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.
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
Project description:This SuperSeries is composed of the following subset Series: GSE27452: Genome-wide maps of polyadenylation sites in control and PABPN1kd cells GSE32302: Genome-wide maps of polyadenylation sites in OPMD-model and control mice Refer to individual Series
Project description:We sequenced the 3' ends of polyadenylated transcripts among total RNA in fission yeast wildtype and rdp1∆ cells and mapped the 3'-most nucleotide prior to polyadenylation. We find that mRNA-coding open reading frames have a low number of polyadenylation sites with most of the reads, whereas pericentromeric dg and dh repeats have a high number of polyadenylation sites throughout the transcribed region, mapping to both strands. direct sequencing of polyadenylated 3' ends of RNA (Helicos)
Project description:Most eukaryotic genes express mRNAs with alternative polyadenylation sites at their 3’ ends. Here we show that polyadenylated 3’ termini in three yeast species (S. cerevisiae, K. lactis, D. hansenii) are remarkably heterogeneous. Instead of a few discrete 3’ ends, the average yeast gene has an “end zone”, a >200 bp window with >60 distinct poly(A) sites, the most utilized of which represents only 20% of the mRNA molecules. The pattern of polyadenylation within this zone varies across species, with D. hansenii possessing a higher focus on a single dominant point closer to the ORF terminus. Some polyadenylation occurs within mRNA coding regions with a strong bias towards the promoter. The polyadenylation pattern is determined by a highly degenerate sequence over a broad region and by a local sequence that relies on A residues after the cleavage point. Many dominant poly(A) sites are predicted to adopt a common secondary structure that may be recognized by the cleavage/polyadenylation machinery. We suggest that the end zone reflects a region permissive for polyadenylation, within which cleavage occurs preferentially at the A-rich sequence. In S. cerevisiae strains, D. hansenii genes adopt the S. cerevisiae polyadenylation profile, indicating that the polyadenylation pattern is mediated primarily by species-specific factors. Four sequencing lanes containing direct RNA sequence from S. cerevisiae (strains JGY2000 and two replicates of AB1380), K. lactis strain CLIB209, D. hansenii strain NCYC2572, and S. cerevisiae strains JYAC06 and JYAC07, each harboring D. hansenii sequences on a YAC. AB1380 is the non-YAC-containing S. cerevisiae parental strain for JYAC06 and JYAC07.
Project description:Cleavage factor I mammalian (CFIm) complex, composed of cleavage and polyadenylation specificity factor CPSF6, regulates alternative polyadenylation (APA). CPSF6 has a RS-like domain which plays role in protein -protein interactions. This interaction might have role in alternative polyadenylation site selection. The phosphorylation of RS- like domain might play role in protein -protein interaction and thus might have a role in alternative polyadenylation site selection. So we did mass specteroanalysis to analyse phosphorylation sites of RS-like domain of CPSF6.