke, 2008; Yu et al., 2015; Zhao et al., 2017; T. Zhou et al., 2009). In some bacteria, lower
eukaryotes (such as S. cerevisiae, C. elegans, and D. melanogaster) and plants (A. thaliana), codon usage has been shown
to be biased towards the most abundant tRNAs (Duret & Mouchiroud, 1999; Grantham et al., 1980; Gutman &
Hatfield, 1989; Ikemura, 1985). Furthermore, codon usage bias is positively correlated with transcript abundance in
these species, leading to the most abundant transcripts being further enriched for codons corresponding to abundant
tRNAs (Duret & Mouchiroud, 1999; Gouy & Gautier, 1982; Ikemura, 1985; Sharp & Li, 1986, 1987). These features sup-
port a “translational selection” model, where codons decoded by the most abundant tRNAs are positively selected
among highly expressed genes, to optimize translational efficiency and accuracy (Akashi, 1994; Moriyama, 1998).
F I G U R E 4 Ribosome-associated quality control mechanisms. Upon ribosome collision induced by problematic mRNAs, EDF1 is
recruited first at the disome interface (Panel 1), and then it stabilizes the interaction of ZNF598, GIGYF2, and 4EHP with collided ribosomes
(Panel 2). These proteins act in two distinct ways to minimize ribosome collision: (1) GIGYF2-4EHP represses initiation of new rounds of
translation by competing with eIF4E for binding to the cap structure and marks problematic mRNAs for decay (Panel 3). (2) ZNF598
ubiquitinates several 40S ribosomal proteins and these ubiquitinations are required for the ribosome-associated quality control involved in
degradation of the nascent peptide and rescue of collided ribosomes. The ubiquitinations can be reversed by the deubiquitinases USP21 and
OTUD3 (Panel 4)
Consistent with this hypothesis, ribosome profiling assays in S. cerevisiae and N. crassa in the absence of translation
inhibitors show a negative correlation between tRNA abundance and ribosome elongation dwell time (Dana &
Tuller, 2014; Gardin et al., 2014; Hanson et al., 2018; Hussmann et al., 2015; Weinberg et al., 2016). Nevertheless, tRNA
levels do not exclusively explain codon decoding rates and other factors including wobble base-pairing, the nature of
the amino acids at the P-site and A-site (defining peptidyl transfer time) and at proximity of the exit tunnel of the ribo-
some have also been shown to modulate the dwell time on a given codon (Gardin et al., 2014; Stadler & Fire, 2011).
To quantify the degree of synonymous codon usage bias within an organism and between species, different metrics
have been developed, each with their own pros and cons. For example, the Codon Adaptation Index (CAI) calculates
the relative synonymous codon usage (RSCU) value from a restricted set of highly expressed transcripts in a given
organism (Sharp & Li, 1987). The obtained values are then used to calculate a score for each gene within that same
organism. It is a simple and powerful metric to monitor RSCU, but it is based on the assumption that translational
selection occurs within the studied organism. To quantify translational selection, the tRNA adaptation index (tAI) was
created (Reis et al., 2004) that takes into account tRNA abundance and the binding strength of the codon–anticodon
coupling to measure how well a given mRNA is adapted to the available tRNA pool (using tRNA gene number as a
proxy for tRNA abundance). Using the tAI, authors were able to study the degree of co-adaptation between codon usage
and tRNA levels in a wide range of organisms and quantify the extent of translational selection acting on codon usage
(Reis et al., 2004). Interestingly, their results indicate that genome size and tRNA gene number act in combination to
determine the extent of translational selection on codon usage bias. Above a certain genome size, translational selection
should no longer be observed. This raises many questions regarding the impact of codon usage and translational selec-
tion in lower eukaryotes with small genomes, and clear evidence of translational selection, compared with higher
eukaryotes with larger genomes and a relatively restricted number of tRNA gene copies.
5.1 | Codon usage is a major determinant of mRNA decay rates in yeast
Evidence for codon usage affecting mRNA stability was first proposed in S. cerevisiae, by manipulating the codon con-
tent of the highly expressed gene PGK1, which is enriched for synonymous codons corresponding to the most abundant
tRNA species (Hoekema et al., 1987). Replacing “optimal” codons (i.e., those corresponding to the most abundant
tRNAs and over-represented in highly expressed genes) within the PGK1 coding region by “non-optimal” synonymous
codons resulted in a 10-fold reduction of protein output and a 3-fold reduction of steady-state mRNA levels, which was
attributed to decreased mRNA stability (Hoekema et al., 1987). Further evidence linking codon usage to mRNA stability
was obtained by direct measurement of mRNA decay rates for 20 different yeast endogenous mRNAs, where authors
found that mRNA half-life was correlated to the percentage of rare codons within the ORF (Herrick et al., 1990). Proof
of a direct role for translation elongation and codon usage was obtained by expressing chimeric mRNAs corresponding
to in-frame fusions of the stable PGK1 or ACT1 transcripts with that of the unstable MATα1 transcript (Caponigro
et al., 1993; Parker & Jacobson, 1990). This approach led to the identification of a stretch of rare codons in the coding
region of MATα1 that was required, but not sufficient, to induce decay of chimeric mRNAs. Both insertion of a stop
codon upstream of the stretch of rare codons from MATα1 or translation inhibition with cycloheximide led to a stabili-
zation of the chimeric mRNAs, implying that ribosomes elongating across the stretch of rare codons are required to
induce mRNA degradation (Caponigro et al., 1993; Parker & Jacobson, 1990). This observation was generalized in a
transcriptome-wide study monitoring mRNA decay in S. cerevisiae (Presnyak et al., 2015). Using a new metric described
as the codon occurrence to mRNA stability correlation coefficient (CSC), that corresponds to the Pearson correlation
between the frequency of each codon within the coding region in mRNAs and their corresponding half-lives, Presnyak
et al were able to identify codons associated with mRNA stability or instability. Interestingly, CSC values in S. cerevisiae
were found to be positively correlated with codon optimality as defined by the tAI metric (Reis et al., 2004), and linked
to the speed of ribosome decoding (Figure 5). More precisely, mRNA stability correlates with the codon-specific elonga-
tion rate at the A-site of the ribosome but not with amino acid identity or codon usage at the P-site and E-site (Hanson
et al., 2018). This indicates that among all steps that can influence the dynamics of translation elongation, the kinetics
of tRNA recruitment at the A-site is the only one that is probed by the cell to induce mRNA decay in yeast. Further dis-
