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. 2001 Jun;7(6):793–805. doi: 10.1017/s1355838201010524

Functional selection of splicing enhancers that stimulate trans-splicing in vitro.

L A Boukis 1, J P Bruzik 1
PMCID: PMC1370131  PMID: 11421358

Abstract

The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element (G/C)GAC(G/C) and also 5' splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5' end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5' splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction.

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Selected References

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  1. Barabino S. M., Blencowe B. J., Ryder U., Sproat B. S., Lamond A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990 Oct 19;63(2):293–302. doi: 10.1016/0092-8674(90)90162-8. [DOI] [PubMed] [Google Scholar]
  2. Berget S. M. Exon recognition in vertebrate splicing. J Biol Chem. 1995 Feb 10;270(6):2411–2414. doi: 10.1074/jbc.270.6.2411. [DOI] [PubMed] [Google Scholar]
  3. Berglund J. A., Chua K., Abovich N., Reed R., Rosbash M. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell. 1997 May 30;89(5):781–787. doi: 10.1016/s0092-8674(00)80261-5. [DOI] [PubMed] [Google Scholar]
  4. Bourgeois C. F., Popielarz M., Hildwein G., Stevenin J. Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation. Mol Cell Biol. 1999 Nov;19(11):7347–7356. doi: 10.1128/mcb.19.11.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bruzik J. P. Splicing glue: a role for SR proteins in trans splicing? Microb Pathog. 1996 Sep;21(3):149–155. doi: 10.1006/mpat.1996.0050. [DOI] [PubMed] [Google Scholar]
  6. Bruzik J. P., Van Doren K., Hirsh D., Steitz J. A. Trans splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature. 1988 Oct 6;335(6190):559–562. doi: 10.1038/335559a0. [DOI] [PubMed] [Google Scholar]
  7. Cavaloc Y., Bourgeois C. F., Kister L., Stévenin J. The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers. RNA. 1999 Mar;5(3):468–483. doi: 10.1017/s1355838299981967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chiara M. D., Reed R. A two-step mechanism for 5' and 3' splice-site pairing. Nature. 1995 Jun 8;375(6531):510–513. doi: 10.1038/375510a0. [DOI] [PubMed] [Google Scholar]
  9. Cohen J. B., Snow J. E., Spencer S. D., Levinson A. D. Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10470–10474. doi: 10.1073/pnas.91.22.10470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coulter L. R., Landree M. A., Cooper T. A. Identification of a new class of exonic splicing enhancers by in vivo selection. Mol Cell Biol. 1997 Apr;17(4):2143–2150. doi: 10.1128/mcb.17.4.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Côté J., Simard M. J., Chabot B. An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection. Nucleic Acids Res. 1999 Jun 15;27(12):2529–2537. doi: 10.1093/nar/27.12.2529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Graveley B. R., Hertel K. J., Maniatis T. A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers. EMBO J. 1998 Nov 16;17(22):6747–6756. doi: 10.1093/emboj/17.22.6747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graveley B. R. Sorting out the complexity of SR protein functions. RNA. 2000 Sep;6(9):1197–1211. doi: 10.1017/s1355838200000960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hannon G. J., Maroney P. A., Denker J. A., Nilsen T. W. Trans splicing of nematode pre-messenger RNA in vitro. Cell. 1990 Jun 29;61(7):1247–1255. doi: 10.1016/0092-8674(90)90689-c. [DOI] [PubMed] [Google Scholar]
  16. Hannon G. J., Maroney P. A., Nilsen T. W. U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro. J Biol Chem. 1991 Dec 5;266(34):22792–22795. [PubMed] [Google Scholar]
  17. Heinrichs V., Baker B. S. The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences. EMBO J. 1995 Aug 15;14(16):3987–4000. doi: 10.1002/j.1460-2075.1995.tb00070.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hertel K. J., Maniatis T. The function of multisite splicing enhancers. Mol Cell. 1998 Feb;1(3):449–455. doi: 10.1016/s1097-2765(00)80045-3. [DOI] [PubMed] [Google Scholar]
