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The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay


Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.


Vyšlo v časopise: The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay. PLoS Genet 9(10): e32767. doi:10.1371/journal.pgen.1003893
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003893

Souhrn

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.


Zdroje

1. DomaMK, ParkerR (2007) RNA quality control in eukaryotes. Cell 131: 660–668 doi:10.1016/j.cell.2007.10.041

2. SchmidM, JensenTH (2008) Quality control of mRNP in the nucleus. Chromosoma 117: 419–429 doi:10.1007/s00412-008-0166-4

3. FaskenMB, CorbettAH (2009) Mechanisms of nuclear mRNA quality control. RNA Biol 6: 237–241.

4. SchmidtM-J, NorburyCJ (2010) Polyadenylation and beyond: emerging roles for noncanonical poly(A) polymerases. Wiley Interdiscip Rev RNA 1: 142–151 doi:10.1002/wrna.16

5. SlomovicS, SchusterG (2011) Exonucleases and endonucleases involved in polyadenylation- assisted RNA decay. Wiley Interdiscip Rev RNA 2: 106–123 doi:10.1002/wrna.45

6. LacavaJ, HouseleyJ, SaveanuC, PetfalskiE, ThompsonE, et al. (2005) RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121: 713–724 doi:10.1016/j.cell.2005.04.029

7. WyersF, RougemailleM, BadisG, RousselleJ-C, DufourM-E, et al. (2005) Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121: 725–737 doi:10.1016/j.cell.2005.04.030

8. SchmidM, JensenTH (2008) The exosome: a multipurpose RNA-decay machine. Trends Biochem Sci 33: 501–510 doi:10.1016/j.tibs.2008.07.003

9. HillerenP, McCarthyT, RosbashM, ParkerR, JensenTH (2001) Quality control of mRNA 3′-end processing is linked to the nuclear exosome. Nature 413: 538–542 doi:10.1038/35097110

10. RougemailleM, GudipatiRK, OlesenJR, ThomsenR, SeraphinB, et al. (2007) Dissecting mechanisms of nuclear mRNA surveillance in THO/sub2 complex mutants. EMBO J 26: 2317–2326 doi:10.1038/sj.emboj.7601669

11. GrzechnikP, KufelJ (2008) Polyadenylation linked to transcription termination directs the processing of snoRNA precursors in yeast. Mol Cell 32: 247–258 doi:10.1016/j.molcel.2008.10.003

12. SaguezC, SchmidM, OlesenJR, GhazyMAE-H, QuX, et al. (2008) Nuclear mRNA surveillance in THO/sub2 mutants is triggered by inefficient polyadenylation. Mol Cell 31: 91–103 doi:10.1016/j.molcel.2008.04.030

13. QuX, Lykke-AndersenS, NasserT, SaguezC, BertrandE, et al. (2009) Assembly of an export-competent mRNP is needed for efficient release of the 3′-end processing complex after polyadenylation. Mol Cell Biol 29: 5327–5338 doi:10.1128/MCB.00468-09

14. SchmidM, PoulsenMB, OlszewskiP, PelechanoV, SaguezC, et al. (2012) Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins. Mol Cell 47: 267–280 doi:10.1016/j.molcel.2012.05.005

15. ShcherbikN, WangM, LapikYR, SrivastavaL, PestovDG (2010) Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells. EMBO Rep doi:10.1038/embor.2009.271

16. SlomovicS, LauferD, GeigerD, SchusterG (2006) Polyadenylation of ribosomal RNA in human cells. Nucleic Acids Res 34: 2966–2975 doi:10.1093/nar/gkl357

17. LubasM, ChristensenMS, KristiansenMS, DomanskiM, FalkenbyLG, et al. (2011) Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell 43: 624–637 doi:10.1016/j.molcel.2011.06.028

18. LeeYJ, GlaunsingerBA (2009) Aberrant herpesvirus-induced polyadenylation correlates with cellular messenger RNA destruction. PLoS Biol 7: e1000107 doi:10.1371/journal.pbio.1000107

19. JensenTH, PatricioK, McCarthyT, RosbashM (2001) A block to mRNA nuclear export in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription. Mol Cell 7: 887–898.

20. HillerenP, ParkerR (2001) Defects in the mRNA export factors Rat7p, Gle1p, Mex67p, and Rat8p cause hyperadenylation during 3′-end formation of nascent transcripts. RNA 7: 753–764.

