Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only ... more Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, com-promises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-de-pendent mRNP assembly i...
Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of ma... more Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 30 end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 50 end maturation. Here, we identify Rrp17p as a previously unidentified 50 –30 exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 30 end formation. Rrp17p is required for efficient exonuclease digestion of the mature 50 ends of 5.8SS and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.
Single-molecule resolution imaging has become an important tool in the study of cell biology. Apt... more Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNP export. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP-NPC intera...
Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which... more Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting ...
The process of creating a translation-competent mRNA is highly complex and involves numerous step... more The process of creating a translation-competent mRNA is highly complex and involves numerous steps including transcription, splicing, addition of modifications, and, finally, export to the cytoplasm. Historically, much of the research on regulation of gene expression at the level of the mRNA has been focused on either the regulation of mRNA synthesis (transcription and splicing) or metabolism (translation and degradation). However, in recent years, the advent of new experimental techniques has revealed the export of mRNA to be a major node in the regulation of gene expression, and numerous large-scale and specific mRNA export pathways have been defined. In this chapter, we will begin by outlining the mechanism by which most mRNAs are homeostatically exported ("bulk mRNA export"), involving the recruitment of the NXF1/TAP export receptor by the Aly/REF and THOC5 components of the TREX complex. We will then examine various mechanisms by which this pathway may be controlled, modified, or bypassed in order to promote the export of subset(s) of cellular mRNAs, which include the use of metazoan-specific orthologs of bulk mRNA export factors, specific cis RNA motifs which recruit mRNA export machinery via specific trans-acting-binding factors, posttranscriptional mRNA modifications that act as "inducible" export cis elements, the use of the atypical mRNA export receptor, CRM1, and the manipulation or bypass of the nuclear pore itself. Finally, we will discuss major outstanding questions in the field of mRNA export heterogeneity and outline how cutting-edge experimental techniques are providing new insights into and tools for investigating the intriguing field of mRNA export heterogeneity.
The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to ... more The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to the cytoplasm. The ability for mRNAs to get exported is linked to various upstream nuclear processes including co‐transcriptional RNP assembly and processing, and only export competent mRNPs are thought to get access to the NPC. While the nuclear pore is generally viewed as a monolithic structure that serves as a mediator of transport driven by transport receptors, more recent evidence suggests that the NPC might be more heterogenous than previously believed, both in its composition or in the selective treatment of cargo that seek access to the pore, providing functional plasticity to mRNA export. In this review, we consider the interconnected processes of nuclear mRNA metabolism that contribute and mediate export competence. Furthermore, we examine different aspects of NPC heterogeneity, including the role of the nuclear basket and its associated complexes in regulating selective and/or efficient binding to and transport through the pore.
RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow... more RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels ...
Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribos... more Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across...
Viruses have transformed our understanding of mammalian RNA processing, including facilitating th... more Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5′ end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5′ end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg–eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-depe...
Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest.... more Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.Lassard et al. demonstrate a relationship between cellular senescence and perturbed ribosome biogenesis and find that the ribosomal protein S14 is an inhibitor of CDK4, inducing an Rb-dependent cell cycle arrest.
To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of sur... more To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of ...
While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has b... more While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a highthroughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.
Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their... more Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3 OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates.
The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surv... more The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of posttranscriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins including many nucleolar proteins showed increased binding to poly(A) RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs which harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within component...
Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are fo... more Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life, and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LS...
Proteomic and RNomic approaches have identified many components of different ribonucleoprotein pa... more Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2016
The nucleolus represents a highly multifunctional intranuclear organelle in which, in addition to... more The nucleolus represents a highly multifunctional intranuclear organelle in which, in addition to the canonical ribosome assembly, numerous processes such as transcription, DNA repair and replication, the cell cycle, and apoptosis are coordinated. The nucleolus is further a key hub in the sensing of cellular stress and undergoes major structural and compositional changes in response to cellular perturbations. Numerous nucleolar proteins have been identified that, upon sensing nucleolar stress, deploy additional, non-ribosomal roles in the regulation of varied cell processes including cell cycle arrest, arrest of DNA replication, induction of DNA repair, and apoptosis, among others. The highly abundant proteins nucleophosmin (NPM1) and nucleolin (NCL) are two such factors that transit to the nucleoplasm in response to stress, and participate directly in the repair of numerous different DNA damages. This review discusses the contributions made by NCL and (or) NPM1 to the different DNA...
Endo- and exonucleases are major contributors to RNA metabolism through their diverse roles in ma... more Endo- and exonucleases are major contributors to RNA metabolism through their diverse roles in maturation and turnover of different species of RNA as well as transcription. Recent data suggests RNA nucleases also affect genome stability programs and act along DNA repair pathways. Here, we describe Nol12 as a multifunctional RNA/DNA endonuclease found in different subcellular compartments - the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles, nucleoli as well as GW/P-bodies. We show that Nol12 is required for a key step in ribosomal RNA processing, separating large and small subunit precursors at site 2, rerouting ribosome biogenesis via an alternative pathway in its absence to ensure ribosome production. Furthermore, loss of Nol12 results in increased oxidized DNA levels followed by a rapid p53-independent ATR-Chk1-mediated apoptotic response, suggesting a role for Nol12 in the prevention or resolution of oxidative DNA damage. Identification of a c...
Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only ... more Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, com-promises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-de-pendent mRNP assembly i...
Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of ma... more Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 30 end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 50 end maturation. Here, we identify Rrp17p as a previously unidentified 50 –30 exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 30 end formation. Rrp17p is required for efficient exonuclease digestion of the mature 50 ends of 5.8SS and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.
Single-molecule resolution imaging has become an important tool in the study of cell biology. Apt... more Single-molecule resolution imaging has become an important tool in the study of cell biology. Aptamer-based approaches (e.g., MS2 and PP7) allow for detection of single RNA molecules in living cells and have been used to study various aspects of mRNA metabolism, including mRNP nuclear export. Here we outline an imaging protocol for the study of interactions between mRNPs and nuclear pore complexes (NPCs) in the yeast S. cerevisiae, including mRNP export. We describe in detail the steps that allow for high-resolution live-cell mRNP imaging and measurement of mRNP interactions with NPCs using simultaneous two-color imaging. Our protocol discusses yeast strain construction, choice of marker proteins to label the nuclear pore complex, as well as imaging conditions that allow high signal-to-noise data acquisition. Moreover, we describe various aspects of postacquisition image analysis for single molecule tracking and image registration allowing for the characterization of mRNP-NPC intera...
Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which... more Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting ...
The process of creating a translation-competent mRNA is highly complex and involves numerous step... more The process of creating a translation-competent mRNA is highly complex and involves numerous steps including transcription, splicing, addition of modifications, and, finally, export to the cytoplasm. Historically, much of the research on regulation of gene expression at the level of the mRNA has been focused on either the regulation of mRNA synthesis (transcription and splicing) or metabolism (translation and degradation). However, in recent years, the advent of new experimental techniques has revealed the export of mRNA to be a major node in the regulation of gene expression, and numerous large-scale and specific mRNA export pathways have been defined. In this chapter, we will begin by outlining the mechanism by which most mRNAs are homeostatically exported ("bulk mRNA export"), involving the recruitment of the NXF1/TAP export receptor by the Aly/REF and THOC5 components of the TREX complex. We will then examine various mechanisms by which this pathway may be controlled, modified, or bypassed in order to promote the export of subset(s) of cellular mRNAs, which include the use of metazoan-specific orthologs of bulk mRNA export factors, specific cis RNA motifs which recruit mRNA export machinery via specific trans-acting-binding factors, posttranscriptional mRNA modifications that act as "inducible" export cis elements, the use of the atypical mRNA export receptor, CRM1, and the manipulation or bypass of the nuclear pore itself. Finally, we will discuss major outstanding questions in the field of mRNA export heterogeneity and outline how cutting-edge experimental techniques are providing new insights into and tools for investigating the intriguing field of mRNA export heterogeneity.
