Project description:Although canonical mRNA degradation is generally recognized to be translation dependent, our understanding of the molecular events that coordinate ribosome movement with the decay machinery is still limited. Here, we show that the 4EHP–GIGYF1/2 complex triggers co-translational mRNA decay as a result of altered ribosome activity during elongation. Human cells lacking 4EHP and GIGYF1 and 2 proteins accumulate transcripts known to be degraded in a translation dependent manner or with prominent ribosome pausing. These include mRNAs encoding secretory and membrane-bound proteins or specific tubulin isotypes, among others. 4EHP–GIGYF1/2 fails to reduce target mRNA levels in the absence of ribosome stalling or upon disruption of its interaction with the cap structure, DDX6 and a GYF domain-associated protein. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.
Project description:N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate correct translation and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.
Project description:N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate correct translation and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.
Project description:N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate correct translation and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.
Project description:Posttranscriptional repression by microRNA (miRNA) occurs through transcript destabilization or translation inhibition. Whereas RNA degradation explains most miRNA-dependent repression, transcript decay occurs co-translationally, raising questions regarding the requirement of target translation to miRNA-dependent transcript destabilization. To assess the contribution of translation to miRNA-mediated RNA destabilization, we decoupled these two molecular processes by dissecting the impact of miRNA loss of function on cytosolic long noncoding RNAs (lncRNAs). We show, that despite interacting with miRNA loaded RNA-induced silencing complex (miRISC), the steady state abundance and degradation rates of these endogenously expressed non-translated transcripts are minimally impacted by miRNA loss. To validate the requirement of translation to miRNA-dependent decay, we fused a miRISC bound lncRNA, whose levels are unaffected by miRNAs, to the 3’end of a protein-coding gene reporter and shown that this results in its miRNA-dependent transcript destabilization. Furthermore, analysis of the few lncRNAs whose levels are regulated by miRNAs revealed these tend to associate with translating ribosomes and are likely misannotated micropeptides, further substantiating the necessity of target translation to miRNA-dependent transcript decay. Our analyses revealed the strict requirement of translation for miRNA-dependent transcript destabilization and demonstrates that the levels of coding and noncoding transcripts are differently affected by miRNAs.
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and organism development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and metabolism, via the recognition by selective binding proteins. In cytoplasm, m6A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 accelerates the decay of m6A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m6A binder of the YTH domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated by YTHDF2. Cells deficient in all of YTHDF proteins experience the most dramatic accumulation of the m6A-methylated transcripts. These results indicate that in cytoplasm, YTHDF proteins act in an integrated and cooperative network to accelerate metabolism of m6A-modified mRNAs. The combinative and dynamic nature of YTHDF proteins may collectively impact fundamental biological processes and diseases related to m6A RNA methylation.
Project description:Although canonical mRNA degradation is generally recognized to be translation dependent, our understanding of the molecular events that coordinate ribosome movement with the decay machinery is still limited. Here, we show that the 4EHP–GIGYF1/2 complex triggers co-translational mRNA decay as a result of altered ribosome activity during elongation. Human cells lacking 4EHP and GIGYF1 and 2 proteins accumulate transcripts known to be degraded in a translation dependent manner or with prominent ribosome pausing. These include mRNAs encoding secretory and membrane-bound proteins or specific tubulin isotypes, among others. 4EHP–GIGYF1/2 fails to reduce target mRNA levels in the absence of ribosome stalling or upon disruption of its interaction with the cap structure, DDX6 and a GYF domain-associated protein. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.
Project description:During meiosis, in Saccharomyces cerevisiae, N6-methyladenosine (m6A) modified transcripts are induced, of which the function is unknown. Here, we uncover the role of the m6A reader Pho92. Cross-linking immunoprecipitation (CLIP) revealed that Pho92 associates with meiotic mRNAs in both m6A dependent and independent manner. Incidentally, Pho92 resides in the nucleus during early meiosis and associates with nascent RNAs, which is mediated through its interaction with Paf1C. Transcripts bound by Pho92 show elevated translational efficiency while cells lacking Pho92 display a small, but notable, increase in mRNA levels but not in protein levels, suggesting role of Pho92 in translation and decay. We show that Pho92 associates with ribosomes where it promotes the decay of m6A modified transcripts, contingent on active translation and the CCR4-NOT complex. We propose that m6A reader Pho92 is loaded co-transcriptionally to promote translation and subsequent decay fate of m6A modified transcripts, which ensures gamete fitness.