Availability of splicing factors in the nucleoplasm can regulate the release of mRNA from the gene after transcription

English version

Autoři: Hodaya Hochberg-Laufer ^aff001; Noa Neufeld ^aff001; Yehuda Brody ^aff001; Shani Nadav-Eliyahu ^aff001; Rakefet Ben-Yishay ^aff001; Yaron Shav-Tal ^aff001
Působiště autorů: The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel ^aff001
Vyšlo v časopise: Availability of splicing factors in the nucleoplasm can regulate the release of mRNA from the gene after transcription. PLoS Genet 15(11): e32767. doi:10.1371/journal.pgen.1008459
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008459

Souhrn

Gene expression dynamics can be measured in single living cells. Using a detectable transcriptionally active gene in living cells, we previously found that an mRNA undergoing several splicing events was retained at this gene after transcription until completion of mRNA processing. To determine the reason for this delay in release and whether mRNA retention on the gene might depend on splicing factor availability, we modulated the levels of splicing factors in the nucleus. Increasing the abundance of the diffusing fraction of splicing factors by their overexpression or by Clk1 kinase overexpression to disassemble nuclear speckles, led to a reduction in splicing factor residence times on the active gene, and the retained mRNA was rapidly released from the gene. Other treatments such as overexpression of a mutant inactive Clk1, the downregulation of MALAT1 lncRNA or of the Son protein, or the overexpression of the splicing factor import factor TNPO3, did not affect the dynamics of mRNA release from the gene. We found that the faster release of the mRNA from the gene mediated by increased availability of splicing factors, was dependent on the RS domain of the splicing factors and its phosphorylation state. We propose that the relative abundancies of splicing factors in the nucleoplasm can affect their availability for the splicing events taking place, and regulate the kinetics of mRNA release from the gene after processing.

Klíčová slova:

DNA transcription – Hyperexpression techniques – Introns – Messenger RNA – Phosphorylation – RNA splicing – Small interfering RNAs – Fluorescence recovery after photobleaching

Zdroje

1. Bentley DL. Coupling mRNA processing with transcription in time and space. Nat Rev Genet. 2014;15(3):163–75. doi: 10.1038/nrg3662 24514444

2. Kornblihtt AR, de la Mata M, Fededa JP, Munoz MJ, Nogues G. Multiple links between transcription and splicing. RNA. 2004;10(10):1489–98. doi: 10.1261/rna.7100104 15383674

3. Maniatis T, Reed R. An extensive network of coupling among gene expression machines. Nature. 2002;416(6880):499–506. doi: 10.1038/416499a 11932736

4. Muller-McNicoll M, Neugebauer KM. How cells get the message: dynamic assembly and function of mRNA-protein complexes. Nat Rev Genet. 2013;14(4):275–87. doi: 10.1038/nrg3434 23478349

5. Dreyfuss G, Kim VN, Kataoka N. Messenger-RNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol. 2002;3(3):195–205. doi: 10.1038/nrm760 11994740

6. Neugebauer KM. On the importance of being co-transcriptional. J Cell Sci. 2002;115(Pt 20):3865–71. doi: 10.1242/jcs.00073 12244124

7. Coulon A, Ferguson ML, de Turris V, Palangat M, Chow CC, Larson DR. Kinetic competition during the transcription cycle results in stochastic RNA processing. eLife. 2014;3.

8. Vargas DY, Shah K, Batish M, Levandoski M, Sinha S, Marras SA, et al. Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell. 2011;147(5):1054–65. doi: 10.1016/j.cell.2011.10.024 22118462

9. Girard C, Will CL, Peng J, Makarov EM, Kastner B, Lemm I, et al. Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion. Nature communications. 2012;3:994. doi: 10.1038/ncomms1998 22871813

10. Lamond AI, Spector DL. Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol. 2003;4(8):605–12. doi: 10.1038/nrm1172 12923522

11. Hall LL, Smith KP, Byron M, Lawrence JB. Molecular anatomy of a speckle. Anat Rec A Discov Mol Cell Evol Biol. 2006;288(7):664–75. doi: 10.1002/ar.a.20336 16761280

12. Galganski L, Urbanek MO, Krzyzosiak WJ. Nuclear speckles: molecular organization, biological function and role in disease. Nucleic Acids Res. 2017;45(18):10350–68. doi: 10.1093/nar/gkx759 28977640

13. Han J, Xiong J, Wang D, Fu XD. Pre-mRNA splicing: where and when in the nucleus. Trends Cell Biol. 2011;21(6):336–43. doi: 10.1016/j.tcb.2011.03.003 21514162

14. Chen Y, Belmont AS. Genome organization around nuclear speckles. Curr Opin Genet Dev. 2019;55:91–9. doi: 10.1016/j.gde.2019.06.008 31394307

