Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation

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Mobile group II introns are bacterial retrotransposons that are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase, which together promote intron mobility to new DNA sites by a mechanism called retrohoming. Although found in bacteria, archaea and eukaryotic organelles, group II introns are absent from eukaryotic nuclear genomes, where host defenses impede their expression and lower intracellular Mg2+ concentrations limit their ribozyme activity. Here, we developed a mobile group II intron expression system that bypasses expression barriers and show that simply adding Mg2+ to culture medium enables group II intron retrohoming into plasmid and chromosomal target sites in human cells at appreciable frequencies. Genetic selections and deep sequencing identified intron RNA mutations that moderately enhance retrohoming in human cells, but not without added Mg2+. Thus, low Mg2+ concentrations in human cells are a natural barrier to efficient retrohoming that is not readily overcome by mutational variation and selection. Our results have implications for group II intron use for gene targeting in higher organisms and highlight the impact of different intracellular environments on intron evolution and gene expression mechanisms in bacteria and eukarya.

Vyšlo v časopise: Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation. PLoS Genet 11(8): e32767. doi:10.1371/journal.pgen.1005422
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1005422

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1. Lambowitz AM, Zimmerly S. Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb Perspect Biol. 2011;3: a003616. doi: 10.1101/cshperspect.a003616 20463000

2. Zimmerly S, Hausner G, Wu X. Phylogenetic relationships among group II intron ORFs. Nucleic Acids Res. 2001;29: 1238–1250. 11222775

3. Martin W, Koonin EV. Introns and the origin of nucleus-cytosol compartmentalization. Nature. 2006;440: 41–45. 16511485

4. Lambowitz AM, Belfort M (2015) Mobile bacterial group II introns at the crux of eukaryotic evolution. Microbiol. Spectrum, 3(1) MDNA3-0050-2014; and In: Craig NL, Gellert M, Lambowitz AM, Chandler M, Rice P, Sandmeyer S, editors (2015). Mobile DNA III. Washington DC: ASM Press.

5. Yang J, Zimmerly S, Perlman PS, Lambowitz AM. Efficient integration of an intron RNA into double-stranded DNA by reverse splicing. Nature. 1996;381: 332–335. 8692273

6. Guo H, Karberg M, Long M, Jones JP, Sullenger B, Lambowitz AM. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science. 2000;289: 452–457. 10903206

7. Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM. Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol. 2001;19: 1162–1167. 11731786

8. Perutka J, Wang W, Goerlitz D, Lambowitz AM. Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol. 2004;336: 421–439. 14757055

9. Enyeart PJ, Mohr G, Ellington AD, Lambowitz AM. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. Mob DNA. 2014;5: 2. doi: 10.1186/1759-8753-5-2 24410776

10. Mastroianni M, Watanabe K, White TB, Zhuang F, Vernon J, Matsuura M et al. Group II intron-based gene targeting reactions in eukaryotes. PLOS One. 2008;3: e3121. doi: 10.1371/journal.pone.0003121 18769669

11. Chalamcharla VR, Curcio MJ, Belfort M. Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry. Genes Dev. 2010;24: 827–836. doi: 10.1101/gad.1905010 20351053

12. Zerbato M, Holic N, Moniot-Frin S, Ingrao D, Galy A, Perea J. The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities. PLOS One. 2013;8: e58263. doi: 10.1371/journal.pone.0058263 23505475

13. Doolittle WF. The trouble with (group II) introns. Proc Natl Acad Sci U S A. 2014;111: 6536–6537. doi: 10.1073/pnas.1405174111 24757059

14. Peebles CL, Perlman PS, Mecklenburg KL, Petrillo ML, Tabor JH, Jarrell KA et al. A self-splicing RNA excises an intron lariat. Cell. 1986;44: 213–223. 3510741

15. Toor N, Keating KS, Taylor SD, Pyle AM. Crystal structure of a self-spliced group II intron. Science. 2008;320: 77–82. doi: 10.1126/science.1153803 18388288

16. Marcia M, Pyle AM. Visualizing group II intron catalysis through the stages of splicing. Cell. 2012;151: 497–507. doi: 10.1016/j.cell.2012.09.033 23101623

17. Robart AR, Chan RT, Peters JK, Rajashankar KR, Toor N. Crystal structure of a eukaryotic group II intron lariat. Nature. 2014;514: 193–197. doi: 10.1038/nature13790 25252982