section of the molecular determinants linking codon usage and mRNA decay in S. cerevisiae identified the DEAD-box
protein Dhh1p (DDX6 in mammals) as a key factor implicated in monitoring the decoding speed of ribosomes and in
inducing translational repression and degradation of transcripts with non-optimal codons (Radhakrishnan et al., 2016;
Sweet et al., 2012).
In addition to Dhh1p, the Ccr4–Not complex is involved in monitoring codon optimality. The yeast Ccr4–Not core
complex, composed of two catalytic subunits (Ccr4 and Caf1) and five Not proteins (Not1 to 5) (H.-Y. Liu et al., 1998),
catalyzes mRNA deadenylation. Furthermore, the Ccr4–Not complex interacts with 80S ribosomes and represses trans-
lation of mRNAs with stalled ribosomes (Preissler et al., 2015). Moreover, the Not4 and Not5 core subunits have been
shown to mediate ubiquitination of the ribosomal protein eS7 and translational control (Panasenko & Collart, 2012;
Villanyi et al., 2014). In a study aimed at understanding the role of Pab1 in regulating the activity of the Ccr4–Not com-
plex, Webster et al (Webster et al., 2018) showed a direct interaction between Pab1 and the Ccr4–Not complex, which is
required for efficient bulk poly(A) shortening mostly catalyzed by Ccr4. In contrast, Caf1 is not necessary for bulk
mRNA deadenylation but is required for deadenylation of mRNAs bearing non-optimal codons (Figure 5(c)). In this
case, Caf1 acts in a translation-dependent manner, as introduction of a stem–loop structure in the 50 -UTR to block ribo-
some loading relieves Caf1-dependent deadenylation of such transcripts. Interestingly, the authors observed a positive
correlation between Pab1 binding and codon optimality, arguing that translation elongation rates could have an impact
on the relative abundance of Pab1 associated with transcripts. In this model, mRNAs with optimal codons and high
Pab1 loading are deadenylated by the Ccr4–Not complex through the action of the Ccr4 nuclease alone, while
deadenylation of mRNAs with non-optimal codons and low Pab1 binding is enhanced by the combined action of Ccr4
and Caf1. This step acts upstream of the activity of Dhh1p, since yeast lacking the DHH1 gene still show Caf1-depen-
dent deadenylation of mRNAs with non-optimal codons. Further refining the role of Ccr4–Not in linking codon
(a) (b)
(c)
(d) (e)
F I G U R E 5 Codon-dependent mRNA decay in yeast and zebrafish. In yeast, the kinetics of tRNA recruitment at the A-site of elongating
ribosomes is a major determinant of mRNA stability. (a) mRNAs with optimal codons (corresponding to abundant tRNAs) mediate efficient
recruitment of tRNAs in the A-site of elongating ribosomes and display long half-lives. (b) If peptidyl transfer is slow but an optimal codon
resides at the ribosome A-site, the ribosome will bear an empty E-site and an accommodated A-site. This conformation allows recruitment of
eIF5A to the E-site to promote peptidyl transfer activity and rescue the stalled ribosome. (c) If a non-optimal codon is present at the
ribosome A-site, tRNA accommodation will take longer than for an optimal codon and this increases the probability of the ribosome to bear
empty E-site and A-site. This specific post-translocation conformation favors the recruitment of the Ccr4–Not complex in the E-site, which
induces eS7 ubiquitination, mRNA deadenylation through the Caf1 subunit and stimulates Dhh1p recruitment to induce mRNA
degradation. (d) In zebrafish, a similar Ccr4–Not-dependent mechanism probes codon optimality to induce mRNA degradation in a
translation-dependent manner. However, in addition to codon usage, amino-acid content can also mediate mRNA destabilization through a
still uncharacterized mechanism. (e) In zebrafish, the distance between the non-optimal codon and the poly(A) tail acts as a buffer against
the deadenylase activity of the Ccr4–Not complex
optimality to mRNA deadenylation and decay in yeast, a second study by Buschauer et al. purified ribosomes bound to
Ccr4–Not to perform cryo-EM analyses (Buschauer et al., 2020). Their results indicate an association of the entire Ccr4–
Not complex with monosomes and polysomes and, more specifically, an interaction of the N-terminal domain (NTD) of
Not5 with the E-site of post-translocation ribosomes carrying a peptidyl-tRNA in the P-site and a vacant A-site
(Figure 5(c)). Incubation of cells with cycloheximide, that favors the pre-translocation state with an accommodated
tRNA in the A-site, decreases Ccr4–Not binding to ribosomes and increases eIF5A binding to the E-site, indicating spe-
cific recruitment of the Ccr4–Not complex to ribosomes with a vacant A-site (Figure 5(c)). Furthermore, ribosome pro-
filing of Ccr4–Not-bound ribosomes revealed an enrichment for non-optimal codons at the A-site of ribosome
footprints, as defined by their CSC or tAI values. Deletion of Not5–NTD impairs Dhh1p recruitment on ribosomes with
empty A-sites and reduces their decapping, therefore linking the role of Ccr4–Not in monitoring translation elongation
rates with the downstream activity of Dhh1p in mediating mRNA decapping leading to decay. Binding of Not5–NTD to
the ribosome E-site was finally shown by authors to depend on the prior ubiquitination of eS7 by Not4 (Figure 5(c)).
Such a mechanism allows cells to monitor translation elongation speed and to discriminate between ribosomes
displaying slow decoding and those showing slow peptidyl transfer: slow-decoding ribosomes, characterized by a
peptidyl-tRNA loaded in the P-site and a vacant A-site, induce Ccr4–Not deadenylation and Dhh1p decapping followed
by 50 -to-30 degradation (Figure 5(c)) whereas ribosomes displaying slow peptidyl transfer, characterized by a peptidyl-
tRNA loaded in the P-site and a tRNA accommodated in the A-site, are rescued by eIF5A (Figure 5(b)), which promotes
peptidyl transferase center activity (Schuller et al., 2017).
5.2 | Codon and amino acid usage coupled to GC content regulate mRNA decay in
metazoans
In addition to yeast, several pieces of evidence support that the role of codon usage in mediating mRNA decay may be
conserved across most eukaryotes, including metazoans. However, the constraints imposed by multicellular organisms
with large genomes suggest a more complex relationship between codon usage, tRNA expression, and mRNA decay
than the one observed in yeast. In particular, additional factors such as amino acid identity, GC content, UTR length,
and RNA binding proteins appear to modulate the impact of codon usage on mRNA stability or even drive translation-
dependent mRNA decay independently from it.
Two studies performed in zebrafish uncovered a role for codon usage in mediating translation-dependent degrada-
tion of maternal mRNAs during the maternal to zygotic transition (MZT), similar to yeast (Bazzini et al., 2016;
Mishima & Tomari, 2016). During the late stages of oogenesis, the maternal genome becomes transcriptionally silent
