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N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

ABSTRACT: N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

PROVIDER: PRJNA647276 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12266500_1.fastq.gz	Fastqsanger.gz
	SRR12266500_2.fastq.gz	Fastqsanger.gz
	SRR12266501_1.fastq.gz	Fastqsanger.gz
	SRR12266501_2.fastq.gz	Fastqsanger.gz
	SRR12266502_1.fastq.gz	Fastqsanger.gz

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N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome [4sU-seq]

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

N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome

Project description:N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome

| PRJNA647274 | ENA

N6-Methyladenosine mediates alternative intron/exon inclusion in the nascent transcriptome

Project description:This SuperSeries is composed of the SubSeries listed below.

2020-09-15 | GSE154709 | GEO

Acute depletion of METTL3 implicates <i>N</i> <sup>6</sup>-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

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

SRSF2 directly inhibits intron splicing to suppresses cassette exon inclusion.

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].

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Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution

Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.

2012-04-25 | E-GEOD-31653 | biostudies-arrayexpress

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Project description:Exon-intron circRNA (EIciRNA) is a subclass of backsplicing-generated circRNAs featured with intron retention, among which some play roles in transcriptional regulation. We aim to characterize EIciRNAs by developing pipeline and investigate the factors involved in regulating EIciRNA biogenesis using CRISPR-Cas9 screen, as well as the physiology functions of EIciRNAs during neuronal differentiation.

2024-03-04 | GSE253233 | GEO

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA.

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.

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Systematic identification and characterization of Exon-intron circRNAs (RNA-Seq)

2024-03-04 | GSE241679 | GEO

Effect of 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq data

Project description:To investigate the effect of increasing 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq, we labeled 3 different cell lines with increasing concentrations of 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.

2024-02-06 | GSE229504 | GEO

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