15. Huang S, Spector DL. Will the real splicing sites please light up? Curr Biol. 1992;2(4):188–90. doi: 10.1016/0960-9822(92)90516-d 15335973

16. Chen Y, Zhang Y, Wang Y, Zhang L, Brinkman EK, Adam SA, et al. Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler. J Cell Biol. 2018; 217(11):4025–48. doi: 10.1083/jcb.201807108 30154186

17. Quinodoz SA, Ollikainen N, Tabak B, Palla A, Schmidt JM, Detmar E, et al. Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus. Cell. 2018;174(3):744–57. doi: 10.1016/j.cell.2018.05.024 29887377

18. Spector DL, Lamond AI. Nuclear speckles. Cold Spring Harb Perspect Biol. 2011;3(2).

19. Phair RD, Misteli T. High mobility of proteins in the mammalian cell nucleus. Nature. 2000;404(6778):604–9. doi: 10.1038/35007077 10766243

20. Kruhlak MJ, Lever MA, Fischle W, Verdin E, Bazett-Jones DP, Hendzel MJ. Reduced mobility of the alternate splicing factor (ASF) through the nucleoplasm and steady state speckle compartments. J Cell Biol. 2000;150(1):41–51. doi: 10.1083/jcb.150.1.41 10893255

21. Rino J, Carvalho T, Braga J, Desterro JM, Luhrmann R, Carmo-Fonseca M. A stochastic view of spliceosome assembly and recycling in the nucleus. PLoS Comput Biol. 2007;3(10):2019–31. doi: 10.1371/journal.pcbi.0030201 17967051

22. Huranova M, Ivani I, Benda A, Poser I, Brody Y, Hof M, et al. The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J Cell Biol. 2010;191(1):75–86. doi: 10.1083/jcb.201004030 20921136

23. Martin RM, Rino J, Carvalho C, Kirchhausen T, Carmo-Fonseca M. Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity. Cell reports. 2013;4(6):1144–55. doi: 10.1016/j.celrep.2013.08.013 24035393

24. Brody Y, Neufeld N, Bieberstein N, Causse SZ, Bohnlein EM, Neugebauer KM, et al. The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing. PLoS Biol. 2011;9(1):e1000573. doi: 10.1371/journal.pbio.1000573 21264352

25. Darzacq X, Singer RH, Shav-Tal Y. Dynamics of transcription and mRNA export. Curr Opin Cell Biol. 2005;17(3):332–9. doi: 10.1016/j.ceb.2005.04.004 15901505

26. Poser I, Sarov M, Hutchins JR, Heriche JK, Toyoda Y, Pozniakovsky A, et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods. 2008;5(5):409–15. doi: 10.1038/nmeth.1199 18391959

27. Sapra AK, Anko ML, Grishina I, Lorenz M, Pabis M, Poser I, et al. SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Mol Cell. 2009;34(2):179–90. doi: 10.1016/j.molcel.2009.02.031 19394295

28. Sacco-Bubulya P, Spector DL. Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing. J Cell Biol. 2002;156(3):425–36. doi: 10.1083/jcb.200107017 11827980

29. Colwill K, Pawson T, Andrews B, Prasad J, Manley JL, Bell JC, et al. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 1996;15(2):265–75. 8617202

30. Lu X, Ng HH, Bubulya PA. The role of SON in splicing, development, and disease. Wiley Interdiscip Rev RNA. 2014;5(5):637–46. doi: 10.1002/wrna.1235 24789761

31. Sharma A, Markey M, Torres-Munoz K, Varia S, Kadakia M, Bubulya A, et al. Son maintains accurate splicing for a subset of human pre-mRNAs. J Cell Sci. 2011;124(Pt 24):4286–98. doi: 10.1242/jcs.092239 22193954

32. Pawellek A, Ryder U, Tammsalu T, King LJ, Kreinin H, Ly T, et al. Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP. eLife. 2017;6.

33. Darzacq X, Shav-Tal Y, de Turris V, Brody Y, Shenoy SM, Phair RD, et al. In vivo dynamics of RNA polymerase II transcription. Nat Struct Mol Biol. 2007;14(9):796–806. doi: 10.1038/nsmb1280 17676063

34. Lai MC, Lin RI, Huang SY, Tsai CW, Tarn WY. A human importin-beta family protein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins. J Biol Chem. 2000;275(11):7950–7. doi: 10.1074/jbc.275.11.7950 10713112

35. Kataoka N, Bachorik JL, Dreyfuss G. Transportin-SR, a nuclear import receptor for SR proteins. J Cell Biol. 1999;145(6):1145–52. doi: 10.1083/jcb.145.6.1145 10366588

36. Duncan PI, Stojdl DF, Marius RM, Bell JC. In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. Mol Cell Biol. 1997;17(10):5996–6001. doi: 10.1128/mcb.17.10.5996 9315658