  19. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  20. Hoffman B. E., Grabowski P. J. U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon. Genes Dev. 1992 Dec;6(12B):2554–2568. doi: 10.1101/gad.6.12b.2554. [DOI] [PubMed] [Google Scholar]
  21. Humphrey M. B., Bryan J., Cooper T. A., Berget S. M. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol Cell Biol. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hwang D. Y., Cohen J. B. Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron. Mol Cell Biol. 1996 Jun;16(6):3012–3022. doi: 10.1128/mcb.16.6.3012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kennedy C. F., Krämer A., Berget S. M. A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point. Mol Cell Biol. 1998 Sep;18(9):5425–5434. doi: 10.1128/mcb.18.9.5425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kreivi J. P., Zerivitz K., Zefrivitz K., Akusjärvi G. A U1 snRNA binding site improves the efficiency of in vitro pre-mRNA splicing. Nucleic Acids Res. 1991 Dec 25;19(24):6956–6956. doi: 10.1093/nar/19.24.6956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kuo H. C., Nasim F. H., Grabowski P. J. Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle. Science. 1991 Mar 1;251(4997):1045–1050. doi: 10.1126/science.1825520. [DOI] [PubMed] [Google Scholar]
  26. Lapham J., Crothers D. M. RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA. 1996 Mar;2(3):289–296. [PMC free article] [PubMed] [Google Scholar]
  27. Lapham J., Yu Y. T., Shu M. D., Steitz J. A., Crothers D. M. The position of site-directed cleavage of RNA using RNase H and 2'-O-methyl oligonucleotides is dependent on the enzyme source. RNA. 1997 Sep;3(9):950–951. [PMC free article] [PubMed] [Google Scholar]
  28. Libri D., Lescure A., Rosbash M. Splicing enhancement in the yeast rp51b intron. RNA. 2000 Mar;6(3):352–368. doi: 10.1017/s1355838200991222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu H. X., Chew S. L., Cartegni L., Zhang M. Q., Krainer A. R. Exonic splicing enhancer motif recognized by human SC35 under splicing conditions. Mol Cell Biol. 2000 Feb;20(3):1063–1071. doi: 10.1128/mcb.20.3.1063-1071.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu H. X., Zhang M., Krainer A. R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 1998 Jul 1;12(13):1998–2012. doi: 10.1101/gad.12.13.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lou H., Yang Y., Cote G. J., Berget S. M., Gagel R. F. An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene. Mol Cell Biol. 1995 Dec;15(12):7135–7142. doi: 10.1128/mcb.15.12.7135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lund M., Tange T. O., Dyhr-Mikkelsen H., Hansen J., Kjems J. Characterization of human RNA splice signals by iterative functional selection of splice sites. RNA. 2000 Apr;6(4):528–544. doi: 10.1017/s1355838200992033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. López-Estraño C., Tschudi C., Ullu E. Exonic sequences in the 5' untranslated region of alpha-tubulin mRNA modulate trans splicing in Trypanosoma brucei. Mol Cell Biol. 1998 Aug;18(8):4620–4628. doi: 10.1128/mcb.18.8.4620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. MacMorris M. A., Zorio D. A., Blumenthal T. An exon that prevents transport of a mature mRNA. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3813–3818. doi: 10.1073/pnas.96.7.3813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mair G., Shi H., Li H., Djikeng A., Aviles H. O., Bishop J. R., Falcone F. H., Gavrilescu C., Montgomery J. L., Santori M. I. A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA. RNA. 2000 Feb;6(2):163–169. doi: 10.1017/s135583820099229x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Maroney P. A., Romfo C. M., Nilsen T. W. Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol Cell. 2000 Aug;6(2):317–328. doi: 10.1016/s1097-2765(00)00032-0. [DOI] [PubMed] [Google Scholar]
