Localization of RNAPII and 3 end formation factor CstF subunits on C. elegans genes and operons
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Alfonso Garrido-Lecca
PROVIDER: E-GEOD-79823 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Transcription termination is mechanistically coupled to pre-mRNA 3â end formation to prevent transcription much beyond the gene 3â end. C. elegans, however, engages in polycistronic transcription of operons in which 3â end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3â ends. These 3â peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3â end formation factor CstF. This pattern occurs at all 3â ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3â end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3â ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3â cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3â end machinery to the transcription complex. RNA-seq analysis of wild type (empty vector) and RNAi CstF50 mRNA (polyA-selected) and rRNA-depleted RNA (total RNA).
2016-04-02 | E-GEOD-79822 | biostudies-arrayexpress
Project description:Transcription termination is mechanistically coupled to pre-mRNA 3' end formation to prevent transcription much beyond the gene 3' end. C. elegans, however, engages in polycistronic transcription of operons in which 3' end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3' ends. These 3' peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3' end formation factor CstF. This pattern occurs at all 3' ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3' end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3' ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3' cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3' end machinery to the transcription complex.
| S-EPMC4984680 | biostudies-literature
Project description:Transcription termination is mechanistically coupled to pre-mRNA 3â end formation to prevent transcription much beyond the gene 3â end. C. elegans, however, engages in polycistronic transcription of operons in which 3â end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3â ends. These 3â peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3â end formation factor CstF. This pattern occurs at all 3â ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3â end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3â ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3â cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3â end machinery to the transcription complex. ChIP-seq examination of RNAPII and CstF subunits using C. elegans in duplicates.
2016-04-02 | E-GEOD-79821 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2016-04-02 | GSE79823 | GEO
Project description:Transcription termination is mechanistically coupled to pre-mRNA 3’ end formation to prevent transcription much beyond the gene 3’ end. C. elegans, however, engages in polycistronic transcription of operons in which 3’ end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3’ ends. These 3’ peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3’ end formation factor CstF. This pattern occurs at all 3’ ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3’ end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3’ ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3’ cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3’ end machinery to the transcription complex.
2016-04-02 | GSE79822 | GEO
Project description:Transcription termination is mechanistically coupled to pre-mRNA 3’ end formation to prevent transcription much beyond the gene 3’ end. C. elegans, however, engages in polycistronic transcription of operons in which 3’ end formation between genes is not accompanied by termination. We have performed RNA polymerase II (RNAPII) and CstF ChIP-seq experiments to investigate at a genome-wide level how RNAPII can transcribe through multiple poly-A signals without causing termination. Our data shows that transcription proceeds in some ways as if operons were composed of multiple adjacent single genes. Total RNAPII shows a small peak at the promoter of the gene cluster and a much larger peak at 3’ ends. These 3’ peaks coincide with maximal phosphorylation of Ser2 within the C-terminal domain (CTD) of RNAPII and maximal localization of the 3’ end formation factor CstF. This pattern occurs at all 3’ ends including those at internal sites in operons where termination does not occur. Thus the normal mechanism of 3’ end formation does not always result in transcription termination. Furthermore, reduction of CstF50 by RNAi did not substantially alter the pattern of CstF64, total RNAPII, or Ser2 phosphorylation at either internal or terminal 3’ ends. However, CstF50 RNAi did result in a subtle reduction of CstF64 binding upstream of the site of 3’ cleavage, suggesting that the CstF50/CTD interaction may facilitate bringing the 3’ end machinery to the transcription complex.
2016-04-02 | GSE79821 | GEO
Project description:Localization of RNAPII and 3 end formation factor CstF subunits on C. elegans genes and operons
| PRJNA317067 | ENA
Project description:The Integrator Complex is a group of proteins responsible for the endonucleolytic cleavage of primary small nuclear RNA (snRNA) transcripts within the nucleus. Integrator subunits 9 and 11 (IntS9/11) are thought to contain the catalytic activity based on their high sequence similarity to CPSF100 and CPSF73, which have been shown to be components of both the poly(A)(+) and histone pre-mRNA cleavage complex. Here we demonstrate that the specific heterodimeric interaction between IntS9 and IntS11 is mediated by a discrete domain present at the extreme C terminus of IntS9 and within the C terminus of IntS11, adjacent to the predicted active site of this endonuclease. This domain is highly conserved within IntS11 but conspicuously absent in CPSF73. Using a cell-based complementation assay that measures Integrator activity, we determined that the IntS9 interaction domain within IntS11 is required for its ability to restore snRNA 3' end processing after RNA interference (RNAi)-mediated depletion of IntS11. Moreover, overexpression of these interaction domains alone elicits snRNA misprocessing through a dominant-negative titration of endogenous Integrator subunits. These data collectively explain the mechanism by which the IntS11/9 and, by analogy, the CPSF73/100 heterodimeric cleavage factors distinguish themselves from each other and demonstrate that the heterodimeric interaction is functionally required for snRNA 3' end formation.
| S-EPMC3295014 | biostudies-literature
Project description:Cleavage stimulation factor (CstF) is a highly conserved protein complex composed of three subunits that recognizes G/U-rich sequences downstream of the polyadenylation signal of eukaryotic mRNAs. While CstF has been identified over 25 years ago, the architecture and contribution of each subunit to RNA recognition have not been fully understood. In this study, we provide a structural basis for the recruitment of CstF-50 to CstF via interaction with CstF-77 and establish that the hexameric assembly of CstF creates a high affinity platform to target various G/U-rich sequences. We further demonstrate that CstF-77 boosts the affinity of the CstF-64 RRM to the RNA targets and CstF-50 fine tunes the ability of the complex to recognize G/U sequences of certain lengths and content.
| S-EPMC5778602 | biostudies-literature
Project description:The 3' ends of mRNAs terminate with a poly(A) tail. This post-transcriptional modification is directed by sequence features present in the 3'-untranslated region (3'-UTR). We have undertaken a computational analysis of 3' end formation in Caenorhabditis elegans. By aligning cDNAs that diverge from genomic sequence at the poly(A) tract, we accurately identified a large set of true cleavage sites. When there are many transcripts aligned to a particular locus, local variation of the cleavage site over a span of a few bases is frequently observed. We find that in addition to the well-known AAUAAA motif there are several regions with distinct nucleotide compositional biases. We propose a generalized hidden Markov model that describes sequence features in C.elegans 3'-UTRs. We find that a computer program employing this model accurately predicts experimentally observed 3' ends even when there are multiple AAUAAA motifs and multiple cleavage sites. We have made available a complete set of polyadenylation site predictions for the C.elegans genome, including a subset of 6570 supported by aligned transcripts.
| S-EPMC443532 | biostudies-literature