Brogna, Saverio

Hebenstreit, Daniel

Singh, A. K.

Zhang, Jie

Warwick Research Archives Portal Repository

 Evidence of slightly increased Pol II pausing in UPF1-depletedDrosophila melanogaster cellsAnand K Singh1, Jie Zhang2, Daniel Hebenstreit2 and Saverio Brogna1§1University of Birmingham, UK  2University of Warwick, UK§To whom correspondence should be addressed: s.brogna@bham.ac.ukFigure 1: Depletion of UPF1 increases Pol II pausing at TSS proximal sites. (A) Metagene line plots of ChIP-seq datashowing average occupancy of unphosphorylated Pol II at gene bodies (scaled as 16 bins of full length) and + 500 bp fromeither end, in normal S2 cells (control, dark blue) or UPF1-RNAi S2 cells (UPF1KD, light blue); based on 3934 (N)transcription units that do not overlap within 500 bp of either end. (B) Metagene plots showing unphosphorylated Pol II atTSS +/- 100 bp, based on 25440 TSSs that are not closer than 200 bp. (C) Metagene plots as in A showing Ser2 Pol IIoccupancy; in normal S2 cells (control, dark orange line) and UPF1-RNAi S2 cells (UPF1KD, light orange line). (D) Asfor B but showing the Ser2 Pol II profiles. (E) Normalised ChIP-seq profile of either Pol II (blue lines) or Ser2 Pol II(orange lines) at the Xrp1 gene, +/- 1000 bp from the TSS (transcript, NM_001275790). The equivalent plots for RpS3A(NM_166714) gene are shown below. Dark lines refer to normal S2 cells, light lines to UPF1-RNAi, as indicated in thelegend below the plots. (F) Schematics of the timeframe (top) of transcription recovery following removal of DRB after 1hour (T0) treatment of S2 cells; protein levels were assayed at T0 and at three time points from DRB removal: 2.5 minutes(T2.5), 5 minutes (T5) and 10 minutes (T10). The panel below shows western blotting of Ser2 Pol II (top row) andunphosphorylated Pol II (middle row) at different time points after DRB washout in normal (N) and UPF1-RNAi (U)cells. The α-tubulin was detected as a loading control (bottom row). Values below lanes are ImageJ quantifications of theSer2 Pol II and unphosphorylated Pol II band/s intensities, which are normalised by the signal of the α-tubulin band in thesame lane and expressed as ratios of the intensity of the corresponding band in lane 1. (G) Line graphs show real-time 10/16/2020 - Open Access PCR quantifications of average Ser2 Pol II enrichment at either Socs36E gene (on left) or Xrp1 gene (on right) followingDRB treatment and removal at different time points as described in F. The PCR primers used are located about 4 kbdownstream of the TSS of both Socs36E and Xrp1 genes.DescriptionUPF1 is an RNA helicase that scans RNA to unwind secondary structures and to displace associated factors (Franks et al.,2010; Fiorini et al., 2015; Lee et al., 2015; Kanaan et al., 2018). UPF1 has been mostly studied for its role in nonsensemediated mRNA decay (NMD) and other translation-dependent RNA quality-control pathways in the cytoplasm (Iskenand Maquat, 2008; Kim and Maquat, 2019). However, we have recently reported that UPF1 associates, genome wide, withnascent transcripts at most Pol II transcription sites in Drosophila (Singh et al., 2019). The association of UPF1 withnascent transcripts appears to be necessary for the release of processed polyadenylated mRNAs from their transcriptionsites and also for their export from nucleus to cytoplasm (Singh et al., 2019).Here we considered whether the association of UPF1 with nascent transcripts influences Pol II transcription, and tested itby ChIP-seq of Pol II in Drosophila S2 cells. We discovered (using two biological replicates) that there is significantlymore unphosphorylated Pol II associated with the start of genes in cells depleted of UPF1 (Figure 1A and 1B). Thisdifference is most apparent 20 to 60 base pair (bp) downstream of the transcription start site (TSS), as shown in theexpanded depiction in Figure 1B which is based on many more individual TSSs. This is measured by quantifying theaggregate Pol II signal over a +/- 100 bp