  37. Maroney P. A., Yu Y. T., Jankowska M., Nilsen T. W. Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs. RNA. 1996 Aug;2(8):735–745. [PMC free article] [PubMed] [Google Scholar]
  38. Merendino L., Guth S., Bilbao D., Martínez C., Valcárcel J. Inhibition of msl-2 splicing by Sex-lethal reveals interaction between U2AF35 and the 3' splice site AG. Nature. 1999 Dec 16;402(6763):838–841. doi: 10.1038/45602. [DOI] [PubMed] [Google Scholar]
  39. Nilsen T. W., Shambaugh J., Denker J., Chubb G., Faser C., Putnam L., Bennett K. Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum. Mol Cell Biol. 1989 Aug;9(8):3543–3547. doi: 10.1128/mcb.9.8.3543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Page-McCaw P. S., Amonlirdviman K., Sharp P. A. PUF60: a novel U2AF65-related splicing activity. RNA. 1999 Dec;5(12):1548–1560. doi: 10.1017/s1355838299991938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Query C. C., McCaw P. S., Sharp P. A. A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract. Mol Cell Biol. 1997 May;17(5):2944–2953. doi: 10.1128/mcb.17.5.2944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Reed R., Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. doi: 10.1016/0092-8674(86)90343-0. [DOI] [PubMed] [Google Scholar]
  43. Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
  44. Reyes J. L., Kois P., Konforti B. B., Konarska M. M. The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome. RNA. 1996 Mar;2(3):213–225. [PMC free article] [PubMed] [Google Scholar]
  45. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  47. Sanford J. R., Bruzik J. P. Developmental regulation of SR protein phosphorylation and activity. Genes Dev. 1999 Jun 15;13(12):1513–1518. doi: 10.1101/gad.13.12.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sanford J. R., Bruzik J. P. SR proteins are required for nematode trans-splicing in vitro. RNA. 1999 Jul;5(7):918–928. doi: 10.1017/s1355838299990234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sawa H., Abelson J. Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11269–11273. doi: 10.1073/pnas.89.23.11269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schaal T. D., Maniatis T. Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA. Mol Cell Biol. 1999 Jan;19(1):261–273. doi: 10.1128/mcb.19.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schaal T. D., Maniatis T. Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences. Mol Cell Biol. 1999 Mar;19(3):1705–1719. doi: 10.1128/mcb.19.3.1705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Seiwert S. D., Steitz J. A. Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing. Mol Cell Biol. 1993 Jun;13(6):3135–3145. doi: 10.1128/mcb.13.6.3135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Selvakumar M., Helfman D. M. Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA. RNA. 1999 Mar;5(3):378–394. doi: 10.1017/s1355838299981050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Shambaugh J. D., Hannon G. E., Nilsen T. W. The spliceosomal U small nuclear RNAs of Ascaris lumbricoides. Mol Biochem Parasitol. 1994 Apr;64(2):349–352. doi: 10.1016/0166-6851(94)00040-9. [DOI] [PubMed] [Google Scholar]
  55. Shi H., Hoffman B. E., Lis J. T. A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52. Mol Cell Biol. 1997 May;17(5):2649–2657. doi: 10.1128/mcb.17.5.2649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Singh R., Valcárcel J., Green M. R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. doi: 10.1126/science.7761834. [DOI] [PubMed] [Google Scholar]