and relies on stored mRNAs and proteins to undergo the meiotic maturation, fertilization and first steps of embryo
development (Tadros & Lipshitz, 2009). Degradation of maternal mRNAs during MZT is an essential step during
embryogenesis that allows reprogramming of gene expression from the maternal genome to the zygotic genome. In
order to identify cis-acting factors involved in maternal mRNA degradation, Bazzini et al. used an elegant approach
relying on the use of synthetic reporter mRNAs with invariable 50 -UTR and 30 -UTR sequence, but with variable codon
composition, to demonstrate that mRNA stability during the MZT was modulated by codon usage (Bazzini et al., 2016).
Similar results were obtained with endogenous mRNAs upon transcriptional inhibition, which allowed them to identify
optimal and non-optimal codons, using the CSC metric described by Presnyak et al. (2015). Consistent with findings
obtained in S. cerevisiae, the authors found a mild but significant correlation between tRNA levels and codon optimality
as well as a clear correlation between codon usage in the transcriptome and codon optimality (Figure 5(d)). Moreover,
mRNAs with non-optimal codons display shorter poly(A) tails and a lower translational efficiency. Finally, the authors
uncovered a translation-dependent effect of specific amino-acids in regulating mRNA stability (Figure 5(d)), thus
suggesting that metazoans, in contrast to S. cerevisiae, have evolved additional mechanisms to probe for translation
elongation dynamics. Mishima and Tomari (2016) also found that codon usage is a major determinant of maternal
mRNA stability during the MZT in zebrafish and that the exonuclease activity of the Ccr4–Not complex is responsible
for co-translational poly(A) tail shortening of mRNAs bearing uncommon codons (Figure 5(e)). Surprisingly, the
authors also found that long 30 -UTRs could protect mRNAs with uncommon codons from degradation. This protective
role was sequence-independent but, instead, relied on the distance between the poly(A) tail and the uncommon codons
(Figure 5(e)). Altogether, these results argue that Ccr4–Not acts as a sensor of ribosomes with vacant A-sites, both in
yeast and metazoans. Further supporting this conclusion, Not5 (CNOT3 in metazoans), which is involved in anchoring
Ccr4–Not to the ribosome is highly conserved across eukaryotes (Buschauer et al., 2020).
Interestingly, although zebrafish has a large genome (1.5 billion base pairs), it does possess an unusual large num-
ber of tRNA genes (>8000) (P. P. Chan & Lowe, 2016), which could be compatible with translational selection acting
on codon usage (Reis et al., 2004), as observed in S. cerevisiae. However, organisms with large genomes and a limited
number of tRNA genes (as it is the case for most mammals) should be less prone to translational selection on codon
usage, thus raising the question of whether codon usage could have an impact on mRNA translation and stability in
those species. Numerous studies have addressed this question in different mammalian species (human, mouse, and Chi-
nese hamster) using different technical approaches to monitor mRNA decay rates and the impact of codon usage
(Forrest et al., 2020; Narula et al., 2019; Shu et al., 2020; Q. Wu et al., 2019). Q. Wu et al. (2019) and Narula et al. (2019)
both used a library of reporter genes with fixed UTRs and variable composition of the coding sequence, combined with
non-invasive metabolic labeling of nascent transcripts or transcription inhibitors, to monitor the impact of codon usage
on mRNA stability in HEK 293T, HeLa, RPE, and K562 cells. Forrest et al. (2020) and Shu et al. (2020) relied on meta-
bolic labeling of nascent transcripts to monitor mRNA stability of endogenous transcripts in HeLa, CHO, and primary
mouse neurons. Interestingly, despite the methodological differences, most studies have yielded similar CSC values,
which validates a role for codon usage in regulating mRNA stability in mammals and indicates a conservation of opti-
mal and non-optimal codons between these organisms, independent of the cell type (Shu et al., 2020). Although the
results are not as robust as those observed in yeast, the authors showed that, in human cells, the effects of codon usage
on mRNA decay rates are explained, to some extent, by the tRNA abundance and speed of codon decoding (Forrest
et al., 2020; Narula et al., 2019; Shu et al., 2020; Q. Wu et al., 2019; Figure 6(a)). In agreement with this, overexpression
of specific tRNAs in metastatic cells was shown to increase translation and stability of mRNAs enriched with cognate
codons (Goodarzi et al., 2016). Similarly to findings in yeast and zebrafish, transcripts with non-optimal codons in
mammals display a shorter poly(A) tail. However, the presence of a poly(A) tail is not required for codon-dependent
mRNA degradation, since transcripts with non-optimal codons bearing a histone tail instead of a poly(A) tail are still
prone to degradation (Q. Wu et al., 2019). Finally, in addition to a role of synonymous codons in inducing mRNA decay,
Forrest et al also identified several amino-acids whose incorporation is associated with mRNA stabilization or destabili-
zation (Forrest et al., 2020) (Figure 6(a)). Therefore, both synonymous codons and specific amino acids are able to mod-
ulate mRNA stability in mammals, similarly to zebrafish but contrary to S. cerevisiae.
Surprisingly, in all but one of the aforementioned studies, a clear bias in the GC content at the wobble position can
be observed among optimal and non-optimal codons (Figure 6(b)), although this specific finding was not highlighted by
authors. Independently of whether endogenous mRNAs or ORFeome reporters were studied, codons enriched for cyti-
dine and guanine at the wobble position (GC3) are typically associated with positive CSCs and classified as optimal
(Figure 6(b), see “Optimal Codons”), while codons enriched for adenine and uridine at the wobble position (AU3) are
typically associated with negative CSCs and classified as non-optimal (Figure 6(b), see “Non-optimal Codons”). GC3
bias is particularly strong in HEK 293T and K562 cells, where up to 100% of optimal codons are GC3 and up to 93% of
non-optimal codons are AU3 (Figure 6(b), Conditions 1–6). Further supporting a role of GC3 content in modulating
translation-dependent mRNA degradation in mammals, a study by Hia et al. performed an analysis of the frequency of
codons across all human coding sequences and found that 22% of the total variance is explained by GC3 content, corre-
lating with CSC values obtained from HEK 293 cells (Hia et al., 2019). They also found that GC3-rich transcripts tend
to be more stable than AU3-rich transcripts, both on a transcriptome-wide scale and when using reporter constructs
with different GC3 content, which agrees with the GC3 bias observed between optimal and non-optimal codons in
other studies. Authors also found that genes with optimal GC3-rich codons display higher translation efficiencies than
transcripts enriched with AU3 codons, as measured by ribosome profiling (Hia et al., 2019).
In agreement with these observations, it has been shown that 70% of synonymous codon usage within human genes
is explained by local GC3 content, which is caused by the process of GC-biased gene conversion (gBGC) that occurs dur-
ing meiotic recombination in mammals, favoring transmission of GC-alleles over AT-alleles (Pouyet et al., 2017).
Accordingly, there should be limited room for other selective processes to act on codon usage at a global scale in mam-
malian genomes. This does not exclude an impact of synonymous codon usage and GC3 content on the regulation of
mRNA translation and decay, as clearly shown by the above-mentioned studies. However, it raises many questions
regarding the mechanisms evolved by mammalian cells to cope with the gBGC-driven content bias.
One possible mechanism could be through the regulated expression or aminoacylation of tRNAs. Indeed, it has been
proposed that genes related to cellular proliferation have different codon usage than those involved in differentiation.
Particularly, proliferation related genes tend to have an A or T at the third codon position, while differentiation related
genes tend to be enriched in C or G (Gingold et al., 2014). In the same study, authors also found evidence of coordina-
tion in the gene expression pattern of tRNAs and mRNAs during cell differentiation and cancer where tRNA
corresponding to the codons enriched in proliferation or differentiation are differentially expressed to match the codon
demand associated with differential transcript expression. A GC content bias in the codon composition of differentially
expressed genes in differentiating and proliferating cells has also been described in other studies although no-associated
changes in tRNA expression were found (Bornelöv et al., 2019; Guimaraes et al., 2020; Rudolph et al., 2016). Neverthe-
less, changes in tRNA charging or editing of specific anticodon loops were found to occur and mediate changes in ribo-
some density that could potentially regulate mRNA stability (Bornelöv et al., 2019; Guimaraes et al., 2020).
A second possible mechanism to explain the impact of GC3 content in regulating mRNA stability could be through
the action of RNA-binding proteins (RBPs) with biased binding specificities. Hia et al identified several RBPs, preferen-
tially bound to either GC3-rich or AU3-rich transcripts (Hia et al., 2019). Among these, ILF2 and ILF3 were shown to
interact with AU3-rich transcripts and induce their decay. The RBP Fragile X mental retardation protein (FMRP) also
functions in regulating mRNA decay through codon usage in mouse neurons (Shu et al., 2020) and CSC values in neu-
rons show a clear bias in favor of GC3 among optimal codons and AU3 among non-optimal codons (Figure 6(c)). Loss
of FMRP induces a complete reshuffling of CSC values in neurons, leading to optimal codons showing a clear bias in
(a)
(b) (c)
F I G U R E 6 GC3 content, amino acid identity, and RBPs modulate translation-dependent mRNA degradation in mammals. (a) Possible
factors implicated in codon-dependent mRNA destabilization in mammals. (b) Barplot representing the percentage of GC3 and AU3 codons
among optimal and non-optimal codons as defined for endogenous mRNAs or ORFeome reporters with fixed UTRs and variable codons in
the CDS from different cell types as obtained from the literature. In most studies, optimal codons are mainly GC3, while non-optimal codons
are enriched in AU3. (c) FMRP deficiency in mouse primary neurons leads to an inversion in the GC3 and AU3 content of optimal and non-
optimal codons
favor or AU3 and non-optimal codons enriched for GC3 (Figure 6(c)). FMRP was shown by authors to bind preferen-
tially to mRNAs with optimal codons, independently of the overall transcript GC content (but not GC3 content), thus
suggesting a requirement for active translation of GC3-rich codons to mediate FMRP-dependent effects (Shu
et al., 2020). Finally, GC and GC3 content have been shown to directly regulate mRNA storage within P-bodies and dic-
tate the decay pathway of AU-rich and GC-rich transcripts in human cells (Courel et al., 2019). AU-rich transcripts are
typically enriched in P-bodies, contain rare codons and are poorly translated, while GC-rich transcripts contain fre-
quent codons and are efficiently translated. Interestingly, in this study, DDX6 (the mammalian homolog of Dhh1p) was
shown to bind to GC-rich transcripts enriched with high usage codons and to regulate their degradation via the XRN1
50 -to-30 exonuclease. On the contrary, DDX6 regulates the translation of AU-rich transcripts that are mainly degraded
by the PAT1B 30 -to-50 exonuclease (Courel et al., 2019). Together, these results indicate an important role for several
RBPs in affecting stability of mammalian transcripts in a codon and GC specific manner. However, their precise mecha-
nism of action remains to be fully characterized. In particular, the role of DDX6 could apparently differ between mam-
mals and yeast. Additionally, it is currently unclear to what extent the differences in translation and stability observed
between GC-rich and AU-rich transcripts are driven by the primary sequence or RNA secondary structures
(GC content and secondary structures being highly correlated). In an attempt to disentangle the two factors, a study
reported that both secondary structure and codon usage appear to modulate translation efficiency and the functional
half-life of transfected synthetic transcripts in a coherent but independent manner (Mauger et al., 2019). Transcripts
with extensive secondary structures display higher ribosome loading and longer functional half-lives. Additional work
will be necessary to better understand the precise role of codon usage, GC content and RNA secondary structure in reg-
ulating translation and decay of mammalian mRNAs.
Altogether, the role of codon usage in modulating mRNA degradation appears much more complex in mammals
than in unicellular organisms. This is likely due to additional layers of gene regulation and differences in the selective
pressures that shape the nucleotide composition at the genomic scale. Nevertheless, in spite of this complexity, there is
evidence that, tRNA availability plays an important role in addition to RNA-binding proteins. In humans, expression of
tRNAs is not perfectly correlated to tRNA copy number (Behrens et al., 2021), thus indicating a potential level of regula-
tion. Accordingly, the expression of tRNA isodecoders has been shown to differ significantly between tissues (Pinkard
et al., 2020; Schmitt et al., 2014). However, in spite of the observed differences in isodecoder expression, the global anti-
codon pool (integrating all isodecoders together) remains very similar across tissues (Pinkard et al., 2020; Schmitt