span at each of 25440 TSSs (P = 0.011). A 1.2-fold or greater increase ofunphosphorylated Pol II in UPF1-depleted cells was detected at 1990 TSSs. The 20-60 bp downstream region correspondsto TSS-proximal Pol II pausing sites in Drosophila (Adelman and Lis, 2012). Conversely, the amount of Ser2 Pol IIassociated with these TSSs was unchanged or marginally reduced in UPF1-depleted cells (Figure 1C and 1D). Theincrease in unphosphorylated Pol II loading downstream of the TSS in UPF1-depleted cells, alongside fairly constant Ser2Pol II loading, is illustrated here by Xrp1 and RpS3A (Figure 1E), two genes that are highly transcribed and show strongUPF1 association in normal cells (Singh et al., 2019).We considered whether this increase in unphosphorylated Pol II at pausing sites might be a consequence of reduced Ser2phosphorylation, resulting in the slow release of Pol II from pausing sites and abnormal transcription elongation. Wetested 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 phosphorylationand should lead to rapid depletion of Ser2 Pol II. As expected, DRB treatment of S2 cells causes a drastic depletion ofSer2 Pol II and an increase of unphosphorylated Pol II (Figure 1F, compare control lanes 9 and 10 with DRB-treatedlanes 1 and 2). Ser2 Pol II levels began to recover soon after DRB removal and were similar to those of untreated controlcells within 10 minutes (Figure 1F, compare lanes 7-8 with control lanes 9-10). However, the recovery in UPF1-depletedcells seemed slower during the first 2.5 minutes and to a lesser extent after 5 minutes (Figure 1F, compare lanes 3 vs. 4and 5 vs. 6). Similar observations were made in another independent replicate of the experiment. A comparable bluntedrecovery of gene-associated Ser2 Pol II in UPF1-depleted cells was detected by ChIP-PCR at the two representativehighly active genes, Socs36E and Xrp1 (Figure 1G), both of which show strong association with UPF1 (Singh et al.,2019). RpS3A was not examined because it is too short (about 2 kb) for this assay.These observations indicate that UPF1 might, by associating with nascent transcripts, influence Ser2 phosphorylation ofPol II at TSS-proximal pausing sites and hence transcription elongation at several Drosophila genes. Alternatively, oradditionally, UPF1 might affect premature transcription termination and Pol II release from DNA downstream of the TSS,which are other, perhaps more important determinants of the high accumulation of Pol II at the beginning of genes(Kamieniarz-Gdula and Proudfoot, 2019) .MethodsRequest a detailed protocolCell culture and RNA interferenceS2 cells (CVCL_Z232, laboratory stock originally purchased from Invitrogen/Thermo Fisher Scientific) were cultured inInsect–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 PCRamplified from S2 cell genomic DNA by using corresponding primer pairs. Along with the desired gene sequence, theprimer pair carried the T7 promoter sequence at their 5’ end. The amplified PCR fragments were purified usingMonarch® PCR and DNA Cleanup Kit (T1030S, NEB) and dsRNA was synthesized using the T7 RiboMAX expressRNAi system (P1700, Promega). To induce RNAi, a six-well culture dish was seeded with 106cells/well in serum-freemedia and mixed with 15 µg of dsRNA/well. Following 1 hour incubation at room temperature, 2 mL of complete mediawas added to each well and the cells were incubated for the next three days to knockdown UPF1 before harvesting.Western blotting and antibodies 10/16/2020 - Open Access Western blotting was performed as described previously (Singh et al., 2019). The antibodies used in western blotting werediluted as follow: Rabbit anti-Ser2 Pol II (Abcam, ab5095, 1:5000), Mouse anti-Pol II (Abcam, ab817 (8WG16), 1:5000)and mouse anti-β-tubulin (Sigma-Aldrich, T5168 1:5000).DRB treatmentS2 cells were treated with DRB (Sigma-Aldrich, 125µM) for 1 hour at room temperature. Ser2-Pol II ChIP was performedas described below and quantified by real-time PCR using Socs36E and Xrp1 