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. 2001 Sep;7(9):1239–1253. doi: 10.1017/s1355838201010603

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element.

A K Amarasinghe 1, R MacDiarmid 1, M D Adams 1, D C Rio 1
PMCID: PMC1370169  PMID: 11565747

Abstract

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. D., Rudner D. Z., Rio D. C. Biochemistry and regulation of pre-mRNA splicing. Curr Opin Cell Biol. 1996 Jun;8(3):331–339. doi: 10.1016/s0955-0674(96)80006-8. [DOI] [PubMed] [Google Scholar]
  2. Adams M. D., Tarng R. S., Rio D. C. The alternative splicing factor PSI regulates P-element third intron splicing in vivo. Genes Dev. 1997 Jan 1;11(1):129–138. doi: 10.1101/gad.11.1.129. [DOI] [PubMed] [Google Scholar]
  3. Berglund J. A., Fleming M. L., Rosbash M. The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence. RNA. 1998 Aug;4(8):998–1006. doi: 10.1017/s1355838298980499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buckanovich R. J., Darnell R. B. The neuronal RNA binding protein Nova-1 recognizes specific RNA targets in vitro and in vivo. Mol Cell Biol. 1997 Jun;17(6):3194–3201. doi: 10.1128/mcb.17.6.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  6. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  7. Chain A. C., Zollman S., Tseng J. C., Laski F. A. Identification of a cis-acting sequence required for germ line-specific splicing of the P element ORF2-ORF3 intron. Mol Cell Biol. 1991 Mar;11(3):1538–1546. doi: 10.1128/mcb.11.3.1538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chou M. Y., Underwood J. G., Nikolic J., Luu M. H., Black D. L. Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing. Mol Cell. 2000 Jun;5(6):949–957. doi: 10.1016/s1097-2765(00)80260-9. [DOI] [PubMed] [Google Scholar]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Engebrecht J. A., Voelkel-Meiman K., Roeder G. S. Meiosis-specific RNA splicing in yeast. Cell. 1991 Sep 20;66(6):1257–1268. doi: 10.1016/0092-8674(91)90047-3. [DOI] [PubMed] [Google Scholar]
  11. Förch P., Puig O., Kedersha N., Martínez C., Granneman S., Séraphin B., Anderson P., Valcárcel J. The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing. Mol Cell. 2000 Nov;6(5):1089–1098. doi: 10.1016/s1097-2765(00)00107-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gurevich V. V. Use of bacteriophage RNA polymerase in RNA synthesis. Methods Enzymol. 1996;275:382–397. doi: 10.1016/s0076-6879(96)75023-1. [DOI] [PubMed] [Google Scholar]
  14. Jensen K. B., Dredge B. K., Stefani G., Zhong R., Buckanovich R. J., Okano H. J., Yang Y. Y., Darnell R. B. Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron. 2000 Feb;25(2):359–371. doi: 10.1016/s0896-6273(00)80900-9. [DOI] [PubMed] [Google Scholar]
  15. Kanamori H., Dodson R. E., Shapiro D. J. In vitro genetic analysis of the RNA binding site of vigilin, a multi-KH-domain protein. Mol Cell Biol. 1998 Jul;18(7):3991–4003. doi: 10.1128/mcb.18.7.3991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laski F. A., Rio D. C., Rubin G. M. Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell. 1986 Jan 17;44(1):7–19. doi: 10.1016/0092-8674(86)90480-0. [DOI] [PubMed] [Google Scholar]
  17. Laski F. A., Rubin G. M. Analysis of the cis-acting requirements for germ-line-specific splicing of the P-element ORF2-ORF3 intron. Genes Dev. 1989 May;3(5):720–728. doi: 10.1101/gad.3.5.720. [DOI] [PubMed] [Google Scholar]
  18. Lewis H. A., Chen H., Edo C., Buckanovich R. J., Yang Y. Y., Musunuru K., Zhong R., Darnell R. B., Burley S. K. Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains. Structure. 1999 Feb 15;7(2):191–203. doi: 10.1016/S0969-2126(99)80025-2. [DOI] [PubMed] [Google Scholar]
