Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Sep;7(9):3131–3137. doi: 10.1128/mcb.7.9.3131

Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract.

A M Kleinschmidt 1, T Pederson 1
PMCID: PMC367947  PMID: 3670307

Abstract

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

Full text

PDF
3131

Images in this article

3132Image
on p.3132
3133Image
on p.3133
3133Image
on p.3133
3134Image
on p.3134
3134Image
on p.3134
3134Image
on p.3134
3135Image
on p.3135
3135Image
on p.3135

Selected References

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

  1. Ares M., Jr, Mangin M., Weiner A. M. Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene. Mol Cell Biol. 1985 Jul;5(7):1560–1570. doi: 10.1128/mcb.5.7.1560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berget S. M., Robberson B. L. U1, U2, and U4/U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation. Cell. 1986 Aug 29;46(5):691–696. doi: 10.1016/0092-8674(86)90344-2. [DOI] [PubMed] [Google Scholar]
  3. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  4. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  5. Black D. L., Steitz J. A. Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein. Cell. 1986 Aug 29;46(5):697–704. doi: 10.1016/0092-8674(86)90345-4. [DOI] [PubMed] [Google Scholar]
  6. Castaño J. G., Ornberg R., Koster J. G., Tobian J. A., Zasloff M. Eukaryotic pre-tRNA 5' processing nuclease: copurification with a complex cylindrical particle. Cell. 1986 Aug 1;46(3):377–385. doi: 10.1016/0092-8674(86)90658-6. [DOI] [PubMed] [Google Scholar]
  7. Chabot B., Black D. L., LeMaster D. M., Steitz J. A. The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein. Science. 1985 Dec 20;230(4732):1344–1349. doi: 10.1126/science.2933810. [DOI] [PubMed] [Google Scholar]
  8. Chandrasekharappa S. C., Smith J. H., Eliceiri G. L. Biosynthesis of small nuclear RNAs in human cells. J Cell Physiol. 1983 Nov;117(2):169–174. doi: 10.1002/jcp.1041170206. [DOI] [PubMed] [Google Scholar]
  9. Contreras R., Cheroutre H., Degrave W., Fiers W. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic Acids Res. 1982 Oct 25;10(20):6353–6362. doi: 10.1093/nar/10.20.6353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Earley J. M., 3rd, Roebuck K. A., Stumph W. E. Three linked chicken U1 RNA genes have limited flanking DNA sequence homologies that reveal potential regulatory signals. Nucleic Acids Res. 1984 Oct 11;12(19):7411–7421. doi: 10.1093/nar/12.19.7411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eliceiri G. L. Formation of low molecular weight RNA species in HeLa cells. J Cell Physiol. 1980 Feb;102(2):199–207. doi: 10.1002/jcp.1041020211. [DOI] [PubMed] [Google Scholar]
  12. Eliceiri G. L. Short-lived, small RNAs in the cytoplasm of HeLa cells. Cell. 1974 Sep;3(1):11–14. doi: 10.1016/0092-8674(74)90031-2. [DOI] [PubMed] [Google Scholar]
  13. Fisher D. E., Conner G. E., Reeves W. H., Wisniewolski R., Blobel G. Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification. Cell. 1985 Oct;42(3):751–758. doi: 10.1016/0092-8674(85)90271-5. [DOI] [PubMed] [Google Scholar]
  14. Fradin A., Jove R., Hemenway C., Keiser H. D., Manley J. L., Prives C. Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs. Cell. 1984 Jul;37(3):927–936. doi: 10.1016/0092-8674(84)90427-6. [DOI] [PubMed] [Google Scholar]
  15. Frederiksen S., Hellung-Larsen P., Gram Jensen E. The differential inhibitory effect of alpha-amanitin on the synthesis of low molecular weight RNA components in BHK cells. FEBS Lett. 1978 Mar 15;87(2):227–231. doi: 10.1016/0014-5793(78)80338-x. [DOI] [PubMed] [Google Scholar]
  16. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  17. Galli G., Hofstetter H., Stunnenberg H. G., Birnstiel M. L. Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini. Cell. 1983 Oct;34(3):823–828. doi: 10.1016/0092-8674(83)90539-1. [DOI] [PubMed] [Google Scholar]