primers shown below.ChIP-seq and data analysisChromatin immunoprecipitation, sequencing and data analysis was done as described previously (Singh et al., 2019). Foreach ChIP, typically we used 5 µg of antibody including Rabbit anti-Ser2 Pol II (Abcam, ab5095) and Mouse anti-Pol II(8WG16, Abcam, ab817). ChIP-signal quantification and metagene analysis were performed as previously described(Singh et al., 2019). All new ChIP-seq raw sequencing FASTQ data and Bedgraph files were deposited in the GEOrepository (Accession No GSE157612), all other datasets used in this study were previously deposited (Accession NoGSE16808).Primers List:UPF1RNAi-FP – 5’-TTAATACGACTCACTATAGGGGAGAGGAGAAGCCAGGCATTGA-3’UPF1RNAi-RP – 5’-TTAATACGACTCACTATAGGGGAGAGACCGTGGCCCAACAGG-3’(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’ReagentsSee MethodsAcknowledgments: We like to thank Hannah L Dixon and Bob Michell for critically reading the manuscript.ReferencesAdelman K, Lis JT. 2012. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat RevGenet 13: 720-31. PMID: 22986266.Fiorini F, Bagchi D, Le Hir H, Croquette V. 2015. Human Upf1 is a highly processive RNA helicase and translocase withRNP remodelling activities. Nat Commun 6: 7581. PMID: 26138914.Franks TM, Singh G, Lykke-Andersen J. 2010. Upf1 ATPase-dependent mRNP disassembly is required for completion ofnonsense- mediated mRNA decay. Cell 143: 938-50. PMID: 21145460.Isken O, Maquat LE. 2008. The multiple lives of NMD factors: balancing roles in gene and genome regulation. Nat RevGenet 9: 699-712. PMID: 18679436.Kamieniarz-Gdula K, Proudfoot NJ. 2019. Transcriptional Control by Premature Termination: A Forgotten Mechanism.Trends Genet 35: 553-564. PMID: 31213387.Kanaan J, Raj S, Decourty L, Saveanu C, Croquette V, Le Hir H. 2018. UPF1-like helicase grip on nucleic acids dictatesprocessivity. Nat Commun 9: 3752. PMID: 30218034.Kim YK, Maquat LE. 2019. UPFront and center in RNA decay: UPF1 in nonsense-mediated mRNA decay and beyond.RNA 25: 407-422. PMID: 30655309.Lee SR, Pratt GA, Martinez FJ, Yeo GW, Lykke-Andersen J. 2015. Target Discrimination in Nonsense-Mediated mRNADecay Requires Upf1 ATPase Activity. Mol Cell 59: 413-25. PMID: 26253027.Singh AK, Choudhury SR, De S, Zhang J, Kissane S, Dwivedi V, Ramanathan P, Petric M, Orsini L, Hebenstreit D,Brogna S. 2019. The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release ofmRNAs from gene loci. Elife 8:e41444. PMID: 30907728. Funding: 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; 10/16/2020 - Open Access BBSRC, BB/L006340/1, Daniel HebenstreitAuthor Contributions: Anand K Singh: Conceptualization, Data curation, Formal analysis, Investigation, Methodology,Validation, Writing - original draft, Writing - review and editing. Jie Zhang: Data curation, Formal analysis. DanielHebenstreit: Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft. Saverio Brogna:Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Projectadministration, Resources, Supervision, Validation, Writing - original draft, Writing - review and editing.Reviewed By: AnonymousHistory: Received September 12, 2020  Revision received October 10, 2020  Accepted October 16, 2020  PublishedOctober 16, 2020Copyright: © 2020 by the authors. This is an open-access article distributed under the terms of the Creative CommonsAttribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original author and source are credited.Citation: Singh, AK; Zhang, J; Hebenstreit, D; Brogna, S (2020). Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells. microPublication Biology. https://doi.org/10.17912/micropub.biology.000319 10/16/2020 - Open Access

Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells

http://wrap.warwick.ac.uk/145234/1/WRAP-evidence-slightly-increased-Pol-II-pausing-UPF1-depleted-Drosophila-melanogaster-cells-Hebenstreit-2020.pdf

Evidence of slightly increased Pol II pausing in UPF1-depleted Drosophila melanogaster cells

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