AS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - original draft, Writing - review and editing
JZ: Data curation, Formal analysis
DH: Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft
SB: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - original draft, Writing - review and editing
UPF1 is an RNA helicase that scans RNA to unwind secondary structures and to displace associated factors (Franks
Here we considered whether the association of UPF1 with nascent transcripts influences Pol II transcription, and tested it by ChIP-seq of Pol II in
We considered whether this increase in unphosphorylated Pol II at pausing sites might be a consequence of reduced Ser2 phosphorylation, resulting in the slow release of Pol II from pausing sites and abnormal transcription elongation. We tested this hypothesis by examining the reappearance of newly phosphorylated Ser2 Pol II following withdrawal of 5, 6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB), which is a transcription inhibitor that blocks Ser2 phosphorylation and should lead to rapid depletion of Ser2 Pol II. As expected, DRB treatment of S2 cells causes a drastic depletion of Ser2 Pol II and an increase of unphosphorylated Pol II (
These observations indicate that UPF1 might, by associating with nascent transcripts, influence Ser2 phosphorylation of Pol II at TSS-proximal pausing sites and hence transcription elongation at several
S2 cells (CVCL_Z232, laboratory stock originally purchased from Invitrogen/Thermo Fisher Scientific) were cultured in Insect–XPRESS media (Lonza) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin-Glutamine mix (P/S/G, Invitrogen) at 27°C. To make the RNAi constructs for UPF1, the specific sequences were PCR amplified from S2 cell genomic DNA by using corresponding primer pairs. Along with the desired gene sequence, the primer pair carried the T7 promoter sequence at their 5’ end. The amplified PCR fragments were purified using Monarch® PCR and DNA Cleanup Kit (T1030S, NEB) and dsRNA was synthesized using the T7 RiboMAX express RNAi system (P1700, Promega). To induce RNAi, a six-well culture dish was seeded with 10 6 cells/well in serum-free media and mixed with 15 µg of dsRNA/well. Following 1 hour incubation at room temperature, 2 mL of complete media was added to each well and the cells were incubated for the next three days to knockdown UPF1 before harvesting.
Western blotting was performed as described previously (Singh
S2 cells were treated with DRB (Sigma-Aldrich, 125µM) for 1 hour at room temperature. Ser2-Pol II ChIP was performed as described below and quantified by real-time PCR using
Chromatin immunoprecipitation, sequencing and data analysis was done as described previously (Singh
UPF1RNAi-FP – 5’-
UPF1RNAi-RP – 5’-
(Bold is T7 promoter sequence)
Socs36E-FP – 5’-CAGAAAACCGCACACAGACA -3’
Socs36E-RP – 5’-CACACATCGGACTAACAGCG-3’
Xrp1-FP – 5’-TCATAATGCTTGTGGGGCCT -3’
Xrp1-RP – 5’-AGGGTCCCTCTAAACAAGCT-3’
See Methods
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Leverhulme Trust, RPG-2014-291, Saverio Brogna; Wellcome Trust, 9340/Z/09/Z, Saverio Brogna; BBSRC, BB/M022757/1, Saverio Brogna; BBSRC , BB/S017984/1, Saverio Brogna; BBSRC, BB/M017982/1, Daniel Hebenstreit; BBSRC, BB/L006340/1, Daniel Hebenstreit