Project description:Motivation: Alternative cleavage and polyadenylation generates mRNA 3´ isoforms in a cell type- and tissue-specific manner. Due to finite available RNA sequencing data of organisms with vast cell type complexity, currently available gene annotation resources are incomplete, which poses significant challenges to the comprehensive interpretation and quantification of transcriptomes. Results: We developed 3'GAmES, a stand-alone analysis pipeline to identify and annotate novel (cell-type-specific) mRNA 3´ isoforms from 3' mRNA sequencing datasets. When applied to mouse embryonic stem cells or Zebrafish embryos, 3'GAmES expands currently available mRNA 3' annotations by 47% and 57%, respectively; and the resulting annotations significantly improve comprehensive gene-tag counting by cost-effective 3' mRNA sequencing to more accurately mirror whole-transcriptome RNAseq measurements. As a stand-alone analysis tool, 3'GAmES systematically augments cell type-specific transcript annotations and increases the robustness of quantitative gene expression profiling by 3' mRNA sequencing.
Project description:The purpose of these experiments was to determine the 5' and 3' transcriptional termini of genes on chromosomes 21-22. Towards this end, we mapped the poly A + RNA isolated from 12 normal tissues and four cell lines. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
Project description:Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA)-Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 (ID2) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3’-UTR termini in highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs.
Project description:Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA)-Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3’ untranslated region (3’-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3’-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 (ID2) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3’-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3’-UTR termini in highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs.
Project description:Phosphorylation plays a central role in coordinating transcription with pre-mRNA maturation, yet the mechanistic understanding of how transcription is regulated by phosphorylation remains limited. The PP1 phosphatase removes CDK9-dependent phosphorylation of Pol II and Spt5, triggering transcription termination. Here, we demonstrate that PNUTS enhances PP1 activity, whereas WDR82 mediates an interaction between PP1 and the pre-mRNA 3’-end processing machinery subunit Symplekin. We present the first structure of a PNUTS-WDR82-Symplekin-Ssu72 complex, revealing that PNUTS and Symplekin bind WDR82 through distinct interfaces using conserved short linear motifs. Mutations that inactivate PP1's catalytic activity or disrupt its interaction with either PNUTS or the 3’-end processing machinery led to impaired transcription elongation, inefficient cleavage of nascent transcripts, altered poly(A) site selection, and widespread transcription termination defects. Our findings demonstrate that the PP1 complex is essential for coupling transcription to pre-mRNA 3’-end maturation, ensuring production of functional mRNA.
Project description:The purpose of these experiments was to determine the 5' and 3' transcriptional termini of genes on chromosomes 21-22. Towards this end, we mapped the poly A + RNA isolated from 12 normal tissues and four cell lines. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf polyA+ RNAs from 11 tissues (Brain Frontal Lobe, Brain Hippocampus, Brain Hypothalamus, Cerebellum, Ovary, Placenta, Prostate, Testis, Fetal Kidney, Fetal Spleen, Fetal Thymus; all from BD Clontech) and 5 cell lines (GM06990, HeLaS3, HepG2, K562, tert-BJ) using the BD SMARTTM RACE cDNA amplification kit (BD Clontech Cat. No.634914). A single array with no replicates was run for each sample with a pool of ~23-24 RACE reactions per sample.