Mrna Translation

We tested the hypothesis that AMP-activated protein kinase (AMPK), an energy sensor, regulates diabetes-induced renal hypertrophy. In kidney glomerular epithelial cells, high glucose (30 mM), but not equimolar mannitol, stimulated de novo protein synthesis and induced hypertrophy in association with increased phosphorylation of eukaryotic initiation factor 4E binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2, regulatory events in mRNA translation. These high-glucose-induced changes in protein synthesis were phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin (mTOR) dependent and transforming growth factor-β independent. High glucose reduced AMPK α-subunit theronine (Thr) 172 phosphorylation, which required Akt activation. Changes in AMP and ATP content could not fully account for high-glucose-induced reductions in AMPK phosphorylation. Metformin and 5-aminoimidazole-4-carboxamide-1β-riboside (AICAR) increased AMPK phosphorylation, in...

Cell polarity is a universal biological phenomenon. While much is known about the establishment and maintenance of cell polarity, its role in aging and age-related diseases remains to be fully addressed. Nonetheless, the exciting findings in the budding yeast indicate that the polar processes are intimately linked to both aging of the mother cell and rejuvenation of the daughter cell. This includes polar segregation of damaged proteins and ERCs due to the septin-based diffusion barrier, asymmetric inheritance of MDR proteins and retrograde protein transport. The principal, still unexplored question is whether the same polar mechanisms work during the asymmetric division of germ and stem cells, allowing their rejuvenation across generations. Further strengthening the links between cell polarity and aging is a large number of common genes associated with both polarity and longevity. Given a strong similarity between mechanisms of cell polarity in yeast and higher eukaryotes, the budding yeast Saccharomyces cerevisiae could serve as a convenient model system for studying the links between the cell polarity, aging and rejuvenation. Consequently, exploring the potential mammalian equivalents of yeast-established polarity mechanisms could be the focus for future biogerontological investigations.

Localization of tau mRNA to the axon requires the axonal localization cis signal (ALS), which is located within the 3' untranslated region, and trans-acting binding proteins, which are part of the observed granular structures in neuronal cells. In this study, using both biochemical and morphological methods, we show that the granules contain tau mRNA, HuD RNA-binding protein, which stabilizes mRNA, and KIF3A, a member of the kinesin microtubule-associated motor protein family involved in anterograde transport. The granules are detected along the axon and accumulate in the growth cone. Inhibition of KIF3A expression caused neurite retraction and inhibited tau mRNA axonal targeting. Taken together, these results suggest that HuD and KIF3A proteins are present in the tau mRNA axonal granules and suggest an additional function for the kinesin motor family in the microtubule-dependent translocation of RNA granules. Localized tau-GFP expression was blocked by a protein synthesis inhibitor, and upon release from inhibition, nascent tau-GFP 'hot spots' were directly observed in the axon and growth cones. These observations are consistent with local protein synthesis in the axon resulting from the transported tau mRNA.

Regulated mRNA translation plays a key role in control of cell cycle progression
in a variety of physiological and pathological processes, including in the self-renewal and
survival of stem cells and cancer stem cells. While targeting mRNA translation presents an
attractive strategy for control of aberrant cell cycle progression, mRNA translation is an
underdeveloped therapeutic target. Regulated mRNAs are typically controlled through
interaction with multiple RNA binding proteins (RBPs) but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly
understood. The challenge now is to gain insight into these mechanisms of coordination
and to identify the molecular mediators that integrate multiple, often conflicting, inputs.
A first step includes the identification of altered mRNA ribonucleoprotein complex
components that assemble on mRNAs bound by multiple, distinct RBPs compared to those
recruited by individual RBPs. This review builds upon our knowledge of combinatorial
control of mRNA translation during the maturation of oocytes from Xenopus laevis,
to address molecular strategies that may mediate RBP diplomacy and conflict resolution
for coordinated control of mRNA translational output. Continued study of regulated
ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational
control and may suggest novel therapeutic strategies for the treatment of disease.

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.

Ana Marques-Ramos, Juliane Menezes, Rafaela Lacerda, Alexandre Teixeira and Luisa Romao Departamento de Genetica Humana, Instituto Nacional de Saude Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal Center for Biodiversity, Functional and Integrative Genomics, Faculdade de Ciencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal Centro de Investigacao em Genetica Molecular Humana, Faculdade de Ciencias Medicas, Universidade Nova de Lisboa, 1349-008 Lisboa, Portugal

Skeletal muscle in the neonate grows at a rapid rate due in part to an enhanced sensitivity to the postprandial rise in amino acids, particularly leucine. To elucidate the molecular mechanism by which leucine stimulates protein synthesis in neonatal muscle, overnight-fasted 7-day-old piglets were treated with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR) complex (mTORC)1] for 1 h and then infused with leucine for 1 h. Fractional rates of protein synthesis and activation of signaling components that lead to mRNA translation were determined in skeletal muscle. Rapamycin completely blocked leucine-induced muscle protein synthesis. Rapamycin markedly reduced raptor-mTOR association, an indicator of mTORC1 activation. Rapamycin blocked the leucine-induced phosphorylation of mTOR, S6 kinase 1 (S6K1), and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1) and formation of the eIF4E·eIF4G complex and increased eIF4E·4E-BP1 complex abundance. Rapamycin had no e...

The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling.

What does it mean when we say a cell’s biochemistry is regulated during changes to the phenotype? While there are a plethora of potential mechanisms and contributions to the final outcome, a more tractable approach is to examine the dynamics of mRNA. This way, we can assess the contributions of both known and unknown decay and aggregation processes for maintaining levels of gene product on a gene-by-gene basis. In this extended protocol, drug treatments that target specific cellular functions (termed mechanism disruption) can be used in tandem with mRNA extraction from the polysome to look at the dynamics of mRNA levels associated with transcription and translation at multiple stages during a physiological perturbation. This is accomplished through validating the polysome isolation method in human cells and comparing fractions of mRNA for each experimental treatment at multiple points in time. First, three different drug treatments corresponding to the arrest of various cellular processes are administered to populations of human cells. For each treatment, the transcriptome and translatome are compared directly at different time points by assaying both cell-type specific and non-specific genes. There are two findings of note. First, extraction of mRNA from the polysome and comparison with the transcriptome can yield interesting information about the regulation of cellular mRNA during a functional challenge to the cell. In addition, the conventional application of such drugs to assess mRNA decay is an incomplete picture of how severely challenged or senescent cells regulate mRNA in response. This extended protocol demonstrates how the gene- and process-variable degradation of mRNA might ultimately require investigations into the course-grained dynamics of cellular mRNA, from transcription to ribosome.