Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1984 May 25;12(10):4111–4126. doi: 10.1093/nar/12.10.4111

The 5'-terminal sequence of U1 RNA complementary to the consensus 5' splice site of hnRNA is single-stranded in intact U1 snRNP particles.

J Rinke, B Appel, H Blöcker, R Frank, R Lührmann
PMCID: PMC318820  PMID: 6203096

Abstract

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.

Full text

PDF
4111

Images in this article

4117Image
on p.4117
4118Image
on p.4118
4120Image
on p.4120
4122Image
on p.4122

Selected References

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

  1. Branlant C., Krol A., Ebel J. P., Lazar E., Gallinaro H., Jacob M., Sri-Widada J., Jeanteur P. Nucleotide sequences of nuclear U1A RNAs from chicken, rat and man. Nucleic Acids Res. 1980 Sep 25;8(18):4143–4154. doi: 10.1093/nar/8.18.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branlant C., Krol A., Ebel J. P., Lazar E., Haendler B., Jacob M. U2 RNA shares a structural domain with U1, U4, and U5 RNAs. EMBO J. 1982;1(10):1259–1265. doi: 10.1002/j.1460-2075.1982.tb00022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bringmann P., Reuter R., Rinke J., Appel B., Bald R., Lührmann R. 5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs. J Biol Chem. 1983 Mar 10;258(5):2745–2747. [PubMed] [Google Scholar]
  4. Bringmann P., Rinke J., Appel B., Reuter R., Lührmann R. Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera. EMBO J. 1983;2(7):1129–1135. doi: 10.1002/j.1460-2075.1983.tb01557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Calvet J. P., Pederson T. Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo. Cell. 1981 Nov;26(3 Pt 1):363–370. doi: 10.1016/0092-8674(81)90205-1. [DOI] [PubMed] [Google Scholar]
  6. Darlix J. L. Stimultaneous purification of Escherichia coli termination factor rho, RNAase III and RNAase H. Eur J Biochem. 1975 Feb 21;51(2):369–376. doi: 10.1111/j.1432-1033.1975.tb03937.x. [DOI] [PubMed] [Google Scholar]
  7. Deimel B., Louis C. H., Sekeris C. E. The presence of small molecular weight RNAs in nuclear ribonucleoprotein particles carrying HnRNA. FEBS Lett. 1977 Jan 15;73(1):80–84. [PubMed] [Google Scholar]
  8. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donis-Keller H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 1979 Sep 11;7(1):179–192. doi: 10.1093/nar/7.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Epstein P., Reddy R., Busch H. Site-specific cleavage by T1 RNase of U-1 RNA in u-1 ribonucleoprotein particles. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1562–1566. doi: 10.1073/pnas.78.3.1562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fritz H. J., Belagaje R., Brown E. L., Fritz R. H., Jones R. A., Lees R. G., Khorana H. G. High-pressure liquid chromatography in polynucleotide synthesis. Biochemistry. 1978 Apr 4;17(7):1257–1267. doi: 10.1021/bi00600a020. [DOI] [PubMed] [Google Scholar]
  12. Gallinaro H., Jacob M. An evaluation of small nuclear RNA in hnRNP. FEBS Lett. 1979 Aug 1;104(1):176–182. doi: 10.1016/0014-5793(79)81110-2. [DOI] [PubMed] [Google Scholar]
  13. Grotjahn L., Frank R., Blöcker H. Ultrafast sequencing of oligodeoxyribonucleotides by FAB-mass spectrometry. Nucleic Acids Res. 1982 Aug 11;10(15):4671–4678. doi: 10.1093/nar/10.15.4671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hernandez N., Keller W. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell. 1983 Nov;35(1):89–99. doi: 10.1016/0092-8674(83)90211-8. [DOI] [PubMed] [Google Scholar]
  15. Lazar E., Jacob M., Krol A., Branlant C. Accessibility of U1 RNA to base pairing with a single-stranded DNA fragment mimicking the intron extremities at the splice junction. Nucleic Acids Res. 1982 Feb 25;10(4):1193–1201. doi: 10.1093/nar/10.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lerner M. R., Boyle J. A., Mount S. M., Wolin S. L., Steitz J. A. Are snRNPs involved in splicing? Nature. 1980 Jan 10;283(5743):220–224. doi: 10.1038/283220a0. [DOI] [PubMed] [Google Scholar]
  17. Lerner M. R., Steitz J. A. Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5495–5499. doi: 10.1073/pnas.76.11.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liautard J. P., Sri-Widada J., Brunel C., Jeanteur P. Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs. J Mol Biol. 1982 Dec 15;162(3):623–643. doi: 10.1016/0022-2836(82)90392-8. [DOI] [PubMed] [Google Scholar]
  19. Luhrmann R., Appel B., Bringmann P., Rinke J., Reuter R., Rothe S., Bald R. Isolation and characterization of rabbit anti-m3 2,2,7G antibodies. Nucleic Acids Res. 1982 Nov 25;10(22):7103–7113. doi: 10.1093/nar/10.22.7103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  21. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  23. Mount S. M., Steitz J. A. Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing. Nucleic Acids Res. 1981 Dec 11;9(23):6351–6368. doi: 10.1093/nar/9.23.6351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nohga K., Reddy R., Busch H. Comparison of RNase T1 fingerprints of U1, U2, and U3 small nuclear RNA's of HeLa cells, human normal fibroblasts, and Novikoff hepatoma cells. Cancer Res. 1981 Jun;41(6):2215–2220. [PubMed] [Google Scholar]
  25. Northemann W., Scheurlen M., Gross V., Heinrich P. C. Circular dichroism of ribonucleoprotein complexes from rat liver nuclei. Biochem Biophys Res Commun. 1977 Jun 20;76(4):1130–1137. doi: 10.1016/0006-291x(77)90973-1. [DOI] [PubMed] [Google Scholar]
  26. Ohtsuka E., Ikehara M., Söll D. Recent developments in the chemical synthesis of polynucleotides. Nucleic Acids Res. 1982 Nov 11;10(21):6553–6570. doi: 10.1093/nar/10.21.6553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Padgett R. A., Mount S. M., Steitz J. A., Sharp P. A. Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. Cell. 1983 Nov;35(1):101–107. doi: 10.1016/0092-8674(83)90212-x. [DOI] [PubMed] [Google Scholar]
  28. Raj N. B., Ro-Choi T. S., Busch H. Nuclear ribonucleoprotein complexes containing U1 and U2 RNA. Biochemistry. 1975 Oct 7;14(20):4380–4385. doi: 10.1021/bi00691a006. [DOI] [PubMed] [Google Scholar]
  29. Rogers J., Wall R. A mechanism for RNA splicing. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1877–1879. doi: 10.1073/pnas.77.4.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stepanova O. B., Metelev V. G., Chichkova N. V., Smirnov V. D., Rodionova N. P., Atabekov J. G., Bogdanov A. A., Shabarova Z. A. Addressed fragmentation of RNA Molecules. FEBS Lett. 1979 Jul 1;103(1):197–199. doi: 10.1016/0014-5793(79)81280-6. [DOI] [PubMed] [Google Scholar]
  31. Stiege W., Glotz C., Brimacombe R. Localisation of a series of intra-RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia coli 50S ribosomal subunits. Nucleic Acids Res. 1983 Mar 25;11(6):1687–1706. doi: 10.1093/nar/11.6.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wieben E. D., Pederson T. Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock. Mol Cell Biol. 1982 Aug;2(8):914–920. doi: 10.1128/mcb.2.8.914. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES