Analysis of m6A RNA methylation in Caenorhabditis elegans.
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ABSTRACT:
SUBMITTER: Sendinc E
PROVIDER: S-EPMC7359367 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:One of the most abundant RNA modifications is N6-methyladenosine (m6A). RNA from all forms of life, including viruses, contain m6A. This modification has been detected in many types of RNAs, such as mRNA, ribosomal RNA, long non-coding RNAs, small nuclear RNAs and microRNAs. Diverse set of proteins have been characterized to methylate, demethylate and specifically bind to this modification in different organisms. C. elegans is a unique model organism with abundant m6A modification, although its genome does not code for orthologs of the well characterized m6A methyltransferase METTL3/METTL14 complex or the demethylases FTO or ALKBH5. Furthermore, orthologs of the YTH family m6A reader proteins seem to be absent from the worm genome as well. To gain insights into how this modification is installed in this organism, we set out to identify enzymes that contribute to the abundant level of m6A in C. elegans. We designed a targeted RNAi screen by which the expression of 22 candidate putative RNA methyltransferase genes are knocked down. We measured global RNA methylation level by HPLC-MS/MS analysis after two generations of RNAi-mediated knock down. The knock down of two candidate methyltransferases resulted in a decrease in global m6A level in total RNA. The first methyltransferase, F33A8.4, is an ortholog of the human ZCCHC4 gene. The second methyltransferase, C38D4.9, is an ortholog of the human METTL5 gene. In order to determine if ZCCHC4 or METTL5 are involved in the deposition of m6A at the mRNA level, m6A-RIP-seq experiments were performed in mRNA derived from WT (N2), ZCCHC4 KO, METTL5 KO and ZCCHC4/METTL5 dKO C. elegans embryos.
2021-04-06 | GSE148786 | GEO
Project description:RNA N6-methyladenosine (m6A) methylation is known to be the most popular RNA modification in animals. Many research reports have elaborated on the effects of m6A regulators in medical practice, such as diagnosis, prognosis, and treatment. M6A modification has evident impacts on many aspects of RNA metabolism, just like RNA splicing, processing, translation, and stability. M6A also has a magnificent role in numerous types of cancers. We analyzed the prostate cancer datasets, from The Cancer Genome Atlas (TCGA) database, for every recognized m6A regulator in their gene expression, DNA methylation status and copy number variations (CNVs). We also systematically analyzed the relationship between different m6A regulators and the prognosis of prostate cancer. The results illustrated considerable differences in the expression of various m6A regulators between the prostate and normal cancer samples. At the same time, there were evident differences in the expression of various m6A regulators in prostate cancers with different Gleason scores. Subsequently, we determined CBLL1, FTO, YTHDC1, HNRNPA2B1 as crucial m6A regulators of prostate cancer. Premised on the expression of CBLL1, we also identified potential therapeutic agents for prostate cancer, and knockdown of HNRNPA2B1 prominently inhibited prostate cells migration and invasion in vitro experiment.
| S-EPMC8661905 | biostudies-literature
Project description:BackgroundNoncoding RNAs (ncRNAs) play important roles in a variety of cellular processes. Characterizing the transcriptional activity of ncRNA promoters is therefore a critical step toward understanding the complex cellular roles of ncRNAs.ResultsHere we present an in vivo transcriptional analysis of three C. elegans ncRNA upstream motifs (UM1-3). Transcriptional activity of all three motifs has been demonstrated, and mutational analysis revealed differential contributions of different parts of each motif. We showed that upstream motif 1 (UM1) can drive the expression of green fluorescent protein (GFP), and utilized this for detailed analysis of temporal and spatial expression patterns of 5 SL2 RNAs. Upstream motifs 2 and 3 do not drive GFP expression, and termination at consecutive T runs suggests transcription by RNA polymerase III. The UM2 sequence resembles the tRNA promoter, and is actually embedded within its own short-lived, primary transcript. This is a structure which is also found at a few plant and yeast loci, and may indicate an evolutionarily very old dicistronic transcription pattern in which a tRNA serves as a promoter for an adjacent snoRNA.ConclusionThe study has demonstrated that the three upstream motifs UM1-3 have promoter activity. The UM1 sequence can drive expression of GFP, which allows for the use of UM1::GFP fusion constructs to study temporal-spatial expression patterns of UM1 ncRNA loci. The UM1 loci appear to act in concert with other upstream sequences, whereas the transcriptional activities of the UM2 and UM3 are confined to the motifs themselves.
| S-EPMC2527325 | biostudies-literature
Project description:In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.
| S-EPMC10018337 | biostudies-literature
Project description:AimTo analyze and compare the mRNA N6-methyladenosine modifications in transverse aortic constriction induced mice hearts and normal mice hearts.Materials and methodsColorimetric quantification was used to probe the changes in m6A modifications in the total RNA. The expression of m6A-related enzymes was analyzed via qRT-PCR and western blotting. RNA-seq and MeRIP-seq were performed to identify genes with differences in m6A modifications or expression in the transcriptome profile.ResultsCompared with the control group, the TAC group exhibited higher m6A methylation levels. FTO and WTAP were downregulated after TAC, while METTL3 was significantly downregulated at the protein level. MeRIP-seq revealed that 1179 m6A peaks were upmethylated and 733 m6A peaks were downmethylated, and biological analysis of these genes exhibited a strong relationship with heart function.ConclusionOur findings provide novel information regarding m6A modification and gene expression changes in cardiac hypertrophy, which may be fundamental for further research.
