A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in

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Survival and adaptation of trypanosomatids to new surroundings requires activation of specific gene networks. This is mainly achieved by post-transcriptional mechanisms, and proteins that bind to specific mRNAs, and influence degradation or translation, are known to be important. However, only few such proteins have been characterized to date. The trypanosome genome encodes over 150 proteins with conserved RNA-binding domains, and it is very likely that additional proteins that do not have such domains could also modulate mRNA fate. Here, we report the results of a genome-wide screen to identify mRNA-fate regulators in Trypanosoma brucei. We used a method called “tethering” to artificially attach protein fragments to an mRNA. Our findings confirmed the role of RNA-binding proteins in the regulation of mRNA fate, and also suggested such roles for many other proteins, including some metabolic enzymes. Our results should serve as a useful resource. Moreover, the tethering screen approach could readily be adapted for use in other organisms.

Vyšlo v časopise: A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in. PLoS Pathog 10(6): e32767. doi:10.1371/journal.ppat.1004178
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004178

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Zdroje

1. PreusserC, JaeN, BindereifA (2012) mRNA splicing in trypanosomes. Int J Med Microbiol 302: 221–224.

2. ClaytonCE (2002) Life without transcriptional control? From fly to man and back again. EMBO J 21: 1881–1888.

3. Muller-McNicollM, NeugebauerKM (2013) How cells get the message: dynamic assembly and function of mRNA-protein complexes. Nat Rev Genet 14: 275–287.

4. CastelloA, FischerB, EichelbaumK, HorosR, BeckmannBM, et al. (2012) Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149: 1393–1406.

5. BaltzAG, MunschauerM, SchwanhausserB, VasileA, MurakawaY, et al. (2012) The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46: 674–690.

6. MitchellSF, JainS, SheM, ParkerR (2013) Global analysis of yeast mRNPs. Nat Struct Mol Biol 20: 127–133.

7. KwonSC, YiH, EichelbaumK, FohrS, FischerB, et al. (2013) The RNA-binding protein repertoire of embryonic stem cells. Nat Struct Mol Biol 20: 1122–1130.

8. KolevNG, UlluE, TschudiC (2014) The emerging role of RNA-binding proteins in the life cycle of Trypanosoma brucei. Cell Microbiol 16: 482–489.

9. WalradPB, CapewellP, FennK, MatthewsKR (2012) The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei. Nucleic Acids Res 40: 2869–2883.

10. DrollD, MiniaI, FaddaA, SinghA, StewartM, et al. (2013) Post-transcriptional regulation of the trypanosome heat shock response by a zinc finger protein. PLoS Pathog 9: e1003286.

11. LingAS, TrotterJR, HendriksEF (2011) A zinc finger protein, TbZC3H20, stabilizes two developmentally regulated mRNAs in trypanosomes. J Biol Chem 286: 20152–20162.

12. ArcherSK, LuuVD, de QueirozRA, BremsS, ClaytonC (2009) Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle. PLoS Pathog 5: e1000565.

13. ManiJ, GuttingerA, SchimanskiB, HellerM, Acosta-SerranoA, et al. (2011) Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery. PLoS One 6: e22463.

14. KishoreS, LuberS, ZavolanM (2010) Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression. Brief Funct Genomics 9: 391–404.

15. D'OrsoI, FraschAC (2002) TcUBP-1, an mRNA destabilizing factor from trypanosomes, homodimerizes and interacts with novel AU-rich element- and Poly(A)-binding proteins forming a ribonucleoprotein complex. J Biol Chem 277: 50520–50528.

16. DallagiovannaB, CorreaA, ProbstCM, HoletzF, SmircichP, et al. (2008) Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi. J Biol Chem 283: 8266–8273.

17. JhaBA, ArcherSK, ClaytonCE (2013) The trypanosome Pumilio domain protein PUF5. PLoS One 8: e77371.

18. WurstM, SeligerB, JhaBA, KleinC, QueirozR, et al. (2012) Expression of the RNA recognition motif protein RBP10 promotes a bloodstream-form transcript pattern in Trypanosoma brucei. Mol Microbiol 83: 1048–1063.

19. SinghA, MiniaI, DrollD, FaddaA, ClaytonC, et al. (2014) Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks. Nucleic Acids Res 42: 4652–4668.

20. CollerJM, GrayNK, WickensMP (1998) mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation. Genes Dev 12: 3226–3235.

21. CollerJ, WickensM (2007) Tethered function assays: an adaptable approach to study RNA regulatory proteins. Methods Enzymol 429: 299–321.

22. De GregorioE, PreissT, HentzeMW (1999) Translation driven by an eIF4G core domain in vivo. EMBO J 18: 4865–4874.

23. Lykke-AndersenJ, ShuMD, SteitzJA (2001) Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science 293: 1836–1839.

24. BertrandE, ChartrandP, SchaeferM, ShenoySM, SingerRH, et al. (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2: 437–445.

25. Baron-BenhamouJ, GehringNH, KulozikAE, HentzeMW (2004) Using the lambdaN peptide to tether proteins to RNAs. Methods Mol Biol 257: 135–154.

26. ScharpfM, StichtH, SchweimerK, BoehmM, HoffmannS, et al. (2000) Antitermination in bacteriophage lambda. The structure of the N36 peptide-boxB RNA complex. Eur J Biochem 267: 2397–2408.

27. GloverL, HornD (2009) Site-specific DNA double-strand breaks greatly increase stable transformation efficiency in Trypanosoma brucei. Mol Biochem Parasitol 166: 194–197.

