GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Series GSE201045 Query DataSets for GSE201045
Status Public on Nov 19, 2022
Title The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [eCLIP]
Organisms Homo sapiens; Human gammaherpesvirus 8
Experiment type Other
Summary Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5ยด-end of the gene. From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
Overall design We reactivated iSLK cells latently infected with wild-type BAC16 KSHV. The virus was reactivated with sodium butyrate and doxycycline and cells were harvested 24 hours later. After lysis and immunoprecipitation with anti-PNUTS ab (A300-439A-T; Bethyl), samples were run on an SDS-PAGE, transferred to PVDF membrate, and cut from the gel from the 115-190kD region. Total lysate was run on the gel and a size-matched input (SMI) was processed in parallel. RNA was isolated from both SMI and IP samples, libraries were made for sequencing. The protocol followed that of van Nostrand et al (2016). Two independent biological replicates were performed.
Contributor(s) Conrad NK, Devlin AM, Hunter OV, Barnes SD
Citation(s) 36460671
Submission date Apr 19, 2022
Last update date Jan 04, 2023
Contact name Nicholas K Conrad
Phone 2146480795
Organization name UT Southwestern Medical Center
Department Microbiology
Street address 6000 Harry Hines Blvd
City Dallas
State/province TX
ZIP/Postal code 75063
Country USA
Platforms (1)
GPL24095 Illumina HiSeq 2500 (Homo sapiens; Human gammaherpesvirus 8)
Samples (4)
GSM6049606 SMI-Rep 1
GSM6049607 IP-Rep1
GSM6049608 SMI-Rep2
This SubSeries is part of SuperSeries:
GSE201046 The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication
BioProject PRJNA828144

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE201045_RAW.tar 61.4 Mb (http)(custom) TAR (of BW)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap