M6A reader CPSF70 controls polyadenylation site choice through m6A-dependent recognition of FUE polyadenylation signal
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE143720 | GEO | 2022/01/15
REPOSITORIES: GEO
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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
Project description:m6A reader CPSF70 controls polyadenylation site choice through m6A-dependent recognition of FUE polyadenylation signal
| PRJNA601435 | ENA
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
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 (“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.
2016-06-24 | GSE66086 | GEO
Project description:Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. We first analyzed HeLa cell transcriptome using PAS-Seq to demonstrate that PAS-Seq not only accurately and comprehensively identifies poly(A) junctions, but also provide quantitative information on the relativel abundance of APA isoforms. Next we analyzed the mouse embryonic stem cells, neural stem/progenitor cells, and neurons by PAS-Seq to characterize the dynamic changes of mRNA polyadenylation during stem cell differentiation.
2011-02-26 | E-GEOD-25450 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6 A) and alternative polyadenylation (APA) are important regulators of gene expression in eukaryotes. Recently, it was found that m6 A is closely related to APA. However, the molecular mechanism of this new APA regulation remains elusive. Here, we show that YTHDC1, a nuclear m6 A reader, can suppress proximal APA sites and produce longer 3' UTR transcripts by binding to their upstream m6 A sites. YTHDC1 can directly interact with the 3' end processing factor FIP1L1 and interfere with its ability to recruit CPSF4. Binding to the m6 A sites can promote liquid-liquid phase separation of YTHDC1 and FIP1L1, which may play an important role in their interaction and APA regulation. Collectively, YTHDC1 as an m6 A "reader" links m6 A modification with pre-mRNA 3' end processing, providing a new mechanism for APA regulation.
2022-03-11 | GSE198143 | GEO
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
Project description:Cleavage and polyadenylation is essential for 3’ end processing of almost all eukaryotic mRNAs. Recent studies have shown widespread alternative cleavage and polyadenylation (APA) events leading to mRNA isoforms with different 3’UTRs and/or coding sequences. Here we present a compendium of conserved cleavage and polyadenylation sites (PASs) in mammalian genes, based on ~1.2 billion 3’ end sequencing reads from over 360 human, mouse and rat samples. We show that ~80% of mammalian mRNA genes contain at least one conserved PAS, and ~50% have conserved APA events. PAS conservation generally reduces promiscuous 3’ end processing, stabling gene expression levels across species. Conservation of APA correlates with gene age, gene expression features, and gene functions. Genes with certain functions, such as cell morphology, cell proliferation, and mRNA metabolism, are particularly enriched with APA events. While tissue-specific genes typically have a low APA rate, brain-specific genes tend to evolve APA. We show enrichment of mRNA destabilizing motifs in alternative 3’UTR sequences, leading to substantial differences in mRNA stability between 3’UTR APA isoforms. Using conserved PASs, we reveal sequence motifs surrounding APA sites and a preference of adenosine at the cleavage site. Mutations of the U-rich motif around the PAS often accompany APA profile changes between species. Analysis of lncRNA PASs indicates a mechanism of PAS fixation involving evolution of A-rich motifs. Taken together, our results present a comprehensive view of PAS evolution in mammals, and a phylogenic understanding of APA functions.
2018-08-08 | GSE111134 | GEO
Project description:Alternative polyadenylation (APA) is strikingly dysregulated in many cancers. Although APA dysregulation is frequently associated with poor prognosis, the biological importance of most APA events remains unclear simply because few have been functionally studied. Here, we performed a CRISPR/Cas9-based screen to assess individual APA events’ contributions to tumor growth in vivo. Forcing use of specific polyadenylation sites altered mRNA and protein levels to modify mouse melanoma growth in an immunocompetent host. Our screen highlighted APA events of potential clinical relevance. For example, forced Atg7 3′ UTR lengthening in mouse melanoma reduced ATG7 protein levels and tumor immune infiltration; similarly, in human melanoma, a long ATG7 3′ UTR and low mRNA levels were significantly associated with reduced anti-tumor T cell activity and failure of immune checkpoint blockade. Our data demonstrate that cancer-associated APA plays a causative role in tumorigenesis and motivate future studies of the therapeutic potential of modulating APA.
2023-06-01 | GSE212278 | GEO
Project description:Alternative polyadenylation (APA) is an important post-transcriptional modification implicated in development. Female germline stem cell (FGSC) is unipotent and capable of giving rise to oocyte. However, whether alternative polyadenylation plays a role in self-renew and cell fate determination of FGSCs remain elusive. Here, we used 3T-Seq developed in our lab to profile genome-wide 3a termini of transcripts and delineate APA sites in mouse FGSCs and explored the biological significance of APA modulation in FGSC identity.
2015-11-20 | E-MTAB-4038 | biostudies-arrayexpress