N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify ...m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%–25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is “hard coded” in cis via a simple and predictable code, accounting for 33%–46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.
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•RNA digestion via m6A sensitive RNase (MAZTER-seq) allows systematic m6A quantitation•MAZTER-seq reveals that antibody-based methods are of limited sensitivity•m6A stoichiometry is “hard coded” by a simple, predictable, and conserved code•MAZTER-seq allows quantitative tracking of m6A in diverse biological settings
A new enzymatic approach for precise mapping and measurement of m6A within mRNAs provides insight into how methylation sites are selected and the functional impact of the modifications.
Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N
-methyladenosine (m
A), which disrupts Watson-Crick ...base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m
A at single-nucleotide resolution. Within the cytosol, m
A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m
A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m
A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m
A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m
A levels was adopted as a potential means of post-transcriptional regulation.
Asymmetric messenger RNA (mRNA) localization facilitates efficient translation in cells such as neurons and fibroblasts. However, the extent and importance of mRNA polarization in epithelial tissues ...are unclear. Here, we used single-molecule transcript imaging and subcellular transcriptomics to uncover global apical-basal intracellular polarization of mRNA in the mouse intestinal epithelium. The localization of mRNAs did not generally overlap protein localization. Instead, ribosomes were more abundant on the apical sides, and apical transcripts were consequently more efficiently translated. Refeeding of fasted mice elicited a basal-to-apical shift in polarization of mRNAs encoding ribosomal proteins, which was associated with a specific boost in their translation. This led to increased protein production, required for efficient nutrient absorption. These findings reveal a posttranscriptional regulatory mechanism involving dynamic polarization of mRNA and polarized translation.
Human herpesvirus-6 (HHV-6) A and B are ubiquitous betaherpesviruses, infecting the majority of the human population. They encompass large genomes and our understanding of their protein coding ...potential is far from complete. Here, we employ ribosome-profiling and systematic transcript-analysis to experimentally define HHV-6 translation products. We identify hundreds of new open reading frames (ORFs), including upstream ORFs (uORFs) and internal ORFs (iORFs), generating a complete unbiased atlas of HHV-6 proteome. By integrating systematic data from the prototypic betaherpesvirus, human cytomegalovirus, we uncover numerous uORFs and iORFs conserved across betaherpesviruses and we show uORFs are enriched in late viral genes. We identified three highly abundant HHV-6 encoded long non-coding RNAs, one of which generates a non-polyadenylated stable intron appearing to be a conserved feature of betaherpesviruses. Overall, our work reveals the complexity of HHV-6 genomes and highlights novel features conserved between betaherpesviruses, providing a rich resource for future functional studies.
Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at ...the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5'-untranslated region (UTR), as determined by polysome fractionation and 5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. SIGNIFICANCE: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 19 pandemic
. Coronaviruses developed varied mechanisms to repress host mRNA translation ...to allow the translation of viral mRNAs and concomitantly block the cellular innate immune response
. Although different SARS-CoV-2 proteins are implicated in host expression shutoff
, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here, we combine RNA-sequencing, ribosome profiling and metabolic labeling of newly synthesized RNA, to comprehensively define the mechanisms that are utilized by SARS-CoV-2 to shutoff cellular protein synthesis. We show that infection leads to a global reduction in translation, but viral transcripts are not preferentially translated. Instead, we find that infection leads to accelerated degradation of cytosolic cellular mRNAs which facilitates viral takeover of the mRNA pool in infected cells. Moreover, we reveal that the translation of transcripts whose expression is induced in response to infection, including innate immune genes, is impaired. We demonstrate this impairment is likely mediated by inhibition of nuclear mRNA export, preventing newly transcribed cellular mRNAs from accessing ribosomes. Overall, our results uncover the multipronged strategy employed by SARS-CoV-2 to commandeer the translation machinery and to suppress host defenses.
The herpesvirus human cytomegalovirus (HCMV) infects the majority of the world’s population and leads to severe diseases in immunocompromised adults and newborns. During infection, the virus ...manipulates gene expression in order to alter cellular processes and to promote its propagation. While the research so far mainly focused on changes at the transcription level, we aimed to uncover post-transcriptional mechanisms by which the virus creates supporting conditions for the infection. N6-Methyladenosine (m6A) is the most common mRNA modification. Recent studies revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. In the first part of my Ph.D., I investigated the role of m6A modification during HCMV infection. I was able to show that the expression of m6 A machinery components is upregulated along the infection and that depletion of these proteins restricts viral replication. Although I could identify viral transcripts that are m6A-modified, their expression levels do not significantly differ from unmodified transcripts, when m6A machinery is deleted. In contrary, my results reveal that transcripts of type-I interferons are destabilized by m6A modification and that the propagation of different DNA and RNA viruses is inhibited in cells deficient of m6A machinery in an interferon signaling dependent manner. Altogether, my findings uncover the role of m6A as negative regulator of interferon response and suggest HCMV uses this regulation to dictate fast turnover of interferon mRNAs and thus to reduce the cellular antiviral response and support its own propagation. In the second part of my work, I studied the dynamics of global translation regulation during HCMV infection. My results show that in contrast to early stages of HCMV infection when eIF2a phosphorylation is prevented by viral factors, at 48 hours post infection (hpi) eIF2a is phosphorylated. Although I could confirm that this phosphorylation leads to translation attenuation during infection, viral growth is not affected and restoration of mRNA translation does not improve HCMV replication. Interestingly, although phosphorylation of eIF2a is known to be followed by stress granule (SG) assembly, my experiments showed that SG formation is blocked at late stages of HCMV infection, despite the presence of phosphorylated eIF2a. By using a reporter for translation efficiency, I showed that putative viral upstream ORFs do not confer the virus with resistance to eIF2a-mediated translation attenuation. Finally, I demonstrated that the sensitivity to translation inhibition by phosphorylated eIF2a is globally comparable between human and viral transcripts. Altogether, this part of my work suggests that in contrary to dogmatic view of translation as being crucial factor for viral propagation, at late stages of HCMV infection the efficiency of protein synthesis does not limit viral growth. Overall, my work adds new layers to our understanding of post-transcriptional mechanisms utilized by HCMV to support its proliferation.
Transcriptome-wide mapping of N1-methyladenosine (m.sup.1A) at single-nucleotide resolution reveals m.sup.1A to be scarce in cytoplasmic mRNA, to inhibit translation, and to be highly dynamic at a ...single site in a mitochondrial mRNA. The basis of m1A modification N.sup.1-methyladenosine (m.sup.1A) modification has been detected on mRNA, but validation of the internal mRNA sites at which it occurs and the functional consequences of it have not been well defined. Schraga Schwartz and colleagues now address these limitations using a method that enables single-nucleotide resolution of such sites in the transcriptome. They show that the level of modification is much lower than reported previously and varies during development and by tissue type. The authors identify a structural motif associated with the modification and define the enzymatic machinery responsible for the methylation. They find that m.sup.1A modification is associated with translational repression, consistent with its tight regulation. Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N.sup.1-methyladenosine (m.sup.1A), which disrupts Watson-Crick base pairing, at internal sites of mRNAs.sup.1,2. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m.sup.1A at single-nucleotide resolution. Within the cytosol, m.sup.1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m.sup.1A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m.sup.1A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m.sup.1A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m.sup.1A levels was adopted as a potential means of post-transcriptional regulation.
Transcriptome-wide mapping of N1-methyladenosine (m.sup.1A) at single-nucleotide resolution reveals m.sup.1A to be scarce in cytoplasmic mRNA, to inhibit translation, and to be highly dynamic at a ...single site in a mitochondrial mRNA.
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