The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to ... more The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to the cytoplasm. The ability for mRNAs to get exported is linked to various upstream nuclear processes including co‐transcriptional RNP assembly and processing, and only export competent mRNPs are thought to get access to the NPC. While the nuclear pore is generally viewed as a monolithic structure that serves as a mediator of transport driven by transport receptors, more recent evidence suggests that the NPC might be more heterogenous than previously believed, both in its composition or in the selective treatment of cargo that seek access to the pore, providing functional plasticity to mRNA export. In this review, we consider the interconnected processes of nuclear mRNA metabolism that contribute and mediate export competence. Furthermore, we examine different aspects of NPC heterogeneity, including the role of the nuclear basket and its associated complexes in regulating selective and/or efficient binding to and transport through the pore.
RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow... more RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels ...
Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribos... more Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across...
Viruses have transformed our understanding of mammalian RNA processing, including facilitating th... more Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5′ end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5′ end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg–eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-depe...
Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest.... more Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.Lassard et al. demonstrate a relationship between cellular senescence and perturbed ribosome biogenesis and find that the ribosomal protein S14 is an inhibitor of CDK4, inducing an Rb-dependent cell cycle arrest.
To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of sur... more To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of ...
While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has b... more While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a highthroughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.
Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their... more Non-coding RNAs have critical roles in biological processes, and RNA chaperones can promote their folding into the native shape required for their function. La proteins are a class of highly abundant RNA chaperones that contact pre-tRNAs and other RNA polymerase III transcripts via their common UUU-3 OH ends, as well as through less specific contacts associated with RNA chaperone activity. However, whether La proteins preferentially bind misfolded pre-tRNAs or instead engage all pre-tRNA substrates irrespective of their folding status is not known. La deletion in yeast is synthetically lethal when combined with the loss of tRNA modifications predicted to contribute to the native pre-tRNA fold, such as the N2, N2-dimethylation of G26 by the methyltransferase Trm1p. In this work, we identify G26 containing pre-tRNAs that misfold in the absence of Trm1p and/or La (Sla1p) in Schizosaccharomyces pombe cells, then test whether La preferentially associates with such tRNAs in vitro and in vivo. Our data suggest that La does not discriminate a native from misfolded RNA target, and highlights the potential challenges faced by RNA chaperones in preferentially binding defective substrates.
The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surv... more The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of posttranscriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins including many nucleolar proteins showed increased binding to poly(A) RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs which harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within component...
Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are fo... more Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life, and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LS...
Proteomic and RNomic approaches have identified many components of different ribonucleoprotein pa... more Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2016
The nucleolus represents a highly multifunctional intranuclear organelle in which, in addition to... more The nucleolus represents a highly multifunctional intranuclear organelle in which, in addition to the canonical ribosome assembly, numerous processes such as transcription, DNA repair and replication, the cell cycle, and apoptosis are coordinated. The nucleolus is further a key hub in the sensing of cellular stress and undergoes major structural and compositional changes in response to cellular perturbations. Numerous nucleolar proteins have been identified that, upon sensing nucleolar stress, deploy additional, non-ribosomal roles in the regulation of varied cell processes including cell cycle arrest, arrest of DNA replication, induction of DNA repair, and apoptosis, among others. The highly abundant proteins nucleophosmin (NPM1) and nucleolin (NCL) are two such factors that transit to the nucleoplasm in response to stress, and participate directly in the repair of numerous different DNA damages. This review discusses the contributions made by NCL and (or) NPM1 to the different DNA...
Endo- and exonucleases are major contributors to RNA metabolism through their diverse roles in ma... more Endo- and exonucleases are major contributors to RNA metabolism through their diverse roles in maturation and turnover of different species of RNA as well as transcription. Recent data suggests RNA nucleases also affect genome stability programs and act along DNA repair pathways. Here, we describe Nol12 as a multifunctional RNA/DNA endonuclease found in different subcellular compartments - the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles, nucleoli as well as GW/P-bodies. We show that Nol12 is required for a key step in ribosomal RNA processing, separating large and small subunit precursors at site 2, rerouting ribosome biogenesis via an alternative pathway in its absence to ensure ribosome production. Furthermore, loss of Nol12 results in increased oxidized DNA levels followed by a rapid p53-independent ATR-Chk1-mediated apoptotic response, suggesting a role for Nol12 in the prevention or resolution of oxidative DNA damage. Identification of a c...
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Papers by Marlene Oeffinger