et al., 2014). More work is necessary to understand how tRNA expression and post-transcriptional modification are
dynamically regulated during cell differentiation and proliferation and how these changes affect translation and mRNA
stability. Furthermore, numerous changes in tRNA expression or sequence are linked to pathological conditions such
as cancer and there is evidence that these changes can affect both translation and stability of mRNAs (Goodarzi
et al., 2016; Lant et al., 2019; Schaffer et al., 2019).
5.3 | Codon pair identity regulates mRNA translation and stability in yeast and
mammals
Similarly to synonymous codon usage bias, synonymous codon pairs display a non-random frequency distribution
that is independent from codon usage bias in many organisms (Buchan et al., 2006; Gutman & Hatfield, 1989).
Codon pair usage has been shown to regulate translation elongation and translation efficiency in yeast (Gamble
et al., 2016). In this process, inhibitory codon pairs were shown to involve wobble decoding to slow-down ribosome
elongation as the expression of non-native tRNAs with exact match to anticodons was able to suppress the inhibi-
tory effect of codon pairs. In addition to the observed effect on translation elongation, inhibitory codon pairs in
yeast are associated with shorter mRNA half-lives, even when taking into account the GC content, individual
codon information and the nature of dipeptides (Harigaya & Parker, 2017). Therefore, individual codons and
codon-pairs are able to modulate mRNA stability independently in yeast, although the molecular mechanism of
the latter has not been characterized yet.
In mammals, modifying codon pair usage to favor rare codon pairs is a common strategy to attenuate virus replica-
tion (Coleman et al., 2008; Shen et al., 2015). Moreover, specific synonymous codon pairs at the P-site and A-site of
elongating ribosomes have been shown to be more frequently associated with ribosome collisions (Arpat et al., 2020).
However, it is unclear from these works whether codon pair bias (CPB) itself or bias in dinucleotide composition (CpG
in particular since they are underrepresented in vertebrates genomes) are responsible for the observed effects. Using a
set of recoded Influenza A virus mutants with variable codon pair scores and CpG content, Groenke et al. were able to
disentangle the contribution of both features on virus attenuation (Groenke et al., 2020). They found that codon pair
deoptimization, but not changes in CpG frequency, are significantly associated with lower expression of viral proteins
and virus attenuation both in cultured cells and in vivo. Codon pair deoptimization is associated with a significant
increase in the degradation rates of viral RNAs, with a concomitant decrease in translation efficiency as measured by
ribosome profiling and metabolic labeling of nascent transcripts. Interestingly, a premature arrest of ribosomes at a dis-
crete position upstream of the stop codon was observed for one of the deoptimized transcripts, suggesting that codon
pair identity can effectively affect ribosome progression and lead to transcript degradation, probably through no-go
decay. Therefore, in addition to codon usage, CPB can also represent an important determinant of mRNA stability in
eukaryotic cells.
6 | I M P A C T O F T H E M6 A E P I T R A N S C R I P T O M I C MO D I F I C A T I O N ON
T R A N S LA T I O N AN D S T A B I L I TY OF m R N A s
It was long thought that the fate of mRNAs was strongly dependent on the nucleotide sequence, which has direct
effects on mRNA structure. In recent years, there is mounting evidence that nucleotides can be modified in ways that
confer additional regulation to mRNAs, leading to an emerging field termed “epitranscriptomics.” Recent advances in
RNA sequencing technologies have greatly helped to define the transcriptome-wide distribution and function of these
dynamic modifications that affect many steps of mRNA metabolism (X. Li et al., 2017). In this section, we aim to high-
light recent progress made in the elucidation of the molecular mechanisms by which the most prevalent mRNA modifi-
cation in eukaryotes, N6-adenosine methylation (m6A), regulate mRNA translation and/or stability.
The m6A mark, estimated to occur in half of human transcripts, is modulated by the coordinated action of multiple
proteins referred to as “writers,” “erasers,” or “readers,” which, respectively, add, remove, or bind a methyl group at the
6-amino position of adenosine to generate N6-methyladenosine (for a recent review see Shi et al., 2019). In brief, m6A is
co-transcriptionally catalyzed by the methyltransferase-like (METTL)3–14 heterodimer, in association with several aux-
iliary proteins (J. Liu et al., 2014). In addition, for a small number of transcripts, methylation is installed by the
METTL16 enzyme (Pendleton et al., 2017). The m6A modification can be reversed by the alkylated DNA repair protein
alkB homolog 5 (ALKBH5) or the demethylase fat mass and obesity-associated (FTO) protein (G. Jia et al., 2011; Zheng
et al., 2013). A variety of m6A-associated molecular events are mediated through multiple m6A readers that can be
roughly divided into three groups depending on their binding mechanism (Shi et al., 2019). One group comprises the
family of YTH domain-containing proteins (YTHDF1/2/3, YTHDC1/2) that directly and robustly bind to m6A marks on
mRNAs. A second category of m6A readers includes proteins that bind around or nearby m6A (HNRNPC, HNRNPG,
and HNRNPA2B1). Binding of these proteins to mRNAs is enhanced because the epitranscriptomic modification
induces a local remodeling of the RNA structure. A third class corresponds to RNA binding proteins that exhibit higher
affinity for m6A-containing transcripts (IGF2BP1/2/3 and FMR1). The exact underlying mechanism is unknown to
date. Additionally, the eIF3 translation initiation factor can bind m6A sites within the 50 -UTR of specific transcripts
(Meyer et al., 2015). Lastly, the METTL3 methylase, which acts as a m6A writer in the nucleus, was also shown to func-
tion as a m6A reader in the cytoplasm by recognizing the mark present on 30 -UTRs (S. Lin et al., 2016).
A number of studies have now provided valuable information on how m6A methylation can impact the fate of
mRNA. Several molecular machineries with different functions are recruited to m6A-modified transcripts via distinct
reader proteins. The emerging view is that m6A marks can promote translation and/or affect stability of certain tran-
scripts, depending on which reader protein is present, the mRNA region enriched in m6A, and the specific cellular con-
text. Regarding the m6A readers belonging to the YTH domain family (YTHDF1/2/3), the three proteins share a set of
common target mRNAs, the fate of which appears to be regulated in a cooperative and integrated manner (Shi