  57. Somasekhar M. B., Mertz J. E. Exon mutations that affect the choice of splice sites used in processing the SV40 late transcripts. Nucleic Acids Res. 1985 Aug 12;13(15):5591–5609. doi: 10.1093/nar/13.15.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Spicher A., Etter A., Bernard V., Tobler H., Müller F. Extremely stable transcripts may compensate for the elimination of the gene fert-1 from all Ascaris lumbricoides somatic cells. Dev Biol. 1994 Jul;164(1):72–86. doi: 10.1006/dbio.1994.1181. [DOI] [PubMed] [Google Scholar]
  59. Tacke R., Chen Y., Manley J. L. Sequence-specific RNA binding by an SR protein requires RS domain phosphorylation: creation of an SRp40-specific splicing enhancer. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1148–1153. doi: 10.1073/pnas.94.4.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Tacke R., Manley J. L. Determinants of SR protein specificity. Curr Opin Cell Biol. 1999 Jun;11(3):358–362. doi: 10.1016/S0955-0674(99)80050-7. [DOI] [PubMed] [Google Scholar]
  61. Tacke R., Manley J. L. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995 Jul 17;14(14):3540–3551. doi: 10.1002/j.1460-2075.1995.tb07360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Talerico M., Berget S. M. Effect of 5' splice site mutations on splicing of the preceding intron. Mol Cell Biol. 1990 Dec;10(12):6299–6305. doi: 10.1128/mcb.10.12.6299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Thomas J. D., Conrad R. C., Blumenthal T. The C. elegans trans-spliced leader RNA is bound to Sm and has a trimethylguanosine cap. Cell. 1988 Aug 12;54(4):533–539. doi: 10.1016/0092-8674(88)90075-x. [DOI] [PubMed] [Google Scholar]
  64. Tian H., Kole R. Selection of novel exon recognition elements from a pool of random sequences. Mol Cell Biol. 1995 Nov;15(11):6291–6298. doi: 10.1128/mcb.15.11.6291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Tian M., Maniatis T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 1994 Jul 15;8(14):1703–1712. doi: 10.1101/gad.8.14.1703. [DOI] [PubMed] [Google Scholar]
  66. Wang J., Manley J. L. Regulation of pre-mRNA splicing in metazoa. Curr Opin Genet Dev. 1997 Apr;7(2):205–211. doi: 10.1016/s0959-437x(97)80130-x. [DOI] [PubMed] [Google Scholar]
  67. Wassarman D. A., Steitz J. A. Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. Science. 1992 Sep 25;257(5078):1918–1925. doi: 10.1126/science.1411506. [DOI] [PubMed] [Google Scholar]
  68. Wu Q., Krainer A. R. U1-mediated exon definition interactions between AT-AC and GT-AG introns. Science. 1996 Nov 8;274(5289):1005–1008. doi: 10.1126/science.274.5289.1005. [DOI] [PubMed] [Google Scholar]
  69. Wu S., Green M. R. Identification of a human protein that recognizes the 3' splice site during the second step of pre-mRNA splicing. EMBO J. 1997 Jul 16;16(14):4421–4432. doi: 10.1093/emboj/16.14.4421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wu S., Romfo C. M., Nilsen T. W., Green M. R. Functional recognition of the 3' splice site AG by the splicing factor U2AF35. Nature. 1999 Dec 16;402(6763):832–835. doi: 10.1038/45590. [DOI] [PubMed] [Google Scholar]
  71. Wyatt J. R., Sontheimer E. J., Steitz J. A. Site-specific cross-linking of mammalian U5 snRNP to the 5' splice site before the first step of pre-mRNA splicing. Genes Dev. 1992 Dec;6(12B):2542–2553. doi: 10.1101/gad.6.12b.2542. [DOI] [PubMed] [Google Scholar]
  72. Zamore P. D., Green M. R. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Zhang W. J., Wu J. Y. Functional properties of p54, a novel SR protein active in constitutive and alternative splicing. Mol Cell Biol. 1996 Oct;16(10):5400–5408. doi: 10.1128/mcb.16.10.5400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Zillmann M., Rose S. D., Berget S. M. U1 small nuclear ribonucleoproteins are required early during spliceosome assembly. Mol Cell Biol. 1987 Aug;7(8):2877–2883. doi: 10.1128/mcb.7.8.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Zorio D. A., Blumenthal T. Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans. Nature. 1999 Dec 16;402(6763):835–838. doi: 10.1038/45597. [DOI] [PubMed] [Google Scholar]

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