  19. Lewis H. A., Musunuru K., Jensen K. B., Edo C., Chen H., Darnell R. B., Burley S. K. Sequence-specific RNA binding by a Nova KH domain: implications for paraneoplastic disease and the fragile X syndrome. Cell. 2000 Feb 4;100(3):323–332. doi: 10.1016/s0092-8674(00)80668-6. [DOI] [PubMed] [Google Scholar]
  20. Lin Q., Taylor S. J., Shalloway D. Specificity and determinants of Sam68 RNA binding. Implications for the biological function of K homology domains. J Biol Chem. 1997 Oct 24;272(43):27274–27280. doi: 10.1074/jbc.272.43.27274. [DOI] [PubMed] [Google Scholar]
  21. Lopez A. J. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet. 1998;32:279–305. doi: 10.1146/annurev.genet.32.1.279. [DOI] [PubMed] [Google Scholar]
  22. Matunis M. J., Michael W. M., Dreyfuss G. Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. Mol Cell Biol. 1992 Jan;12(1):164–171. doi: 10.1128/mcb.12.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Min H., Turck C. W., Nikolic J. M., Black D. L. A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes Dev. 1997 Apr 15;11(8):1023–1036. doi: 10.1101/gad.11.8.1023. [DOI] [PubMed] [Google Scholar]
  24. Misra S., Rio D. C. Cytotype control of Drosophila P element transposition: the 66 kd protein is a repressor of transposase activity. Cell. 1990 Jul 27;62(2):269–284. doi: 10.1016/0092-8674(90)90365-l. [DOI] [PubMed] [Google Scholar]
  25. Puig O., Gottschalk A., Fabrizio P., Séraphin B. Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection. Genes Dev. 1999 Mar 1;13(5):569–580. doi: 10.1101/gad.13.5.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Siebel C. W., Admon A., Rio D. C. Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing. Genes Dev. 1995 Feb 1;9(3):269–283. doi: 10.1101/gad.9.3.269. [DOI] [PubMed] [Google Scholar]
  27. Siebel C. W., Fresco L. D., Rio D. C. The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding. Genes Dev. 1992 Aug;6(8):1386–1401. doi: 10.1101/gad.6.8.1386. [DOI] [PubMed] [Google Scholar]
  28. Siebel C. W., Kanaar R., Rio D. C. Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI. Genes Dev. 1994 Jul 15;8(14):1713–1725. doi: 10.1101/gad.8.14.1713. [DOI] [PubMed] [Google Scholar]
  29. Siebel C. W., Rio D. C. Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression. Science. 1990 Jun 8;248(4960):1200–1208. doi: 10.1126/science.2161558. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Siomi H., Matunis M. J., Michael W. M., Dreyfuss G. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res. 1993 Mar 11;21(5):1193–1198. doi: 10.1093/nar/21.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smith C. W., Valcárcel J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci. 2000 Aug;25(8):381–388. doi: 10.1016/s0968-0004(00)01604-2. [DOI] [PubMed] [Google Scholar]
  33. Spingola M., Ares M., Jr A yeast intronic splicing enhancer and Nam8p are required for Mer1p-activated splicing. Mol Cell. 2000 Aug;6(2):329–338. doi: 10.1016/s1097-2765(00)00033-2. [DOI] [PubMed] [Google Scholar]
  34. Tian Q., Streuli M., Saito H., Schlossman S. F., Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629–639. doi: 10.1016/0092-8674(91)90536-8. [DOI] [PubMed] [Google Scholar]
  35. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  36. Yang Y. Y., Yin G. L., Darnell R. B. The neuronal RNA-binding protein Nova-2 is implicated as the autoantigen targeted in POMA patients with dementia. Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13254–13259. doi: 10.1073/pnas.95.22.13254. [DOI] [PMC free article] [PubMed] [Google Scholar]

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