et al., 2017). Specifically, YTHDF1 binds preferentially m6A within the 30 -UTR and increases translation efficiency by
interacting with the eIF3 initiation factor (Wang et al., 2015). YTHDF3 also recognizes m6A sites in 30 -UTRs and exerts
a translation-promoting effect by functioning together with YTHDF1 (Shi et al., 2017). Additionally, YTHDF3 promotes
mRNA decay, either by recruiting the PAN2-PAN3 deadenylase complex (J. Liu et al., 2020) or by binding to YTHDF2
(Shi et al., 2017), which emerges as the main factor involved in decay of m6A-modified transcripts. Recent reports have
shown that YTHDF2 can promote mRNA degradation in two distinct ways. Firstly, YTHDF2 recruits the CCR4–Not
deadenylase complex to m6A-modified mRNAs, via its direct interaction with the CNOT1 component, thereby trigger-
ing deadenylation and subsequent 30 -to-50 decay of m6A-containing transcripts (Du et al., 2016; J. Liu et al., 2020). Sec-
ondly, degradation of m6A transcripts bound by YTHDF2 occurs via their association with the RNase P and its close
relative RNase MRP, two ribonucleoprotein complexes that function as endoribonucleases (O. H. Park et al., 2019). The
authors uncovered that the association of YTHDF2 with RNase P/MRP complex is bridged by the adaptor protein
HRSP12. Consequently, the YTHDF2–HRSP12–RNase P/MRP complex triggers the cleavage of m6A transcripts and the
resulting RNA fragments are subsequently processed by 50 -to-30 and 30 -to-50 exoribonucleases. Currently, it is not
known which particular cellular contexts preferentially lead to the activation of one or the other of these two
YTHDF2-mediated decay pathways.
In addition to the aforementioned combinatorial control between YTHDF1, YTHDF2, and YTHDF3 on stability of
certain m6A mRNAs, METTL3 binds to m6A marks in 30 -UTRs and promotes translation by interacting with the eIF3
initiation factor (Choe et al., 2018; S. S. Lin et al., 2016). Alternatively, eIF3 can directly bind to m6A marks in 50 -UTRs,
particularly under various stress conditions associated with severe attenuation of cap-dependent translation, and with a
large redistribution of m6A bases from 30 -UTRs to 50 -UTRs (Meyer et al., 2015). In line with this, 50 -UTR m6A
methylation is strongly increased in response to heat shock stress (J. Zhou et al., 2015). Furthermore, in response to
amino acid starvation, translation of the ATF4 transcript containing uORFs is up-regulated, in part, by the dynamic
removal of m6A in the 50 -UTR (J. Zhou et al., 2018). m6A in the 50 -UTR of ATF4 acts by controlling ribosome scanning
and alternative start codon selection. Together, these studies highlight the functional importance of m6A modification
in the regulation of translation initiation. Because the initiation step is a primary determinant of mRNA stability in
eukaryotes (see the Section 3 about translation initiation and mRNA decay), it is reasonable to think that these m6A-
related molecular mechanisms which have an impact on the initiation of translation are also very likely to influence
the stability of target mRNAs. Conversely, a m6A-linked stabilizing effect on transcripts also seems to confer a higher
translation efficiency. The m6A readers IGF2BP1/2/3, which bind to m6A sites predominantly in the 30 -UTR of thou-
sands of transcripts, increase the stability of their target RNAs and this stabilizing effect was found to enhance their
translatability (Huang et al., 2018).
In addition to the m6A-related effects mediated by the reader proteins, it has been suggested that m6A modifications
within the mRNA coding region could directly and locally influence the dynamics of translation elongation by affecting
the mRNA decoding process, meaning that the ribosome itself could be a m6A “reader-like.” Using a single molecule-
based in vitro translation system reconstituted from E. coli, Choi et al. found that codons with m6A engenders non-
optimal interactions with cognate anticodons in the ribosomal A-site, thus leading to a slower translation elongation
dynamics (Choi et al., 2016). Another study in mammalian cells also found that the presence of m6A sites within the
coding region of endogenous transcripts delayed translation elongation but, unexpectedly, if the formation of the m6A
marks was prevented, a supplemental decrease of translation was observed (Mao et al., 2019). In exploring this counter-
intuitive finding further, the authors found that m6A methylation within the coding region facilitates translation
elongation by resolving mRNA secondary structures. Moreover, YTHDC2, which is the only m6A reader with an RNA
helicase activity, is essential for destabilization of these mRNA structures. Based on their results, Mao et al proposed
that m6A residues located in coding regions reduce translation efficiency of unstructured mRNAs but promote that of
structured mRNAs, by resolving stable secondary structures. Consistent with this, it was previously reported that
YTHDC2 increases the efficiency of translation and subsequent degradation of its m6A target transcripts (Hsu
et al., 2017). Mechanistically, the effects of YTHDC2 on translation and decay of structured transcripts could be
explained by the interaction of YTHDC2 with the two helicases MOV10 and UPF1, and also the exoribonuclease XRN1
(Hsu et al., 2017; Kretschmer et al., 2018). Collectively, all the results presented in this section and illustrated on
Figure 7 emphasize that the m6A modification increases the level of sophistication in the tight interplay between
mRNA translation and decay.
7 | MICRORNAS, RIBOSOME DYNAMICS, AND mRNA DEGRADATION
The microRNAs (miRNAs) are a conserved class of small RNAs (~22 nt) present in metazoans that associate with
Argonaute proteins to mediate post-transcriptional regulation of mRNA targets (for a recent review see Bartel, 2018).
miRNAs recognize and bind their targets through Watson–Crick base pairing, either in a full complementarity manner
that induces target mRNA cleavage, or in a partial complementary manner allowing the recruitment of effector proteins
that mediate translational repression and mRNA degradation (Bartel, 2018).
The precise mechanism by which miRNAs mediate gene silencing is still a source of debate (for a comprehensive
review see Wilczynska & Bushell, 2015). Some studies suggest a two-step process where miRNAs first induce transla-
tional repression and then mRNA decay (Bazzini et al., 2012; Béthune et al., 2012; Djuranovic et al., 2012; Fabian
et al., 2009; Huntzinger et al., 2013; Larsson & Nadon, 2013; Meijer et al., 2013; Y. Mishima et al., 2012; Selbach
et al., 2008), while others suggest that miRNAs predominantly act through mRNA destabilization (Bagga et al., 2005;
Eisen et al., 2020; Eulalio et al., 2008; Guo et al., 2010). It is therefore possible that miRNAs could repress gene expres-
sion through a variety of mechanisms depending on the RNA target and miRNA-associated partners. Nevertheless, at
least for some miRNA-targeted mRNAs, there is strong evidence for a co-translational degradation, in a process