21. WestS, GromakN, NorburyCJ, ProudfootNJ (2006) Adenylation and exosome-mediated degradation of cotranscriptionally cleaved pre-messenger RNA in human cells. Mol Cell 21: 437–443 doi:10.1016/j.molcel.2005.12.008

22. ConradNK, SteitzJA (2005) A Kaposi's sarcoma virus RNA element that increases the nuclear abundance of intronless transcripts. EMBO J 24: 1831–1841 doi:10.1038/sj.emboj.7600662

23. ConradNK, ShuM-D, UyhaziKE, SteitzJA (2007) Mutational analysis of a viral RNA element that counteracts rapid RNA decay by interaction with the polyadenylate tail. Proc Natl Acad Sci USA 104: 10412–10417 doi:10.1073/pnas.0704187104

24. ConradNK, MiliS, MarshallEL, ShuM-D, SteitzJA (2006) Identification of a rapid mammalian deadenylation-dependent decay pathway and its inhibition by a viral RNA element. Mol Cell 24: 943–953 doi:10.1016/j.molcel.2006.10.029

25. Mitton-FryRM, DeGregorioSJ, WangJ, SteitzTA, SteitzJA (2010) Poly(A) tail recognition by a viral RNA element through assembly of a triple helix. Science 330: 1244–1247 doi:10.1126/science.1195858

26. ChengH, DufuK, LeeC-S, HsuJL, DiasA, et al. (2006) Human mRNA export machinery recruited to the 5′ end of mRNA. Cell 127: 1389–1400 doi:10.1016/j.cell.2006.10.044

27. ValenciaP, DiasAP, ReedR (2008) Splicing promotes rapid and efficient mRNA export in mammalian cells. Proc Natl Acad Sci USA 105: 3386–3391 doi:10.1073/pnas.0800250105

28. EckmannCR, RammeltC, WahleE (2011) Control of poly(A) tail length. Wiley Interdiscip Rev RNA 2: 348–361 doi:10.1002/wrna.56

29. de KlerkE, VenemaA, AnvarSY, GoemanJJ, HuO, et al. (2012) Poly(A) binding protein nuclear 1 levels affect alternative polyadenylation. Nucleic Acids Res doi:10.1093/nar/gks655

30. JenalM, ElkonR, Loayza-PuchF, van HaaftenG, KühnU, et al. (2012) The poly(a)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites. Cell 149: 538–553 doi:10.1016/j.cell.2012.03.022

31. LemieuxC, MargueratS, LafontaineJ, BarbezierN, BählerJ, et al. (2011) A Pre-mRNA degradation pathway that selectively targets intron-containing genes requires the nuclear poly(A)-binding protein. Mol Cell 44: 108–119 doi:10.1016/j.molcel.2011.06.035

32. YamanakaS, YamashitaA, HarigayaY, IwataR, YamamotoM (2010) Importance of polyadenylation in the selective elimination of meiotic mRNAs in growing S. pombe cells. EMBO J 29: 2173–2181 doi:10.1038/emboj.2010.108

33. LemayJ-F, D'AmoursA, LemieuxC, LacknerDH, St-SauveurVG, et al. (2010) The nuclear poly(A)-binding protein interacts with the exosome to promote synthesis of noncoding small nucleolar RNAs. Mol Cell 37: 34–45 doi:10.1016/j.molcel.2009.12.019

34. ChenH-M, FutcherB, LeatherwoodJ (2011) The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover. PLoS ONE 6: e26804 doi:10.1371/journal.pone.0026804

35. PerreaultA, LemieuxC, BachandF (2007) Regulation of the nuclear poly(A)-binding protein by arginine methylation in fission yeast. J Biol Chem 282: 7552–7562 doi:10.1074/jbc.M610512200

36. HectorRE, NykampKR, DheurS, AndersonJT, NonPJ, et al. (2002) Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export. EMBO J 21: 1800–1810 doi:10.1093/emboj/21.7.1800

37. BenoitB, MitouG, ChartierA, TemmeC, ZaessingerS, et al. (2005) An Essential Cytoplasmic Function for the Nuclear Poly(A) Binding Protein, PABP2, in Poly(A) Tail Length Control and Early Development in Drosophila. Dev Cell 9: 511–522 doi:10.1016/j.devcel.2005.09.002

38. ApponiLH, LeungSW, WilliamsKR, ValentiniSR, CorbettAH, et al. (2010) Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis. Hum Mol Genet 19: 1058–1065 doi:10.1093/hmg/ddp569

39. SahinBB, PatelD, ConradNK (2010) Kaposi's sarcoma-associated herpesvirus ORF57 protein binds and protects a nuclear noncoding RNA from cellular RNA decay pathways. PLoS Pathog 6: e1000799 doi:10.1371/journal.ppat.1000799

40. LoflinPT, ChenCY, XuN, ShyuAB (1999) Transcriptional pulsing approaches for analysis of mRNA turnover in mammalian cells. Methods 17: 11–20 doi:10.1006/meth.1998.0702

41. LeiH, DiasAP, ReedR (2011) Export and stability of naturally intronless mRNAs require specific coding region sequences and the TREX mRNA export complex. Proc Natl Acad Sci USA 108 ((44)): 17985–90 doi:10.1073/pnas.1113076108

42. ConradNK, SteitzJA (2005) A Kaposi's sarcoma virus RNA element that increases the nuclear abundance of intronless transcripts. EMBO J 24: 1831–1841 doi:10.1038/sj.emboj.7600662

43. KyriakopoulouCB, NordvargH, VirtanenA (2001) A novel nuclear human poly(A) polymerase (PAP), PAP gamma. J Biol Chem 276: 33504–33511 doi:10.1074/jbc.M104599200

44. TopalianSL, KanekoS, GonzalesMI, BondGL, WardY, et al. (2001) Identification and functional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors. Mol Cell Biol 21: 5614–5623 doi:10.1128/MCB.21.16.5614-5623.2001

45. KerwitzY, KühnU, LilieH, KnothA, ScheuermannT, et al. (2003) Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA. EMBO J 22: 3705–3714 doi:10.1093/emboj/cdg347

46. KuhnU, GundelM, KnothA, KerwitzY, RudelS, et al. (2009) Poly(A) Tail Length Is Controlled by the Nuclear Poly(A)-binding Protein Regulating the Interaction between Poly(A) Polymerase and the Cleavage and Polyadenylation Specificity Factor. Journal of Biological Chemistry 284: 22803–22814 doi:10.1074/jbc.M109.018226

47. KühnU, NemethA, MeyerS, WahleE (2003) The RNA binding domains of the nuclear poly(A)-binding protein. J Biol Chem 278: 16916–16925 doi:10.1074/jbc.M209886200

48. HolbeinS, WengiA, DecourtyL, FreimoserFM, JacquierA, et al. (2009) Cordycepin interferes with 3′ end formation in yeast independently of its potential to terminate RNA chain elongation. RNA 15: 837–849 doi:10.1261/rna.1458909

49. PrekerP, NielsenJ, KammlerS, Lykke-AndersenS, ChristensenMS, et al. (2008) RNA exosome depletion reveals transcription upstream of active human promoters. Science 322: 1851–1854 doi:10.1126/science.1164096

50. TomeckiR, DziembowskiA (2010) Novel endoribonucleases as central players in various pathways of eukaryotic RNA metabolism. RNA 16 ((9)): 1692–724 doi:10.1261/rna.2237610

51. KissDL, AndrulisED (2010) The exozyme model: A continuum of functionally distinct complexes. RNA 17 ((1)): 1–13 doi:10.1261/rna.2364811

52. GrammelM, HangH, ConradNK (2012) Chemical reporters for monitoring RNA synthesis and poly(A) tail dynamics. ChemBioChem 13: 1112–1115 doi:10.1002/cbic.201200091

53. JaoCY, SalicA (2008) Exploring RNA transcription and turnover in vivo by using click chemistry. Proc Natl Acad Sci USA 105: 15779–15784 doi:10.1073/pnas.0808480105

54. WiluszJE, FreierSM, SpectorDL (2008) 3′ end processing of a long nuclear-retained noncoding RNA yields a tRNA-like cytoplasmic RNA. Cell 135: 919–932 doi:10.1016/j.cell.2008.10.012

55. WiluszJE, JnBaptisteCK, LuLY, KuhnCD, Joshua-TorL, et al. (2012) A triple helix stabilizes the 3′ ends of long noncoding RNAs that lack poly(A) tails. Genes Dev 26 ((21)): 2392–407 doi:10.1101/gad.204438.112

56. BrownJA, ValensteinML, YarioTA, TycowskiKT, SteitzJA (2012) Formation of triple-helical structures by the 3′-end sequences of MALAT1 and MENβ noncoding RNAs. Proceedings of the National Academy of Sciences 109 ((47)): 19202–7 doi:10.1073/pnas.1217338109

57. BeaulieuYB, KleinmanCL, Landry-VoyerA-M, MajewskiJ, BachandF (2012) Polyadenylation-Dependent Control of Long Noncoding RNA Expression by the Poly(A)-Binding Protein Nuclear 1. PLoS Genet 8: e1003078 doi:10.1371/journal.pgen.1003078.g007

58. WasmuthEV, LimaCD (2012) Exo- and endoribonucleolytic activities of yeast cytoplasmic and nuclear RNA exosomes are dependent on the noncatalytic core and central channel. Mol Cell 48: 133–144 doi:10.1016/j.molcel.2012.07.012