18. Carignani G, Groudinsky O, Frezza D, Schiavon E, Bergantino E, Slonimski PP. An mRNA maturase is encoded by the first intron of the mitochondrial gene for the subunit I of cytochrome oxidase in S. cerevisiae. Cell. 1983;35: 733–742. 6317200

19. Matsuura M, Saldanha R, Ma H, Wank H, Yang J, Mohr G et al. A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron. Genes Dev. 1997;11: 2910–2924. 9353259

20. Matsuura M, Noah JW, Lambowitz AM. Mechanism of maturase-promoted group II intron splicing. EMBO J. 2001;20: 7259–7270. 11743002

21. Saldanha R, Chen B, Wank H, Matsuura M, Edwards J, Lambowitz AM. RNA and protein catalysis in group II intron splicing and mobility reactions using purified components. Biochemistry. 1999;38: 9069–9083. 10413481

22. Singh NN, Lambowitz AM. Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference. J Mol Biol. 2001;309: 361–386. 11371159

23. Zimmerly S, Guo H, Perlman PS, Lambowitz AM. Group II intron mobility occurs by target DNA-primed reverse transcription. Cell. 1995;82: 545–554. 7664334

24. Zimmerly S, Guo H, Eskes R, Yang J, Perlman PS, Lambowitz AM. A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell. 1995;83: 529–538. 7585955

25. Smith D, Zhong J, Matsuura M, Lambowitz AM, Belfort M. Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming. Genes Dev. 2005;19: 2477–2487. 16230535

26. Yao J, Truong DM, Lambowitz AM. Genetic and biochemical assays reveal a key role for replication restart proteins in group II intron retrohoming. PLOS Genet. 2013;9: e1003469. doi: 10.1371/journal.pgen.1003469 23637634

27. Sharp PA. “Five easy pieces”. Science. 1991;254: 663. 1948046

28. Keating KS, Toor N, Perlman PS, Pyle AM. A structural analysis of the group II intron active site and implications for the spliceosome. RNA. 2010;16: 1–9. doi: 10.1261/rna.1791310 19948765

29. Gordon PM, Sontheimer EJ, Piccirilli JA. Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns. RNA. 2000;6: 199–205. 10688359

30. Fica SM, Tuttle N, Novak T, Li NS, Lu J, Koodathingal P et al. RNA catalyses nuclear pre-mRNA splicing. Nature. 2013;503: 229–234. doi: 10.1038/nature12734 24196718

31. Fica SM, Mefford MA, Piccirilli JA, Staley JP. Evidence for a group II intron-like catalytic triplex in the spliceosome. Nat Struct Mol Biol. 2014;21: 464–471. doi: 10.1038/nsmb.2815 24747940

32. Galej WP, Oubridge C, Newman AJ, Nagai K. Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature. 2013;493: 638–643. doi: 10.1038/nature11843 23354046

33. Dlakic M, Mushegian A. Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase. RNA. 2011;17: 799–808. doi: 10.1261/rna.2396011 21441348

34. Cavalier-Smith T. Intron phylogeny: a new hypothesis. Trends Genet. 1991;7: 145–148. 2068786

35. Qu G, Dong X, Piazza CL, Chalamcharla VR, Lutz S, Curcio MJ et al. RNA-RNA interactions and pre-mRNA mislocalization as drivers of group II intron loss from nuclear genomes. Proc Natl Acad Sci U S A. 2014;111: 6612–6617. doi: 10.1073/pnas.1404276111 24722636

36. Truong DM, Sidote DJ, Russell R, Lambowitz AM. Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core. Proc Natl Acad Sci U S A. 2013;110: E3800–E3809. doi: 10.1073/pnas.1315742110 24043808

37. Gregan J, Kolisek M, Schweyen RJ. Mitochondrial Mg2+ homeostasis is critical for group II intron splicing in vivo. Genes Dev. 2001;15: 2229–2237. 11544180

38. Gunther T. Concentration, compartmentation and metabolic function of intracellular free Mg2+. Magnes Res. 2006;19: 225–236. 17402290

39. Costa M, Fontaine JM, Loiseaux-de Goer S, Michel F. A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation. J Mol Biol. 1997;274: 353–364. 9405145

40. Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996;6: 315–324. 8805248

41. Boshart M, Weber F, Jahn G, Dorsch-Hasler K, Fleckenstein B, Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985;41: 521–530. 2985280

42. Cui X. RNA/protein interactions during group II intron splicing and toward group II intron targeting in mammalian cells. Ph.D. Dissertation. The University of Texas at Austin. 2006. Available: http://repositories.lib.utexas.edu