involving deadenylation followed by decapping and 50 -to-30 degradation by XRN1 (Antic et al., 2015; Tat et al., 2016).
Furthermore, the miRNA-induced silencing complex (miRISC) has been found to be associated with ribosomes (Jannot
et al., 2011; Maroney et al., 2006; Nottrott et al., 2006), thus suggesting a role for ribosomes in the degradation of
miRNA-targeted mRNAs. Interestingly, a recent report by Biasini et al. (2020) has shown that translation is required for
degradation of miRNA-targeted transcripts in mammalian cells. Indeed, the authors showed that cytoplasmic long non-
coding RNAs, although they are bound by Argonaute proteins, are not efficiently degraded by miRNAs contrary to
F I G U R E 7 Impact of the m6A methylation on mRNA fate. The m6A modification can regulate the fate of modified mRNAs in different
ways, that is, enhanced translation, mRNA decay, and mRNA stabilization, depending on which reader protein is present, the mRNA region
enriched in m6A, and the specific cellular context. The m6A readers that promote translation are YTHDF1, YTHDF3, METTL3, YTHDC2,
and eIF3. The m6A readers that promote mRNA decay are YTHDF2, YTHDF3, and YTHDC2. YTHDF2 induces mRNA decay by recruiting
either the CCR4–Not complex or the endonuclease RNase P/MRP via HRSP12. YTHDF3 promotes mRNA degradation by recruiting the
PAN2–PAN3 deadenylase complex. YTHDC2 exerts a dual function of stimulating both translation and decay of its targeted transcripts.
Also, YTHDF1/2/3 share common target mRNAs and their fate seems to be regulated in a coordinated manner. The ribosome can serve as a
scaffold for the m6A reader YTHDC2. Lastly, IGF2BP1/2/3 stabilize their target mRNAs
protein-coding mRNAs. However, when placed downstream of a protein-coding sequence, the long non-coding RNAs
are able to destabilize the mRNA in a miRNA-dependent manner. Finally, in zebrafish, codon usage was recently
shown to modulate the extent of miRNA-mediated mRNA degradation (Medina-Muñoz et al., 2021), thus suggesting a
cross-talk between these two co-translational regulatory pathways. Nevertheless, more work is needed to understand
the dynamics of these processes.
Besides the active role of miRNAs in inducing mRNA translational repression and degradation, translation can also
have an impact in modulating miRNA/siRNA target accessibility. Using single-molecule tracking of translating ribo-
somes and that of a reporter mRNA bearing siRNA-targets in its coding sequence, Ruijtenberg et al. have recently
shown that ribosome progression across the coding sequence can transiently dissolve RNA secondary structures, which
facilitates siRNA binding and subsequent RNA cleavage (Ruijtenberg et al., 2020). They also show that translation can
alleviate RNA secondary structures immediately downstream of the stop codon and improve siRNA accessibility and
cleavage of targets located in the 30 UTR. This is consistent with a previous report indicating that miRNAs are more
effective in inducing target mRNA repression when bound shortly downstream the stop codon (Grimson et al., 2007).
A similar mechanism involving an interplay between translation and targeting of coding regions by small RNAs has
been shown to mediate clearance of maternal mRNAs during the MZT in C. elegans (Quarato et al., 2021). In
C. elegans, the Argonaute protein CSR-1 and its associated guide RNAs (22G-RNAs) are maternally inherited in
embryos and are essential for embryonic development. CSR-1 is implicated in the clearance of maternally inherited
mRNAs through its slicing activity. Interestingly, a large fraction of CSR-1-associated 22G-RNAs are directed against
the coding sequence of maternally inherited mRNAs and the efficiency of mRNA slicing by CSR-1 is negatively corre-
lated with the translational efficiency of maternal mRNAs. Elongating ribosomes are therefore responsible for delaying
the degradation of maternal mRNAs by CSR-1 and could then act as a molecular clock to define the precise timing of
clearance through a mechanism that could be coordinated with 30 -UTR-dependent regulation of translation.
Taken together, these results open new perspectives regarding the role of translation in regulating the binding of
small RNAs and proteins within the coding sequence of mRNAs.
8 | R IB O S O M E - G U I D ED EN D O N U C L E O L Y T I C C L EA V A G E O F p i R N A
P R E C U R S O R TR A N S C R I P T S
As described throughout this review, the dynamics of ribosome progression is under constant scrutiny by the cell in
order to trigger rapid clearance of aberrant mRNAs and also to fine tune the half-life of functional transcripts, which
(a)
(b)
(c)
(d)
(e)
F I G U R E 8 Ribosome-dependent piRNA processing during mouse spermatogenesis. (a) piRNA precursors are capped and
polyadenylated RNAs that reach the cytoplasm and associate with the translational machinery. Ribosomes translate the 50 proximal short
ORFs to produce short peptides of unknown function. (b) Upon reaching the stop codon of short ORFs, ribosomes fail to dissociate from the
piRNA precursor and instead translocate downstream in a MOV10L1-dependent non-canonical manner. (c) The endonuclease PLD6 cleaves
the 50 end of the ribosome-protected piRNA precursor, followed by the non-canonical translocation of the ribosome to the next downstream
PLD6 cleavage site. (d) A PIWI protein is recruited at the 50 -extremity of the PLD6-generated fragment while a second ribosome-protected
PLD6-dependent cleavage event occurs downstream. (e) The 50 PIWI-bound RNA fragment is trimmed at its 30 end by PNLDC1 to produce
the mature piRNA sequence that is further chemically modified. The 30 ribosome-bound fragment associates with a new PIWI protein while
the ribosome translocates downstream to mark the next PLD6 cleavage site
depends on their sequence as well as structural and epitranscriptomic features. All the aforementioned mechanisms
participate in the catabolism of protein-coding mRNAs. However, a recent study has highlighted an unconventional
mechanism of ribosome-guided endonucleolytic cleavage that is involved in small RNA biogenesis (Sun et al., 2020).
PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs (21–35 nucleotides) that are generally
expressed in germ cells of animals and participate in transposon silencing through their association with PIWI proteins
of the Argonaute family (for a recent review see Ozata et al., 2019). Mouse piRNA genes are transcribed by RNA poly-
merase II to generate capped, spliced, and polyadenylated precursor transcripts that were thought to be translationally
silent (X. Z. Li et al., 2013). Instead, Sun et al. showed that a specific class of piRNA precursors expressed at the pachy-
tene stage of meiosis during spermatogenesis in mammals and birds (namely pachytene piRNAs) co-sediment with
actively translating ribosomes (Sun et al., 2020). Through ribosome profiling experiments, the authors showed that
