The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylated tRNAs were substantially reduced, and loss of Dnmt2 and NSun2 was further associated with reduced rates of overall protein synthesis. These results establish a biologically important function for cytosine-C5 tRNA methylation in mammals and suggest that this modification promotes mouse development by supporting protein synthesis.

Chaperone-driven protein disaggregation directly coordinates timing of translation reinitiation with protein folding capacity during cellular protein quality surveillance, enabling efficient protein homeostasis.

SUMMARY Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.

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Cells respond to stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs). Cold shock is identified as a novel trigger of SG assembly in yeast and mammals. Cells actively suppress protein synthesis by parallel pathways to induce SG formation and ensure cellular survival at low temperatures.

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In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3′ untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA–binding protein homologous to mammalian tristetraprolin, was found to target ARE–containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE–containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila.

Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress.