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M6A level and isoform characterization sequencing (m6A-LAIC-seq) reveal the census and complexity of the m6A epitranscriptome

ABSTRACT: N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (“m6A levels”), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3’ untranslated regions (3’-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE66086 | GEO | 2016/06/24

SECONDARY ACCESSION(S): PRJNA275864

REPOSITORIES: GEO

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m6A level and isoform characterization sequencing (m6A-LAIC-seq) reveal the census and complexity of the m6A epitranscriptome

Project description:N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (âm6A levelsâ), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3â untranslated regions (3â-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. m6A-LAIC-seq of H1-ESC and GM12878 cell lines, each cell line has two replicates

2016-06-24 | E-GEOD-66086 | biostudies-arrayexpress

m6A reader CPSF70 controls polyadenylation site choice through m6A-dependent recognition of FUE polyadenylation signal

Project description:We conduct herein a systematic study of mRNA recognition and consequent polyadenylation processing of the Arabidopsis mRNA by m6A reader protein CPSF70. Transcriptome-wide characterization of CPSF70-binding sites supporting the recognition m6A-methylated mRNA with CPSF70, and the results of which linked polyadenylation signals recognition. We then perform 3’end sequencing with A-seq2 to identify CPSF70-dependent APA process, showing that CPSF70 modulate m6A–dependent polyadenylation with FUE recognition.

2022-01-15 | GSE143720 | GEO

Alternative cleavage and polyadenylation analysis in an isoform-resolved way by Second- and Third-Generation Sequencing technologies

Project description:Alternative cleavage and polyadenylation (APA) is emerging as an important mechanism of gene regulation in eukaryotes and plays important regulatory roles in human development and diseases. Despite the widespread application of Second Generation Sequencing (SGS) technology for polyadenylation site identification, matching each identified polyadenylation site within a gene to its derived isoform remains a major challenge. To achieve the isoform-resolved APA analysis, we developed a tool termed “IDP-APA” that constructs truly expressed isoforms and identifies polyadenylation sites by integrating the respective strengths of Third Generation Sequencing (TGS) long reads and SGS short reads. Compared to existing tools, IDP-APA demonstrated superior performance in both isoform reconstruction and polyadenylation site identification. Applications to human embryonic stem cells, breast cancer cells and brain tissue from a patient with Alzheimer’s disease revealed prevalent APA events and cell-/tissue-specific APA patterns, especially in an isoform-resolved way.

2017-06-01 | GSE97688 | GEO

The m6A reader CPSF30-L regulates alternative polyadenylation affecting nitrate signaling in Arabidopsis

Project description:As the most ubiquitous internal modification of eukaryotic mRNA, m6A (N6-methyladenosine) modification plays a vital role in almost every aspect of mRNA metabolism. However, the evidence of m6A in regulating the alternative polyadenylation (APA) is limited. APA is controlled by a large protein-RNA complex with many components including CPSF30. Arabidopsis CPSF30 has two isoforms, the longer one named CPSF30-L containing an additional YTH (YT512-B Homology)-domain in the C terminus, which is unique in plant. In this study, we revealed that m6A modification directly regulated APA, by proving the capability of Arabidopsis CPSF30-L YTH domain binding to m6A substrate by using in vitro assay and structural studies. We observed that extensive genes shifted their poly(A) sites in cpsf30-2 and found that numerous genes altered polyadenylation site (PAS) that correlated well with previously identified m6A peaks, indicating that genes carry m6A modification are prone to be regulated by APA. Moreover, we found that several important genes involved in nitrate metabolism are amongst those genes with APA site alteration in cpsf30-2.And we could rescue these APA and nitrate metabolism defects by introducing the wild-type CPSF30-L, but not by m6A-binding defective mutants (W259A, W310A or Y319A), which explained the nitrogen signaling defects of cpsf30-2 discovered previously. Taken together, our results demonstrated that m6A modification could directly regulate APA in Arabidopsis and revealed the function of m6A reader CPSF30-L in nitrate signaling by controlling APA regulation. This study will shed new lights on the roles of m6A modification during RNA 3’-end processing in plant development.