59. MakinoDL, BaumgärtnerM, ContiE (2013) Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex. Nature 495: 1–7 doi:10.1038/nature11870

60. BonneauF, BasquinJ, EbertJ, LorentzenE, ContiE (2009) The yeast exosome functions as a macromolecular cage to channel RNA substrates for degradation. Cell 139: 547–559 doi:10.1016/j.cell.2009.08.042

61. SagawaF, IbrahimH, MorrisonAL, WiluszCJ, WiluszJ (2011) Nucleophosmin deposition during mRNA 3′ end processing influences poly(A) tail length. EMBO J 30: 3994–4005 doi:10.1038/emboj.2011.272

62. PalaniswamyV, MoraesKCM, WiluszCJ, WiluszJ (2006) Nucleophosmin is selectively deposited on mRNA during polyadenylation. Nat Struct Mol Biol 13: 429–435 doi:10.1038/nsmb1080

63. RothKM, ByamJ, FangF, ButlerJS (2009) Regulation of NAB2 mRNA 3′-end formation requires the core exosome and the Trf4p component of the TRAMP complex. RNA 15: 1045–1058 doi:10.1261/rna.709609

64. RothKM, WolfMK, RossiM, ButlerJS (2005) The nuclear exosome contributes to autogenous control of NAB2 mRNA levels. Mol Cell Biol 25: 1577–1585 doi:10.1128/MCB.25.5.1577-1585.2005

65. WinstallE, SadowskiM, KühnU, WahleE, SachsAB (2000) The Saccharomyces cerevisiae RNA-binding protein Rbp29 functions in cytoplasmic mRNA metabolism. J Biol Chem 275: 21817–21826 doi:10.1074/jbc.M002412200

66. DheurS, NykampKR, ViphakoneN, SwansonMS, Minvielle-SebastiaL (2005) Yeast mRNA Poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent Poly(A) nuclease activity. J Biol Chem 280: 24532–24538 doi:10.1074/jbc.M504720200

67. ApponiLH, KellySM, HarremanMT, LehnerAN, CorbettAH, et al. (2007) An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA processing/export and mRNA stability. Mol Cell Biol 27: 6569–6579 doi:10.1128/MCB.00881-07

68. PakC, GarshasbiM, KahriziK, GrossC, ApponiLH, et al. (2011) Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans. Proceedings of the National Academy of Sciences 108: 12390–12395 doi:10.1073/pnas.1107103108

69. KellySM, PabitSA, KitchenCM, GuoP, MarfatiaKA, et al. (2007) Recognition of polyadenosine RNA by zinc finger proteins. Proc Natl Acad Sci USA 104: 12306–12311 doi:10.1073/pnas.0701244104

70. St-AndréO, LemieuxC, PerreaultA, LacknerDH, BählerJ, et al. (2010) Negative regulation of meiotic gene expression by the nuclear poly(a)-binding protein in fission yeast. Journal of Biological Chemistry 285: 27859–27868 doi:10.1074/jbc.M110.150748

71. JugeF, ZaessingerS, TemmeC, WahleE, SimoneligM (2002) Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila. EMBO J 21: 6603–6613.

72. BenoitB, NemethA, AulnerN, KühnU, SimoneligM, et al. (1999) The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro. Nucleic Acids Res 27: 3771–3778.

73. StubbsSH, HunterOV, HooverA, ConradNK (2012) Viral factors reveal a role for REF/Aly in nuclear RNA stability. Mol Cell Biol 32: 1260–1270 doi:10.1128/MCB.06420-11

74. HautbergueGM, HungM-L, WalshMJ, SnijdersAPL, ChangC-T, et al. (2009) UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA. Curr Biol 19: 1918–1924 doi:10.1016/j.cub.2009.09.041

75. ZhaoC, HamiltonT (2007) Introns regulate the rate of unstable mRNA decay. J Biol Chem 282: 20230–20237 doi:10.1074/jbc.M700180200

76. WahleE (1995) Poly(A) tail length control is caused by termination of processive synthesis. J Biol Chem 270: 2800–2808.

77. BhattacharjeeRB, BagJ (2012) Depletion of Nuclear Poly(A) Binding Protein PABPN1 Produces a Compensatory Response by Cytoplasmic PABP4 and PABP5 in Cultured Human Cells. PLoS ONE 7: e53036 doi:10.1371/journal.pone.0053036.g006

78. HosodaN, LejeuneF, MaquatLE (2006) Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails. Mol Cell Biol 26: 3085–3097 doi:10.1128/MCB.26.8.3085-3097.2006

79. ChurchGM, GilbertW (1984) Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995.

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