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Genome-wide mapping of UPF1 and Pol II [ChIP-seq]

ABSTRACT: ChIP with NMD core componenet UPF1 antibody in normal S2 cells and ChIP with Pol II Ser2 antibodies in S2 cells. UPF1 is an ATP-driven RNA helicase required for efficient nonsense mediated mRNA decay in eukaryotes. Although it is currently understood that UPF1 primarily acts on the 3’UTR of translating mRNAs in the cytoplasm, our data indicates that this is a highly dynamic protein that is rapidly shuttling between nucleus and cytoplasm in Drosophila. Additionally, ChIP-seq analysis in different cell types demonstrates genome-wide association of UPF1 with nascent RNAs at most of the active Pol II transcription sites, and at some specific Pol III genes. Notably, intron recognition appears to interfere with association and translocation of UPF1 on nascent transcripts. Cells depleted of UPF1 show defects in nuclear processes such as mRNA export and transcription site retention. These data, thus redefine UPF1 as a global player in mRNA based processes in the nucleus as well as the cytoplasm.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE116806 | GEO | 2019/04/01

REPOSITORIES: GEO

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Nascent RNA and Poly(A) RNA Seq from S2 cells [RNA-seq]

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Genome-wide chromosomal associationof Upf1 is linked toPol II transcriptionin Schizosaccharomyces pombe [ChIP-chip]

2021-03-24 | GSE169422 | GEO

Genome-wide mapping of RNA Pol II

Project description:ChIP with Pol II Ser2 or Pol II antibodies in normal and UPF1-RNAi S2 cells.

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UPF1 is required for gene expression in mitochondria and for the elimination of paternal mtDNA

Project description:UPF1 is a well-conserved RNA helicase in eukaryotes, involved in nuclear and cytoplasmic processes of gene expression. This study presents ChIP-seq evidence for its RNA-dependent association with mtDNA transcription sites in Drosophila S2 cells, and RNA-seq data indicating its requirement for the correct expression of mtDNA genes. UPF1 localisation in mitochondria was observed by immunostaining and GFP-tagging in different fly tissues and cell types. During spermatogenesis, depletion of UPF1 but not of other NMD factors, resulted in major defects in meiosis and cytokinesis, leading to complete sterility. Notably, the few spermatids produced show abnormalities in the formation of the nebenkern and, unlike the wild type, retain the mtDNA.

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PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II

Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans. ChIP-seq of Pol II of different forms, SEC subunits, PAFc subunits and H2Bub in human cell lines targeted by PAF1 or scramble shRNA. ChIP-seq of total Pol II in HCT116 cells targeted by BRE1A or scramble shRNA. ChIP-seq of total Pol II in S2 cells targeted by Paf1 or LacZ RNAi. Total RNA-seq, nascent RNA-seq and GRO-seq in HCT116 cells targeted by PAF1 or scramble shRNA.

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