37. Ninomiya K, Kataoka N, Hagiwara M. Stress-responsive maturation of Clk1/4 pre-mRNAs promotes phosphorylation of SR splicing factor. J Cell Biol. 2011;195(1):27–40. doi: 10.1083/jcb.201107093 21949414

38. Li P, Carter G, Romero J, Gower KM, Watson J, Patel NA, et al. Clk/STY (cdc2-like kinase 1) and Akt regulate alternative splicing and adipogenesis in 3T3-L1 pre-adipocytes. PLoS One. 2013;8(1):e53268. doi: 10.1371/journal.pone.0053268 23308182

39. Anko ML, Muller-McNicoll M, Brandl H, Curk T, Gorup C, Henry I, et al. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes. Genome Biol. 2012;13(3):R17. doi: 10.1186/gb-2012-13-3-r17 22436691

40. Pandit S, Zhou Y, Shiue L, Coutinho-Mansfield G, Li H, Qiu J, et al. Genome-wide analysis reveals SR protein cooperation and competition in regulated splicing. Mol Cell. 2013;50(2):223–35. doi: 10.1016/j.molcel.2013.03.001 23562324

41. Dvinge H, Kim E, Abdel-Wahab O, Bradley RK. RNA splicing factors as oncoproteins and tumour suppressors. Nat Rev Cancer. 2016;16(7):413–30. doi: 10.1038/nrc.2016.51 27282250

42. Sveen A, Kilpinen S, Ruusulehto A, Lothe RA, Skotheim RI. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes. Oncogene. 2016;35(19):2413–27. doi: 10.1038/onc.2015.318 26300000

43. Karni R, de Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol. 2007;14(3):185–93. Epub 2007/02/21. doi: 10.1038/nsmb1209 17310252

44. Miyagawa R, Tano K, Mizuno R, Nakamura Y, Ijiri K, Rakwal R, et al. Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles. RNA. 2012;18(4):738–51. doi: 10.1261/rna.028639.111 22355166

45. Sharma A, Takata H, Shibahara K, Bubulya A, Bubulya PA. Son is essential for nuclear speckle organization and cell cycle progression. Mol Biol Cell. 2010;21(4):650–63. doi: 10.1091/mbc.E09-02-0126 20053686

46. Fei J, Jadaliha M, Harmon TS, Li ITS, Hua B, Hao Q, et al. Quantitative analysis of multilayer organization of proteins and RNA in nuclear speckles at super resolution. J Cell Sci. 2017;130(24):4180–92. doi: 10.1242/jcs.206854 29133588

47. Zhou Z, Fu XD. Regulation of splicing by SR proteins and SR protein-specific kinases. Chromosoma. 2013;122(3):191–207. doi: 10.1007/s00412-013-0407-z 23525660

48. Cho S, Hoang A, Sinha R, Zhong XY, Fu XD, Krainer AR, et al. Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly. Proc Natl Acad Sci U S A. 2011;108(20):8233–8. doi: 10.1073/pnas.1017700108 21536904

49. Hochberg-Laufer H, Schwed-Gross A, Neugebauer KM, Shav-Tal Y. Uncoupling of nucleo-cytoplasmic RNA export and localization during stress. Nucleic Acids Res. 2019;47(9):4778–97. doi: 10.1093/nar/gkz168 30864659

50. Ajiro M, Jia R, Yang Y, Zhu J, Zheng ZM. A genome landscape of SRSF3-regulated splicing events and gene expression in human osteosarcoma U2OS cells. Nucleic Acids Res. 2016;44(4):1854–70. doi: 10.1093/nar/gkv1500 26704980

51. Jumaa H, Nielsen PJ. The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation. EMBO J. 1997;16(16):5077–85. doi: 10.1093/emboj/16.16.5077 9305649

52. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 24157548

53. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008;5(10):877–9. doi: 10.1038/nmeth.1253 18806792

54. Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science. 2003;300(5628):2061–5. doi: 10.1126/science.1084398 12791999

55. Neufeld N, Brody Y, Shav-Tal Y. Quantifying the ratio of spliceosome components assembled on pre-mRNA. Methods Mol Biol. 2014;1126:257–69. doi: 10.1007/978-1-62703-980-2_19 24549670

56. Shav-Tal Y, Blechman J, Darzacq X, Montagna C, Dye BT, Patton JG, et al. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol Biol Cell. 2005;16(5):2395–413. doi: 10.1091/mbc.E04-11-0992 15758027

57. Brody Y, Shav-Tal Y. Measuring the kinetics of mRNA transcription in single living cells. J Vis Exp. 2011;(54):e2898. doi: 10.3791/2898 21904295

58. Boireau S, Maiuri P, Basyuk E, de la Mata M, Knezevich A, Pradet-Balade B, et al. The transcriptional cycle of HIV-1 in real-time and live cells. J Cell Biol. 2007;179(2):291–304. doi: 10.1083/jcb.200706018 17954611