Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions

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The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Vyšlo v časopise: Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions. PLoS Genet 10(1): e32767. doi:10.1371/journal.pgen.1004090
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004090

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Zdroje

1. FudaNJ, ArdehaliMB, LisJT (2009) Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461: 186–192 doi:10.1038/nature08449

2. KwakH, FudaNJ, CoreLJ, LisJT (2013) Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339: 950–953 doi:10.1126/science.1229386

3. GilchristDA, Santos dosG, FargoDC, XieB, GaoY, et al. (2010) Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation. Cell 143: 540–551 doi:10.1016/j.cell.2010.10.004

4. MinIM, WaterfallJJ, CoreLJ, MunroeRJ, SchimentiJ, et al. (2011) Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev 25: 742–754 doi:10.1101/gad.2005511

5. CoreLJ, WaterfallJJ, LisJT (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322: 1845–1848 doi:10.1126/science.1162228

6. MarshallNF, PriceDH (1995) Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J Biol Chem 270: 12335–12338.

7. YamaguchiY, TakagiT, WadaT, YanoK, FuruyaA, et al. (1999) NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97: 41–51.

8. WadaT, TakagiT, YamaguchiY, FerdousA, ImaiT, et al. (1998) DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes Dev 12: 343–356.

9. MarshallNF, PengJ, XieZ, PriceDH (1996) Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J Biol Chem 271: 27176–27183.

10. LisJT, MasonP, PengJ, PriceDH, WernerJ (2000) P-TEFb kinase recruitment and function at heat shock loci. Genes Dev 14: 792–803.

11. BoehmAK, SaundersA, WernerJ, LisJT (2003) Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol 23: 7628–7637.

12. FujinagaK, IrwinD, HuangY, TaubeR, KurosuT, et al. (2004) Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol Cell Biol 24: 787–795.

13. LeeC, LiX, HechmerA, EisenM, BigginMD, et al. (2008) NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila. Mol Cell Biol 28: 3290–3300 doi:10.1128/MCB.02224-07

14. RahlPB, LinCY, SeilaAC, FlynnRA, McCuineS, et al. (2010) c-Myc regulates transcriptional pause release. Cell 141: 432–445 doi:10.1016/j.cell.2010.03.030

15. ZeitlingerJ, StarkA, KellisM, HongJ-W, NechaevS, et al. (2007) RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat Genet 39: 1512–1516 doi:10.1038/ng.2007.26

16. GilchristDA, NechaevS, LeeC, GhoshSKB, CollinsJB, et al. (2008) NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes Dev 22: 1921–1933 doi:10.1101/gad.1643208

17. CoreLJ, WaterfallJJ, GilchristDA, FargoDC, KwakH, et al. (2012) Defining the status of RNA polymerase at promoters. Cell Rep 2: 1025–1035 doi:10.1016/j.celrep.2012.08.034

18. NiZ, SaundersA, FudaNJ, YaoJ, SuarezJ-R, et al. (2008) P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo. Mol Cell Biol 28: 1161–1170 doi:10.1128/MCB.01859-07

19. YamaguchiY, InukaiN, NaritaT, WadaT, HandaH (2002) Evidence that negative elongation factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA. Mol Cell Biol 22: 2918–2927.

20. NaritaT, YamaguchiY, YanoK, SugimotoS, ChanaratS, et al. (2003) Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex. Mol Cell Biol 23: 1863–1873.

21. MissraA, GilmourDS (2010) Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex. Proc Natl Acad Sci USA 107: 11301–11306 doi:10.1073/pnas.1000681107

22. OttM, GeyerM, ZhouQ (2011) The control of HIV transcription: keeping RNA polymerase II on track. Cell Host Microbe 10: 426–435 doi:10.1016/j.chom.2011.11.002

23. KarnJ, StoltzfusCM (2012) Transcriptional and Posttranscriptional Regulation of HIV-1 Gene Expression. Cold Spring Harb Perspect Med 2: 1–17 doi:10.1101/cshperspect.a006916

24. RaoJN, SchweimerK, WenzelS, WöhrlBM, RöschP (2008) NELF-E RRM undergoes major structural changes in flexible protein regions on target RNA binding. Biochemistry 47: 3756–3761 doi:10.1021/bi702429m

25. RaoJN, NeumannL, WenzelS, SchweimerK, RöschP, et al. (2006) Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription. Biochem J 400: 449–456 doi:10.1042/BJ20060421

26. LatulippeDR, SzetoK, OzerA, DuarteFM, KellyCV, et al. (2013) Multiplexed microcolumn-based process for efficient selection of RNA aptamers. Anal Chem doi:10.1021/ac400105e

27. BaileyTL, ElkanC (1994) Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2: 28–36.

28. BaileyTL, WilliamsN, MislehC, LiWW (2006) MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res 34: W369–W373 doi:10.1093/nar/gkl198

