Analysis of m6A RNA methylation in Caenorhabditis elegans.
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ABSTRACT:
SUBMITTER: Sendinc E
PROVIDER: S-EPMC7359367 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Analysis of m6A RNA methylation in <i>Caenorhabditis elegans</i>.
Sendinc Erdem E Valle-Garcia David D Jiao Alan A Shi Yang Y
Cell discovery 20200714 1
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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.
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Project description:BackgroundN6-methyladenosine (m6A) is the most pervasive modification of RNA methylation in eukaryotic cells. m6A modification plays a pivotal role in tumorigenesis and progression in many types of cancers. Until now, the role of m6A modification in esophageal carcinoma (ESCA) has remained obscure. The aim of the study was to construct and validate prognostic signatures based on m6A regulators for ESCA.MethodsTranscriptomic data, somatic mutations and clinical information were obtained from The Cancer Genome Atlas (TCGA). Copy number variations were obtained from the UCSC (University of California, Santa Cruz) Xena database. We curated 21 m6A regulators and performed consensus clustering analysis to quantify the m6A modification pattern.ResultsOf the 184 patients, 23 (12.5%) were genetically altered in m6A regulators, with the highest frequency of mutations in ZC3H13 and LRPPRC. We constructed a m6A score system to investigate the prognosis of ESCA. The m6A score was closely related to immune cell infiltration in the tumor immune microenvironment. Patients with a high m6A score had an unfavorable prognosis. The combination of tumor mutation burden and m6A score would improve the prognostic value.ConclusionsOur study established and validated a strong prognostic signature based on m6A regulators. This can be used to accurately predict the prognosis of ESCA.
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Project description:RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.
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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.
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Project description:RNA m6A methylome of Caenorhabditis elegans
| PRJNA625719 | ENA
Project description:Development of the early embryo is thought to be mainly driven by maternal gene products and post-transcriptional gene regulation. Here, we used metabolic labeling to show that RNA can be transferred by sperm into the oocyte upon fertilization. To identify genes with paternal expression in the embryo, we performed crosses of males and females from divergent Caenorhabditis elegans strains. RNA sequencing of mRNAs and small RNAs in the 1-cell hybrid embryo revealed that about one hundred sixty paternal mRNAs are reproducibly expressed in the embryo and that about half of all assayed endogenous siRNAs and piRNAs are also of paternal origin. Together, our results suggest an unexplored paternal contribution to early development.
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Project description:MotivationThe post-transcriptional epigenetic modification on mRNA is an emerging field to study the gene regulatory mechanism and their association with diseases. Recently developed high-throughput sequencing technology named Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) enables one to profile mRNA epigenetic modification transcriptome wide. A few computational methods are available to identify transcriptome-wide mRNA modification, but they are either limited by over-simplified model ignoring the biological variance across replicates or suffer from low accuracy and efficiency.ResultsIn this work, we develop a novel statistical method, based on an empirical Bayesian hierarchical model, to identify mRNA epigenetic modification regions from MeRIP-seq data. Our method accounts for various sources of variations in the data through rigorous modeling and applies shrinkage estimation by borrowing information from transcriptome-wide data to stabilize the parameter estimation. Simulation and real data analyses demonstrate that our method is more accurate, robust and efficient than the existing peak calling methods.Availability and implementationOur method TRES is implemented as an R package and is freely available on Github at https://github.com/ZhenxingGuo0015/TRES.Supplementary informationSupplementary data are available at Bioinformatics online.
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