Project description:Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites, within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single-molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.
Project description:<p>The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analyzed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyze the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA</p><p>modifications in viral RNA genomes.</p>
Project description:We report the identification and quantification of Watson-Crick modifications in tRNA and rRNA through the use of high throughput sequencing. We apply the recently published DM-tRNA-Seq method to generate demethylase treated and untreated 293T samples, and using computational methods we are able to flag sites using a modification index. This index allows us to generate site-resolved information about modification that we can use to identify and quantify Watson-Crick face modifications in tRNA and rRNA. With the demethylase treated samples, we are able to validate numerous nucleotide modifications from demethylase substrates, and the absence of demethylase action also serves to aid in identification. We find numerous novel modification sites in tRNA as well as striking comparisons between tissues cultures lines. Our study reports a comprehensive analysis of the tRNA modification landscape by identifying sites of modification as well as quantifying modification levels.
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
Project description:Tumor recurrence is main pattern of treatment failure for early-stage hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying disease recurrence remain poorly understood. Here, we showed that 18S rRNA N6-methyladenosine (m6A1832) modification and its methyltransferase complex METTL5/TRMT112 were upregulated in HCC and correlated with poor prognosis. Loss-of-function and gain-of-function assays demonstrated that METTL5/TRMT112 mediated 18S rRNA m6A1832 modification promotes HCC tumorigenesis in vitro and in vivo. Mechanistically, 18S rRNA m6A1832 modification selectively regulated the translation of mRNAs with long 5’UTR and short 3’UTR through affecting the assembly of 80S subunit at translation initiation and its dissociation at translation termination which was executed by weakening the interaction of ABCE1 with eRF1 and eRF3. Moreover, METTL5-mediated 18S rRNA m6A1832 modification regulated β-oxidation of long-chain fatty acid through ACSL4 to promote HCC progression. Our work uncovered a novel layer of mRNA translation regulation mechanism at ribosome 80S subunit assembly and dissociation step mediated by 18S rRNA m6A1832 modification and revealed a new crosslink between RNA epigenetic modification and fatty acid metabolism in HCC.
Project description:RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.