piRNA precursors display ribosome occupancy with a clear 3-nucleotide periodicity on predicted ORFs near the 50 -end
(Figure 8(a)). Surprisingly, they could also detect ribosome occupancy downstream of annotated ORFs but without any
clear periodicity (Figure 8(b)). Instead, they noticed that the 50 -extremity of ribosome-protected fragments originating
downstream from ORFs coincided with the 50 -ends of mature piRNAs bound to PIWI proteins, thus suggesting that the
PLD6 endonuclease (an essential endonuclease in the piRNA processing pathway) uses ribosome protection to generate
the 50 -end of mature piRNAs (Figure 8(c,d)). Further understanding of the mechanism of ribosome-mediated piRNA
biogenesis identified the RNA helicase MOV10L1 as being essential for ribosome translocation downstream of 50 -
proximal ORFs upon their translation (Figure 8(c)). Consistent with this, incubation with puromycin or inactivation of
MOV10L1 expression within testis leads to a decrease in ribosome occupancy downstream of ORFs and impairs mature
piRNA expression. Upon ribosome-templated RNA cleavage by PLD6, 50 -cleaved RNA fragments are loaded onto PIWI
proteins and their 30 extremity further trimmed by PNLDC1 to produce a fully mature PIWI-bound piRNA
(Figure 8(e)).
The molecular details responsible for ribosome translocation downstream of ORFs and piRNA loading from ribo-
somes to PIWI-proteins are not yet understood, nor what distinguishes piRNA precursors from canonical protein-
coding mRNAs in order to trigger ribosome-guided endonucleolytic cleavage. Nevertheless, these results highlight the
multiple roles played by ribosomes beyond translation and open up exciting new questions about the regulation of these
processes within cells.
9 | C ON C L U S I ON
For many years, it was believed that translation-dependent mRNA decay was restricted to aberrant mRNAs, and that
the bulk of mRNA turnover occurred mainly through a two-step process, first with repression of translation coupled
with ribosome dissociation, followed by decapping and exonucleolytic degradation of RNAs within P-bodies (Franks &
Lykke-Andersen, 2008; Parker & Sheth, 2007). However, since the discovery of co-translational decapping and
exonucleolytic degradation, first in yeast and later in mammals, it has become very clear that translation-dependent
mRNA degradation is a major mechanism of regulation of mRNA half-lives at the transcriptome level. From unicellular
eukaryotic organisms to metazoans, the translation process appears to be constantly scrutinized by the cell, in order to
trigger mRNA degradation through a myriad of pathways capable of monitoring different aspects of ribosome progres-
sion. It is now known that pathways that were initially thought to participate uniquely in mRNA quality control are
also implicated in the fine tuning of a large spectrum of functional transcripts. These widespread ribosome-dependent
RNA decay mechanisms have even been harnessed by cells in order to couple translation-dependent RNA decay with
the biogenesis of functional small RNAs.
Not surprisingly, ribosomes themselves are emerging as central players in this process, acting as hubs for the recruit-
ment of mRNA decay factors or as signaling substrates to orchestrate the various steps required for conflict resolution
or mRNA degradation. However, although the molecular mechanisms that trigger translation-dependent mRNA decay
are now well characterized, more work is required to fully understand their regulations. Also, how the cell discrimi-
nates between benign situations that can be resolved from those requiring mRNA degradation awaits further investiga-
tions. Furthermore, since many of these pathways converge on similar effectors, including XRN1 or Ccr4–Not complex,
it will be important to characterize their interplay, as recently done by the Bazzini laboratory for the use of codons,
m6A marks, and microRNAs (Medina-Muñoz et al., 2021). Finally, although co-translational mRNA degradation is
emerging as a major pathway to regulate the levels of functional transcripts, mRNA degradation does not occur exclu-
sively in association with ribosomes. How cells coordinate mRNA degradation in a translation-dependent or
translational-independent manner remains an open question.
A C K N O WL E D G M E N T S
Work in our laboratory is supported by the European Research Council (ERC-StG-LS6-805500) under the
European Union's Horizon 2020 research and innovation programs; ATIP-Avenir program; Fondation FINOVI;
Agence Nationale des Recherches sur le SIDA et les Hépatites Virales (ANRS-ECTZ3306) and Agence Nationale
de la Recherche (ANR-20-CE15-0025).
CONFLICT OF INTEREST
The authors have declared no conflicts of interest for this article.
A U T H O R C ON T R I B U T I O NS
Christelle Morris: Writing-original draft; writing-review & editing. David Cluet: Writing-review & editing. Emiliano
Ricci: Conceptualization; funding acquisition; writing-original draft; writing-review & editing.
DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.
FURTHER READING
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ORCID
Christelle Morris https://orcid.org/0000-0003-1575-4609
David Cluet https://orcid.org/0000-0002-8018-8765
Emiliano P. Ricci https://orcid.org/0000-0002-9789-5837
R EL A TE D WIR Es AR TI CL ES
Interrelations between translation and general mRNA degradation in yeast
Controlling translation via modulation of tRNA levels
Ribosome-based quality control of mRNA and nascent peptides
Regulation of cytoplasmic RNA stability: Lessons from Drosophila
Roles of Puf proteins in mRNA degradation and translation
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How to cite this article: Morris C, Cluet D, Ricci EP. Ribosome dynamics and mRNA turnover, a complex
relationship under constant cellular scrutiny. WIREs RNA. 2021;12:e1658. https://doi.org/10.1002/wrna.1658

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Received: 30 January 2021 Revised: 26 March 2021 Accepted: 26 March 2021

Ribosome dynamics and mRNA turnover, a complex

Christelle Morris | David Cluet | Emiliano P. Ricci

Laboratory of Biology and Modeling of the

This article is categorized under:

WIREs RNA. 2021;12:e1658. wires.wiley.com/rna 1 of 34

2 | TR ANSLATION- DEPENDENT m RNA S URVEILLANCE P ATHWAYS

2.1 | Nonsense-mediated decay

2.2 | No-go decay

2.3 | Non-stop decay

3 | TR ANSLATION I NITIATION A ND mRNA DECAY

4 | THE RIBOSOME A S A HUB FOR mR NA DECAY, QUALITY CONTROL,

4.1 | The ribosome and mRNA decay

4.2 | The ribosome and selective co-translational degradation of functional mRNAs

4.3 | The ribosome and associated quality control mechanisms

4.4 | The ribosome and associated stress responses

5.1 | Codon usage is a major determinant of mRNA decay rates in yeast

7 | MICRORNAS, RIBOSOME DYNAMICS, AND mRNA DEGRADATION

DATA AVAILABILITY STATEMENT

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