43. Brisson M, He Y, Li S, Yang JP, Huang L. A novel T7 RNA polymerase autogene for efficient cytoplasmic expression of target genes. Gene Ther. 1999;6: 263–270. 10435111

44. Yarovoi SV, Pederson T. Human cell lines expressing hormone regulated T7 RNA polymerase localized at distinct intranuclear sites. Gene. 2001;275: 73–81. 11574154

45. Hanson JH. DNA target site recognition and toward gene targeting in mammalian cells by the Ll.LtrB group II intron RNP. Ph.D. Dissertation. The University of Texas at Austin. 2013. Available: http://repositories.lib.utexas.edu

46. Nisa-Martinez R, Laporte P, Jimenez-Zurdo JI, Frugier F, Crespi M, Toro N. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments. PLOS One. 2013;8: e84056. doi: 10.1371/journal.pone.0084056 24391881

47. San Filippo J, Lambowitz AM. Characterization of the C-terminal DNA-binding/DNA endonuclease region of a group II intron-encoded protein. J Mol Biol. 2002;324: 933–951. 12470950

48. Nicholson P, Yepiskoposyan H, Metze S, Zamudio Orozco R, Kleinschmidt N, Muhlemann O. Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. Cell Mol Life Sci. 2010;67: 677–700. doi: 10.1007/s00018-009-0177-1 19859661

49. Cui X, Matsuura M, Wang Q, Ma H, Lambowitz AM. A group II intron-encoded maturase functions preferentially in cis and requires both the reverse transcriptase and X domains to promote RNA splicing. J Mol Biol. 2004;340: 211–231. 15201048

50. Carapuca E, Azzoni AR, Prazeres DM, Monteiro GA, Mergulhao FJ. Time-course determination of plasmid content in eukaryotic and prokaryotic cells using real-time PCR. Mol Biotechnol. 2007;37: 120–126. 17914172

51. Lam AP, Dean DA. Progress and prospects: nuclear import of nonviral vectors. Gene Ther. 2010;17: 439–447. doi: 10.1038/gt.2010.31 20200566

52. Gilbert N, Lutz S, Morrish TA, Moran JV. Multiple fates of L1 retrotransposition intermediates in cultured human cells. Mol Cell Biol. 2005;25: 7780–7795. 16107723

53. Charras GT. A short history of blebbing. J Microsc. 2008;231: 466–478. doi: 10.1111/j.1365-2818.2008.02059.x 18755002

54. Truong DM. Mobile group II intron: host factors, directed evolution, and gene targeting in human cells. Ph.D. Dissertation. The University of Texas at Austin. 2014. Available: http://repositories.lib.utexas.edu

55. Travers KJ, Chin CS, Rank DR, Eid JS, Turner SW. A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Res. 2010;38: e159. doi: 10.1093/nar/gkq543 20571086

56. Lahr DJ, Katz LA. Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. Biotechniques. 2009;47: 857–866. doi: 10.2144/000113219 19852769

57. Shao W, Boltz VF, Spindler JE, Kearney MF, Maldarelli F, Mellors JW et al. Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of low-frequency drug resistance mutations in HIV-1 DNA. Retrovirology. 2013;10: 18. doi: 10.1186/1742-4690-10-18 23402264

58. Singh RN, Saldanha RJ, D’Souza LM, Lambowitz AM. Binding of a group II intron-encoded reverse transcriptase/maturase to its high affinity intron RNA binding site involves sequence-specific recognition and autoregulates translation. J Mol Biol. 2002;318: 287–303. 12051838

59. Watanabe K, Lambowitz AM. High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA. RNA. 2004;10: 1433–1443. 15273321

60. Mohr G, Smith D, Belfort M, Lambowitz AM. Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences. Genes Dev. 2000;14: 559–573. 10716944

61. Chapman KA, Burgess RR. Construction of bacteriophage T7 late promoters with point mutations and characterization by in vitro transcription properties. Nucleic Acids Res. 1987;15: 5413–5432. 3299271

62. Sandig V, Lieber A, Bahring S, Strauss M. A phage T7 class-III promoter functions as a polymerase II promoter in mammalian cells. Gene. 1993;131: 255–259. 8406019

63. Lieber A, Sandig V, Strauss M. A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells. Eur J Biochem. 1993;217: 387–394. 8223577

64. Ness JE, Kim S, Gottman A, Pak R, Krebber A, Borchert TV et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat Biotechnol. 2002;20: 1251–1255. 12426575

65. Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995;164: 49–53. 7590320

66. Popp MW, Maquat LE. Organizing principles of mammalian nonsense-mediated mRNA decay. Annu Rev Genet. 2013;47: 139–165. doi: 10.1146/annurev-genet-111212-133424 24274751

67. Rubin H. The logic of the Membrane, Magnesium, Mitosis (MMM) model for the regulation of animal cell proliferation. Arch Biochem Biophys. 2007;458: 16–23. 16750508

68. Cousineau B, Smith D, Lawrence-Cavanagh S, Mueller JE, Yang J, Mills D et al. Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination. Cell. 1998;94: 451–462. 9727488

69. Strick R, Strissel PL, Gavrilov K, Levi-Setti R. Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes. J Cell Biol. 2001;155: 899–910. 11739403

70. Kubo S, Seleme MC, Soifer HS, Perez JL, Moran JV, Kazazian HHJ et al. L1 retrotransposition in nondividing and primary human somatic cells. Proc Natl Acad Sci U S A. 2006;103: 8036–8041. 16698926

71. Kinsey JA. Transnuclear retrotransposition of the Tad element of Neurospora. Proc Natl Acad Sci U S A. 1993;90: 9384–9387. 8415711

72. Goodwin TJ, Ormandy JE, Poulter RT. L1-like non-LTR retrotransposons in the yeast Candida albicans. Curr Genet. 2001;39: 83–91. 11405100

73. Zhuang F, Mastroianni M, White TB, Lambowitz AM. Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation. Proc Natl Acad Sci U S A. 2009;106: 18189–18194. doi: 10.1073/pnas.0910277106 19833873

74. White TB, Lambowitz AM. The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and-independent mechanisms. PLOS Genet. 2012;8: e1002534. doi: 10.1371/journal.pgen.1002534 22359518

75. Suzuki J, Yamaguchi K, Kajikawa M, Ichiyanagi K, Adachi N, Koyama H et al. Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition. PLOS Genet. 2009;5: e1000461. doi: 10.1371/journal.pgen.1000461 19390601

76. Chapman KB, Boeke JD. Isolation and characterization of the gene encoding yeast debranching enzyme. Cell. 1991;65: 483–492. 1850323

77. Hayden EJ, Ferrada E, Wagner A. Cryptic genetic variation promotes rapid evolutionary adaptation in an RNA enzyme. Nature. 2011;474: 92–95. doi: 10.1038/nature10083 21637259

78. Hayden EJ, Wagner A. Environmental change exposes beneficial epistatic interactions in a catalytic RNA. Proc Biol Sci. 2012;279: 3418–3425. doi: 10.1098/rspb.2012.0956 22719036

79. Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013;31: 397–405. doi: 10.1016/j.tibtech.2013.04.004 23664777

80. Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods. 2013;10: 957–963. doi: 10.1038/nmeth.2649 24076990

81. Lusk JE, Williams RJ, Kennedy EP. Magnesium and the growth of Escherichia coli. J Biol Chem. 1968;243: 2618–2624. 4968384

82. Romani AM. Magnesium homeostasis in mammalian cells. Front Biosci. 2007;12: 308–331. 17127301

83. Johnson EE, Wessling-Resnick M. Iron metabolism and the innate immune response to infection. Microbes Infect. 2012;14: 207–216. doi: 10.1016/j.micinf.2011.10.001 22033148

84. Mishmar D, Ruiz-Pesini E, Brandon M, Wallace DC. Mitochondrial DNA-like sequences in the nucleus (NUMTs): insights into our African origins and the mechanism of foreign DNA integration. Hum Mutat. 2004;23: 125–133. 14722916

85. Sambrook J, Russell DW. Transformation of E. coli by electroporation. CSH Protoc. 2006;2006:

86. Wank H, SanFilippo J, Singh RN, Matsuura M, Lambowitz AM. A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA. Mol Cell. 1999;4: 239–250. 10488339

87. Choi VW, Asokan A, Haberman RA, Samulski RJ. Production of recombinant adeno-associated viral vectors for in vitro and in vivo use. Curr Protoc Mol Biol. 2007;Chapter 16: Unit 16.25.

88. Milne I, Bayer M, Cardle L, Shaw P, Stephen G, Wright F et al. Tablet—next generation sequence assembly visualization. Bioinformatics. 2010;26: 401–402. doi: 10.1093/bioinformatics/btp666 19965881