M6A reader CPSF70 controls polyadenylation site choice through m6A-dependent recognition of FUE polyadenylation signal
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PROVIDER: PRJNA601435 | ENA |
REPOSITORIES: ENA
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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
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: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:The m6A reader CPSF30-L regulates alternative polyadenylation affecting nitrate signaling in Arabidopsis
| PRJNA606046 | ENA
Project description:Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four, CPSF160, CPSF30, hFip1 and WDR33, are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73 and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV-crosslinked to such RNAs, as can be CPSF30. Transcriptome-wide identification of WDR33 targets by PAR-CLIP shows that WDR33 specifically binds the AAUAAA signal in vivo in contrast to any of the five other CPSF subunits tested before. Thus, our data indicate that the CPSF subunit that is mainly responsible for recognizing the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies. Transcriptome-wide profiling of WDR33 binding sites using PAR-CLIP in HEK293 cells.
2014-09-25 | E-GEOD-61123 | biostudies-arrayexpress
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.
2014-09-18 | GSE58352 | GEO
Project description:Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four, CPSF160, CPSF30, hFip1 and WDR33, are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73 and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV-crosslinked to such RNAs, as can be CPSF30. Transcriptome-wide identification of WDR33 targets by PAR-CLIP shows that WDR33 specifically binds the AAUAAA signal in vivo in contrast to any of the five other CPSF subunits tested before. Thus, our data indicate that the CPSF subunit that is mainly responsible for recognizing the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.
2014-09-25 | GSE61123 | GEO
Project description:In this study, we report on our findings in T. gondii of an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at certain loci such as stage-specific genes. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. We bring Nanopore direct RNA sequencing-based evidence of the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.
2021-07-15 | GSE168155 | GEO
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:The mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human acute myeloid leukemias (AML). To understand the role of YTHDF2 in AML, we generated m6A meRIP-seq libraries form Ythdf2fl/fl (Ythdf2CTL) pre-leukemic cells.
2019-04-05 | E-MTAB-6791 | biostudies-arrayexpress