2021-03-16 | GSE145109 | GEO

Alternative 3’UTRs play a widespread role in translation-independent mRNA association with endoplasmic reticulum

Project description:Transcripts encoding membrane and secreted proteins are known to undergo translation on endoplasmic reticulum (ER). Translation-independent ER association (TiERA) has been reported for certain mRNAs, but the phenomenon is poorly understood. Here, using cell fractionation, polysome profiling, and 3’ end sequencing, we examine TiERA of poly(A)+ RNAs in mouse C2C12 myoblast cells. We identify transcript features that facilitate TiERA, including transcript size and GG and GC content. Consistent with the feature analysis, alternative polyadenylation (APA) isoforms differ substantially in TiERA, with long 3’UTR isoforms generally having a higher TiERA potential than short 3’UTR isoforms. Importantly, the widespread 3’UTR lengthening taking place in cell differentiation leads to greater transcript association with ER in differentiated myotubes, despite that the TiERA potential being generally maintained. In addition, we show that TiERA correlates negatively with mRNA stability, highlighting 3’UTR-mediated mRNA decay on ER. Together, our data indicate that sequence and size features impact ER association independent of translation, leading to distinct mRNA metabolism for different transcript groups, and APA can alter transcript stability and translation through isoform-specific ER association.

2021-07-20 | GSE162971 | GEO

m6A in 5â untranslated regions promotes cap-independent translation of mRNA

Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We present eIF3a PAR-iCLIP data which demonstrate that eIF3 predominantly binds mRNAs within the 5'UTR. eIF3 binding sites are also in proximity to m6A residues within the 5'UTR of cellular mRNAs. Two replicates of eIF3a PAR-iCLIP in HEK293T cells.

2015-10-26 | E-GEOD-73405 | biostudies-arrayexpress

Structural basis of YTH domain as m6A reader

Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.

2014-09-18 | E-GEOD-58352 | biostudies-arrayexpress

m6A RNA methylation promotes XIST-mediated transcriptional repression

Project description:XIST is a long non-coding RNA (lncRNA) that mediates transcriptional silencing of X chromosome genes. Here we show that XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues, a reversible base modification whose function in lncRNAs is unknown. We show that m6A formation in XIST, as well as cellular mRNAs, is mediated by RBM15 and its paralog RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in methylation of adenosines in adjacent m6A consensus motifs. Furthermore, knockdown of RBM15 and RBM15B, or knockdown of the m6A methyltransferase METTL3 impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTHDC1 preferentially recognizes m6A in XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression. Three to four biological HEK293T replicates were used to perform iCLIP of endogenous YTH proteins, RBM15, and RBM15B. Crosslinking induced truncations were identified using CIMS-CITS pipeline.

2016-09-07 | E-GEOD-78030 | biostudies-arrayexpress

Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products

Project description:Alternative polyadenylation (APA) produces transcript 3’ untranslated regions (3’UTRs) with distinct sequences, lengths, stability, and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3’UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3’UTR products of APA, leading to their systematic downregulation. Further, we find that many APA events consistently observed in multiple tumor types are controlled by NMD. Additionally, PTBP1, previously implicated in direct modulation of co-transcriptional polyA site choice, regulates the balance of short and long 3’UTR isoforms by inhibiting NMD. Our data suggest that PTBP1 binding near polyA sites can drive production of long 3’UTR APA products in the nucleus and/or protect them from decay in the cytoplasm. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

2020-05-28 | GSE138736 | GEO

Alternative Polyadenylation by Sequential Activation of Distal and Proximal PolyA Sites

Project description:Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to result from competition between proximal and distal polyA sites. By Fractionation-seq, we unexpectedly identified several hundred APA genes where their distal polyA isoforms are retained in chromatin/nuclear matrix and proximal polyA isoforms released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-seq provided further evidence that the strong distal polyA sites are first processed and the resulting transcripts are anchored in chromatin/nuclear matrix for further processing at proximal polyA sites and removal of certain slowly spliced introns. By engineering an autocleavable ribozyme between the proximal and distal polyA sites, we demonstrated that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, rather than competitively, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells.

2021-11-26 | GSE165742 | GEO

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