| S-EPMC9373449 | biostudies-literature
Project description:RNA m6A methylome of Caenorhabditis elegans
| PRJNA625719 | ENA
Project description:N6-methyladenosine is the most prominent RNA modification in mammals. Here, we study mouse skin embryogenesis to tackle m6A's functions and physiological importance. We first landscape the m6A modifications on skin epithelial progenitor mRNAs. Contrasting with in vivo ribosomal profiling, we unearth a correlation between m6A modification in coding sequences and enhanced translation, particularly of key morphogenetic signaling pathways. Tapping physiological relevance, we show that m6A loss profoundly alters these cues and perturbs cellular fate choices and tissue architecture in all skin lineages. By single-cell transcriptomics and bioinformatics, both signaling and canonical translation pathways show significant downregulation after m6A loss. Interestingly, however, many highly m6A-modified mRNAs are markedly upregulated upon m6A loss, and they encode RNA-methylation, RNA-processing and RNA-metabolism factors. Together, our findings suggest that m6A functions to enhance translation of key morphogenetic regulators, while also destabilizing sentinel mRNAs that are primed to activate rescue pathways when m6A levels drop.
| S-EPMC7535931 | biostudies-literature
Project description:Type 2 diabetes is characterized by chronic hyperglycemia associated with impaired insulin action and secretion. Although the heritability of type 2 diabetes is high, the environment, including blood components, could play a major role in the development of the disease. Amongst environmental effects, epitranscriptomic modifications have been recently shown to affect gene expression and glucose homeostasis. The epitranscriptome is characterized by reversible chemical changes in RNA, with one of the most prevalent being the m6A methylation of RNA. Since pancreatic β cells fine tune glucose levels and play a major role in type 2 diabetes physiopathology, we hypothesized that the environment, through variations in blood glucose or blood free fatty acid concentrations, could induce changes in m6A methylation of RNAs in pancreatic β cells. Here we observe a significant decrease in m6A methylation upon high glucose concentration, both in mice and human islets, associated with altered expression levels of m6A demethylases. In addition, the use of siRNA and/or specific inhibitors against selected m6A enzymes demonstrate that these enzymes modulate the expression of genes involved in pancreatic β-cell identity and glucose-stimulated insulin secretion. Our data suggest that environmental variations, such as glucose, control m6A methylation in pancreatic β cells, playing a key role in the control of gene expression and pancreatic β-cell functions. Our results highlight novel causes and new mechanisms potentially involved in type 2 diabetes physiopathology and may contribute to a better understanding of the etiology of this disease.
| S-EPMC8773766 | biostudies-literature
Project description:Glioma is considered to be the most common brain malignancy in the central nervous system. At present, the aetiology of glioma is not clear. Due to its rapidly growth and easily recurrence, the prognosis of patients with glioma is very poor. N6-methyladenosine (m6A) methylation is an internal reversible modification in most RNAs, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). Recent studies have shown that the m6A regulators are abnormal expressed, and are extensively involved in the progression of glioma by targeting ncRNAs. Moreover, as the most important epigenetic regulators, ncRNAs can also affect the function of m6A regulators in glioma. This review summarized the expression and function of certain common m6A regulators in glioma. Also, the current review sum up the mutual interactions between m6A regulators and ncRNAs in glioma.
| S-EPMC9187709 | biostudies-literature
Project description:N6‑methyladenosine (m6A) is the most prevalent and abundant type of internal post‑transcriptional RNA modification in eukaryotic cells. Multiple types of RNA, including mRNAs, rRNAs, tRNAs, long non‑coding RNAs and microRNAs, are involved in m6A methylation. The biological function of m6A modification is dynamically and reversibly mediated by methyltransferases (writers), demethylases (erasers) and m6A binding proteins (readers). The methyltransferase complex is responsible for the catalyzation of m6A modification and is typically made up of methyltransferase‑like (METTL)3, METTL14 and Wilms tumor 1‑associated protein. Erasers remove methylation by fat mass and obesity‑associated protein and ALKB homolog 5. Readers play a role through the recognition of m6A‑modified targeted RNA. The YT521‑B homology domain family, heterogeneous nuclear ribonucleoprotein and insulin‑like growth factor 2 mRNA‑binding protein serve as m6A readers. The m6A methylation on transcripts plays a pivotal role in the regulation of downstream molecular events and biological functions, such as RNA splicing, transport, stability and translatability at the post‑transcriptional level. The dysregulation of m6A modification is associated with cancer, drug resistance, virus replication and the pluripotency of embryonic stem cells. Recently, a number of studies have identified aberrant m6A methylation in cardiovascular diseases (CVDs), including cardiac hypertrophy, heart failure, arterial aneurysm, vascular calcification and pulmonary hypertension. The aim of the present review article was to summarize the recent research progress on the role of m6A modification in CVD and give a brief perspective on its prospective applications in CVD.
| S-EPMC7595665 | biostudies-literature