29. KleinDJ (2001) The kink-turn: a new RNA secondary structure motif. EMBO J 20: 4214–4221 doi:10.1093/emboj/20.15.4214

30. TurnerB, LilleyDMJ (2008) The importance of G.A hydrogen bonding in the metal ion- and protein-induced folding of a kink turn RNA. Journal of Molecular Biology 381: 431–442 doi:10.1016/j.jmb.2008.05.052

31. PaganoJM, ClingmanCC, RyderSP (2011) Quantitative approaches to monitor protein-nucleic acid interactions using fluorescent probes. RNA 17: 14–20 doi:10.1261/rna.2428111

32. WeiP, GarberME, FangSM, FischerWH, JonesKA (1998) A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92: 451–462.

33. FengS, HollandEC (1988) HIV-1 tat trans-activation requires the loop sequence within tar. Nature 334: 165–167 doi:10.1038/334165a0

34. SzetoK, LatulippeDR, OzerA, PaganoJM, WhiteBS, et al. (2013) RAPID-SELEX for RNA Aptamers. PLoS ONE doi:10.1371/journal.pone.0082667

35. DsouzaM, LarsenN, OverbeekR (1997) Searching for patterns in genomic data. Trends Genet 13: 497–498.

36. MarisC, DominguezC, AllainFH-T (2005) The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J 272: 2118–2131 doi:10.1111/j.1742-4658.2005.04653.x

37. JiX, ZhouY, PanditS, HuangJ, LiH, et al. (2013) SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase. Cell 153: 855–868 doi:10.1016/j.cell.2013.04.028

38. DingwallC, ErnbergI, GaitMJ, GreenSM, HeaphyS, et al. (1989) Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc Natl Acad Sci USA 86: 6925–6929.

39. DingwallC, ErnbergI, GaitMJ, GreenSM, HeaphyS, et al. (1990) HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. EMBO J 9: 4145–4153.

40. SaundersA, CoreLJ, LisJT (2006) Breaking barriers to transcription elongation. Nat Rev Mol Cell Biol 7: 557–567 doi:10.1038/nrm1981

41. NaritaT, YungTMC, YamamotoJ, TsuboiY, TanabeH, et al. (2007) NELF interacts with CBC and participates in 3′ end processing of replication-dependent histone mRNAs. Mol Cell 26: 349–365 doi:10.1016/j.molcel.2007.04.011

42. SheffieldP, GarrardS, DerewendaZ (1999) Overcoming expression and purification problems of RhoGDI using a family of “parallel” expression vectors. Protein Expr Purif 15: 34–39 doi:10.1006/prep.1998.1003

43. PaganoJM, FarleyBM, McCoigLM, RyderSP (2007) Molecular basis of RNA recognition by the embryonic polarity determinant MEX-5. J Biol Chem 282: 8883–8894 doi:10.1074/jbc.M700079200

44. ZukerM (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: 3406–3415.

45. PaganoJM, FarleyBM, EssienKI, RyderSP (2009) RNA recognition by the embryonic cell fate determinant and germline totipotency factor MEX-3. Proc Natl Acad Sci USA 106: 20252–20257 doi:10.1073/pnas.0907916106

46. ZearfossNR, RyderSP (2012) End-labeling oligonucleotides with chemical tags after synthesis. Methods Mol Biol 941: 181–193 doi:_10.1007/978-1-62703-113-4_14

47. MarquardtDW (1963) An algorithm for least-squares estimation of nonlinear parameters. Journal of the Society for Industrial & Applied Mathematics 11: 431–441.

48. LoveJD, MintonKW (1985) Screening of λ library for differentially expressed genes using in vitro transcripts. Analytical Biochemistry 150: 429–441 doi:10.1016/0003-2697(85)90532-9

49. MartinM (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17: 10–12.

50. AdamsMD (2000) The Genome Sequence of Drosophila melanogaster. Science 287: 2185–2195 doi:10.1126/science.287.5461.2185

51. FujitaPA, RheadB, ZweigAS, HinrichsAS, KarolchikD, et al. (2010) The UCSC Genome Browser database: update 2011. Nucleic Acids Res 39: D876–D882 doi:10.1093/nar/gkq963

52. LangmeadB, TrapnellC, PopM, SalzbergSL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25.1–R25.10.

53. NechaevS, FargoDC, Santos dosG, LiuL, GaoY, et al. (2010) Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327: 335–338 doi:10.1126/science.1181421

54. SchneiderTD, StephensRM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18: 6097–6100.

55. ColeC, BarberJD, BartonGJ (2008) The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36: W197–W201 doi:10.1093/nar/gkn238

56. CuffJA, BartonGJ (2000) Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40: 502–511 doi:;10.1002/1097-0134(20000815)40:3<502::AID-PROT170>3.0.CO;2-Q

57. HenikoffS, HenikoffJG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 89: 10915–10919.