Project description:This SuperSeries is composed of the following subset Series: GSE37037: Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq GSE37398: Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: CLIP Refer to individual Series
Project description:Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: CLIP
Project description:In eukaryotes, the 3' ends of RNA polymerase II-generated transcripts are made in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. By alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control of 3’ UTR length and drastic changes in 3’ UTR length of mRNAs upon induction of proliferation in resting cells. To understand the dynamics of polyadenylation site usage, we recently identified binding sites of the major pre-mRNA 3’ end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped cleaved polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3’ UTR length. More specifically, we demonstrate that the loss-of-function of CF Im68 and CF Im25 but not of CF Im59 leads to a transcriptome-wide increase of the use of proximal polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in HEK 293 cells under normal conditions, and in HEK 293 cells depleted of 3' end processing factors CF Im25, CF Im59, and CF Im68.
Project description:Genome‐wide analysis of pre‐mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length: A-seq
Project description:In eukaryotes, the 3' ends of RNA polymerase II-generated transcripts are made in the majority of cases by site-specific endonucleolytic cleavage, followed by the addition of a poly(A) tail. By alternative polyadenylation, a gene can give rise to multiple mRNA isoforms that differ in the length of their 3' UTRs and hence in their susceptibility to post-transcriptional regulatory factors such as microRNAs. A series of recently conducted high-throughput studies of poly(A) site usage revealed an extensive tissue-specific control of 3’ UTR length and drastic changes in 3’ UTR length of mRNAs upon induction of proliferation in resting cells. To understand the dynamics of polyadenylation site usage, we recently identified binding sites of the major pre-mRNA 3’ end processing factors - cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), and cleavage factor Im (CF Im) - and mapped cleaved polyadenylation sites in HEK293 cells. Our present study extends previous findings on the role of CF Im in alternative polyadenylation and reveals that subunits of the CF Im complex generally control 3’ UTR length. More specifically, we demonstrate that the loss-of-function of CF Im68 and CF Im25 but not of CF Im59 leads to a transcriptome-wide increase of the use of proximal polyadenylation sites.
Project description:Processing of 3’ untranslated region (3’-UTR) of precursor messenger RNAs (pre-mRNA) is a fundamental step in mRNA maturation, where the mRNA transcript is cleaved at a specific site, located downstream the open reading frame of the encoded gene. The position of the cleavage site is determined by cis-acting RNA sequence elements, which are recognized with high specificity by large 3’ end processing multiprotein complexes. Two main categories of metazoan 3’ pre-mRNA processing have been identified so far, describing elaboration of either canonical mRNAs – occurring during all phases of the cell cycle and containing a characteristic Poly-Adenylation Signal (PAS) element – or histone mRNAs – prevalently occurring during the S phase and containing a Histone Downstream Element (HDE). In this work, we isolate both human endogenous canonical and histone 3’ pre-mRNA processing complexes from human nuclear extracts by using a pre-mRNA containing the 3’UTR sequence of replication-dependent histone H2A.2. This H2A.2 pre-mRNA (H2A_4m), containing both an HDE sequence and a PAS element in a native overlaid position, was modified with a 5’ photocleavable biotin tag, and with modification of few bases to allow complex assembly and purification by affinity chromatography on streptavidin-agarose beads, elution by UV irradiation and subsequent mass spectrometric identification of bound 3’ mRNA processing factors.
Project description:Processing of 3’ untranslated region (3’-UTR) of precursor messenger RNAs (pre-mRNA) is a fundamental step in mRNA maturation, where the mRNA transcript is cleaved at a specific site, located downstream the open reading frame of the encoded gene. The position of the cleavage site is determined by cis-acting RNA sequence elements, which are recognized with high specificity by large 3’ end processing multiprotein complexes. Two main categories of metazoan 3’ pre-mRNA processing have been identified so far, describing elaboration of either canonical mRNAs – occurring during all phases of the cell cycle and containing a characteristic Poly-Adenylation Signal (PAS) element – or histone mRNAs – prevalently occurring during the S phase and containing a Histone Downstream Element (HDE). In this work, we isolate both human endogenous canonical and histone 3’ pre-mRNA processing complexes from human nuclear extracts by using a pre-mRNA containing the 3’UTR sequence of replication-dependent histone H2A.2. This H2A.2 pre-mRNA (H2A_4m), containing both an HDE sequence and a PAS element in a native overlaid position, was modified with a 5’ photocleavable biotin tag, and with modification of few bases to allow complex assembly and purification by affinity chromatography on streptavidin-agarose beads, elution by UV irradiation and subsequent mass spectrometric identification of bound 3’ mRNA processing factors.
Project description:Alternative polyadenylation (APA) produces mRNA isoforms with different 3’UTR lengths. Previous studied indicated that 3’ end processing and mRNA nuclear export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to expression of long 3’UTR isoforms. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal a number of gene features that impact NXF1-dependent APA, including 3’UTR size, gene size and AT content. Surprisingly, downregulation of NXF1 results in accumulation of RNAP II at the 3’ end of genes, correlating with its role in APA regulation. Moreover, we show that NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3’UTR isoform with UGUA motifs. Together, our work reveals several important roles of NXF1 in coordinating RNAPII distribution, 3’ end processing, and mRNA export of long 3’UTR transcripts, implicating NXF1 as the nexus of gene expression regulation.
Project description:Termination of RNAPII transcription is associated with RNA 3â end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3â end processing factors, is enriched at the 3â end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3â UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3â end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation. Two biological replicates of Seb1 and Control (parental strain) CRAC experiments
Project description:During S-phase of the cell cycle production of the core histone proteins is precisely balanced with DNA replication. Metazoan mRNAs encoding replication dependent (RD) histones lack polyA tail normally formed by 3’ end cleavage and coupled polyadenylation of the pre-mRNA. Instead, they undergoes to endonucleolytic cleavage on the 3ʹ side of an RNA hairpin (stem loop) producing mRNA with a 3´-stem loop (SL), which is exported from the nucleus for use in translation. The same endonuclease that is involved in normal protein-coding pre-mRNA cleavage, i.e. cleavage and poyladenylation specificity factor 73 (CPSF73), is proposed to catalyse RD pre-histone mRNA cleavage. Additional factors specific to RD pre-histone mRNA processing, including stem loop binding protein (SLBP) and the U7 small nuclear ribonucleoprotein (U7snRNP) that binds to a histone downstream element (HDE) are thought to be involved in CPSF73 targeting to RD pre-histone mRNA. We report that a different histone specific endonuclease (HSE), which like CPSF73 is a metallo β lactamase (MBL) fold protein, is specific for RD pre-histone mRNA cleavage10,11. Crystallographic and biochemical studies reveal HSE has a di-zinc ion containing active site related to that of CPSF73, but which has distinct overall fold. Notably HSE depletion from cells leads to the production of unprocessed RD pre-histone mRNA due to inefficient 3ʹ end processing. The consequent depletion of core histone proteins correlates with a cell cycle defect due to a delay in entering/progressing through S-phase. HSE thus may represent a new type of S-phase specific cancer target.