Similar Datasets
Project description:N6-methyladenosines (m6A) are stoichiometrically deposited on exons of nearly one third of RNA Pol II transcriptome, mainly catalysed by METTL3/14 complex. However, neither the intronic methylation pattern and its functional relevance nor the immediate response upon m6A loss have been fully understood. Here we applied MeRIP-seq on mESC nascent transcriptome and thus revealed that approximately 6-10% of m6A peaks occurred at intronic regions, preferentially the conserved and alternative exon/intron part of longer introns, proximately to 5’-splice sites. These intronic m6A deposition correlates with both Rbm15 binding and H3K36me3. Moreover, coupled 4sU-seq with METTL3 dTAG system in mESC, we found that m6A mediates alternative exon/intron inclusive in nascent transcriptome. Intriguingly, m6A self-regulation including writers and readers are early response and partially contributed by alternative splicing changes. Collectively, our study presents a unified model that m6A mediates alternative splicing, and dTAG METTL3 opens an avenue to interrogate the direct response of functional m6A disruption.
2020-09-15 | GSE154705 | GEO
Project description:N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome
| PRJNA647274 | ENA
Project description:N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]
| PRJNA647276 | ENA
Project description:RNA N 6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5' splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5' splice site that is suppressed in the presence of m6A and upstream of a 5' splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.
| S-EPMC8327914 | biostudies-literature
Project description:N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.
| S-EPMC361031 | biostudies-other
Project description:N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective 'reader' proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.
| S-EPMC5648532 | biostudies-literature
Project description:SRSF2, a Serine-Arginine rich (SR) protein, is a splicing activator that mediates exon inclusion and exclusion events equally well. Here we show SRSF2 directly suppresses intron splicing to suppress cassette exon inclusion in SMN premRNA. Through a serial mutagenesis, we demonstrate that a 10 nt RNA sequence surrounding the branch-point (BP), is important for SRSF2-mediated inhibition of cassette exon inclusion through directly interacting with SRSF2. We conclude that SRSF2 inhibits intron splicing to promote exon exclusion. [BMB Reports 2017; 50(8): 423-428].
| S-EPMC5595172 | biostudies-literature
Project description:The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2?. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2? signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). While depleting m6A demethylases represses ATF4 reinitiation, knocking down m6A methyltransferases promotes ATF4 translation. We demonstrate that m6A in the 5' UTR controls ribosome scanning and subsequent start codon selection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by dynamic mRNA methylation. Consistently, Fto transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of m6A in translational regulation of ISR at cellular and organismal levels.
| S-EPMC5816726 | biostudies-literature
Project description:Despite significant advances in high-throughput DNA sequencing, many important species remain understudied at the genome level. In this study we addressed a question of what can be predicted about the genome-wide characteristics of less studied species, based on the genomic data from completely sequenced species. Using NCBI databases we performed a comparative genome-wide analysis of such characteristics as alternative splicing, number of genes, gene products and exons in 36 completely sequenced model species. We created statistical regression models to fit these data and applied them to loblolly pine (Pinus taeda L.), an example of an important species whose genome has not been completely sequenced yet. Using these models, the genome-wide characteristics, such as total number of genes and exons, can be roughly predicted based on parameters estimated from available limited genomic data, e.g. exon length and exon/gene ratio.
| S-EPMC3064661 | biostudies-literature
Project description:Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA-RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5' splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5' splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.
| S-EPMC25003 | biostudies-literature