28. DelhiP, QueirozR, InchausteguiD, CarringtonM, ClaytonC (2011) Is there a classical nonsense-mediated decay pathway in trypanosomes? PLoS One 6: e25112.

29. FarberV, ErbenE, SharmaS, StoecklinG, ClaytonC (2013) Trypanosome CNOT10 is essential for the integrity of the NOT deadenylase complex and for degradation of many mRNAs. Nucleic Acids Res 41: 1211–1222.

30. BlattnerJ, HelfertS, MichelsP, ClaytonC (1998) Compartmentation of phosphoglycerate kinase in Trypanosoma brucei plays a critical role in parasite energy metabolism. Proc Natl Acad Sci USA 95: 11596–11600.

31. AlsfordS, TurnerDJ, ObadoSO, Sanchez-FloresA, GloverL, et al. (2011) High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 21: 915–924.

32. SiegelTN, HekstraDR, WangX, DewellS, CrossGA (2010) Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites. Nucleic Acids Res 38: 4946–4957.

33. KimS, CoulombePA (2010) Emerging role for the cytoskeleton as an organizer and regulator of translation. Nat Rev Mol Cell Biol 11: 75–81.

34. EstevezAM (2008) The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes. Nucl Acids Res 36: 4573–4586.

35. SternMZ, GuptaSK, Salmon-DivonM, HahamT, BardaO, et al. (2009) Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism. RNA 15: 648–665.

36. DasA, MoralesR, BandayM, GarciaS, HaoL, et al. (2012) The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism. RNA 18: 1968–1983.

37. FreireER, DhaliaR, MouraDM, da Costa LimaTD, LimaRP, et al. (2011) The four trypanosomatid eIF4E homologues fall into two separate groups, with distinct features in primary sequence and biological properties. Mol Biochem Parasitol 176: 25–36.

38. ErbenE, ChakrabortyC, ClaytonC (2014) The CAF1-NOT complex of trypanosomes. Front Genet 4: 299.

39. LiCH, IrmerH, Gudjonsdottir-PlanckD, FreeseS, SalmH, et al. (2006) Roles of a Trypanosoma brucei 5′-3′ exoribonuclease homolog in mRNA degradation. RNA 12: 2171–2186.

40. BraunJE, TruffaultV, BolandA, HuntzingerE, ChangCT, et al. (2012) A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5′ exonucleolytic degradation. Nat Struct Mol Biol 19: 1324–1331.

41. NajafabadiHS, LuZ, MacPhersonC, MehtaV, AdoueV, et al. (2013) Global identification of conserved post-transcriptional regulatory programs in trypanosomatids. Nucleic Acids Res 41: 8591–8600.

42. QuenaultT, LithgowT, TravenA (2011) PUF proteins: repression, activation and mRNA localization. Trends Cell Biol 21: 104–112.

43. ZinovievA, LegerM, WagnerG, ShapiraM (2011) A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways. Nucleic Acids Res 39: 8404–8415.

44. HentzeMW, PreissT (2010) The REM phase of gene regulation. Trends Biochem Sci 35: 423–426.

45. CieslaJ (2006) Metabolic enzymes that bind RNA: yet another level of cellular regulatory network? Acta Biochim Pol 53: 11–32.

46. HuY, RolfsA, BhullarB, MurthyTV, ZhuC, et al. (2007) Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae. Genome Res 17: 536–543.

47. YangX, BoehmJS, Salehi-AshtianiK, HaoT, ShenY, et al. (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods 8: 659–661.

48. TsvetanovaNG, KlassDM, SalzmanJ, BrownPO (2010) Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PLoS One 5: e12671.

49. SiprashviliZ, WebsterD, KretzM, JohnstonD, RinnJ, et al. (2012) Identification of proteins binding coding and non-coding human RNAs using protein microarrays. BMC Genomics 13: 633.

50. MonyBM, MacGregorP, IvensA, RojasF, CowtonA, et al. (2014) Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei. Nature 505: 681–685.

51. TaiN, SchmitzJC, LiuJ, LinX, BaillyM, et al. (2004) Translational autoregulation of thymidylate synthase and dihydrofolate reductase. Front Biosci 9: 2521–2526.

52. BiebingerS, WirtzLE, LorenzP, ClaytonC (1997) Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei. Mol Biochem Parasitol 85: 99–112.

53. WirtzE, HartmannC, ClaytonC (1994) Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes. Nucleic Acids Res 22: 3887–3894.

54. Schumann BurkardG, JutziP, RoditiI (2011) Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Mol Biochem Parasitol 175: 91–94.

55. FaddaA, FarberV, DrollD, ClaytonC (2013) The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation. RNA 19: 937–947.

56. LiH, HandsakerB, WysokerA, FennellT, RuanJ, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079.

57. AngenendtP, KreutzbergerJ, GloklerJ, HoheiselJD (2006) Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR products. Mol Cell Proteomics 5: 1658–1666.

58. HendriksEF, RobinsonDR, HinkinsM, MatthewsKR (2001) A novel CCCH protein which modulates differentiation of Trypanosoma brucei to its procyclic form. EMBO J 20: 6700–6711.

59. PaterouA, WalradP, CraddyP, FennK, MatthewsK (2006) Identification and stage-specific association with the translational apparatus of TbZFP3, a CCCH protein that promotes trypanosome life-cycle development. J Biol Chem 281: 39002–39013.

60. MittraB, RayDS (2004) Presence of a poly(A) binding protein and two proteins with cell cycle-dependent phosphorylation in Crithidia fasciculata mRNA cycling sequence binding protein II. Eukaryot Cell 3: 1185–1197.