  18. Gick O., Krämer A., Keller W., Birnstiel M. L. Generation of histone mRNA 3' ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction. EMBO J. 1986 Jun;5(6):1319–1326. doi: 10.1002/j.1460-2075.1986.tb04362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  20. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  21. Gurney T., Jr, Eliceiri G. L. Intracellular distribution of low molecular weight RNA species in HeLa cells. J Cell Biol. 1980 Nov;87(2 Pt 1):398–403. doi: 10.1083/jcb.87.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hellung-Larsen P., Kulamowicz I., Frederiksen S. Synthesis of low molecular weight RNA components in cells with a temperature-sensitive polymerase II. Biochim Biophys Acta. 1980 Aug 26;609(1):201–204. doi: 10.1016/0005-2787(80)90213-0. [DOI] [PubMed] [Google Scholar]
  23. Hernandez N. Formation of the 3' end of U1 snRNA is directed by a conserved sequence located downstream of the coding region. EMBO J. 1985 Jul;4(7):1827–1837. doi: 10.1002/j.1460-2075.1985.tb03857.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hernandez N., Weiner A. M. Formation of the 3' end of U1 snRNA requires compatible snRNA promoter elements. Cell. 1986 Oct 24;47(2):249–258. doi: 10.1016/0092-8674(86)90447-2. [DOI] [PubMed] [Google Scholar]
  25. Hoffman M. L., Korf G. M., McNamara K. J., Stumph W. E. Structural and functional analysis of chicken U4 small nuclear RNA genes. Mol Cell Biol. 1986 Nov;6(11):3910–3919. doi: 10.1128/mcb.6.11.3910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jensen E. G., Hellung-Larsen P., Frederiksen S. Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells. Nucleic Acids Res. 1979 Jan;6(1):321–330. doi: 10.1093/nar/6.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  28. Korf G. M., Stumph W. E. Chicken U2 and U1 RNA genes are found in very different genomic environments but have similar promoter structures. Biochemistry. 1986 Apr 22;25(8):2041–2047. doi: 10.1021/bi00356a031. [DOI] [PubMed] [Google Scholar]
  29. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  30. Krol A., Lund E., Dahlberg J. E. The two embryonic U1 RNA genes of Xenopus laevis have both common and gene-specific transcription signals. EMBO J. 1985 Jun;4(6):1529–1535. doi: 10.1002/j.1460-2075.1985.tb03813.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Krämer A., Keller W., Appel B., Lührmann R. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell. 1984 Aug;38(1):299–307. doi: 10.1016/0092-8674(84)90551-8. [DOI] [PubMed] [Google Scholar]
  32. Kunkel G. R., Maser R. L., Calvet J. P., Pederson T. U6 small nuclear RNA is transcribed by RNA polymerase III. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8575–8579. doi: 10.1073/pnas.83.22.8575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kunkel G. R., Pederson T. Transcription boundaries of U1 small nuclear RNA. Mol Cell Biol. 1985 Sep;5(9):2332–2340. doi: 10.1128/mcb.5.9.2332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Laski F. A., Fire A. Z., RajBhandary U. L., Sharp P. A. Characterization of tRNA precursor splicing in mammalian extracts. J Biol Chem. 1983 Oct 10;258(19):11974–11980. [PubMed] [Google Scholar]
  35. Madore S. J., Wieben E. D., Kunkel G. R., Pederson T. Precursors of U4 small nuclear RNA. J Cell Biol. 1984 Sep;99(3):1140–1144. doi: 10.1083/jcb.99.3.1140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Madore S. J., Wieben E. D., Pederson T. Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes. J Biol Chem. 1984 Feb 10;259(3):1929–1933. [PubMed] [Google Scholar]
  37. Madore S. J., Wieben E. D., Pederson T. Intracellular site of U1 small nuclear RNA processing and ribonucleoprotein assembly. J Cell Biol. 1984 Jan;98(1):188–192. doi: 10.1083/jcb.98.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mangin M., Ares M., Jr, Weiner A. M. Human U2 small nuclear RNA genes contain an upstream enhancer. EMBO J. 1986 May;5(5):987–995. doi: 10.1002/j.1460-2075.1986.tb04313.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Marzluff W. F., Brown D. T., Lobo S., Wang S. S. Isolation and characterization of two linked mouse U1b small nuclear RNA genes. Nucleic Acids Res. 1983 Sep 24;11(18):6255–6270. doi: 10.1093/nar/11.18.6255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mattaj I. W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986 Sep 12;46(6):905–911. doi: 10.1016/0092-8674(86)90072-3. [DOI] [PubMed] [Google Scholar]
  41. Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
  42. Moore C. L., Sharp P. A. Accurate cleavage and polyadenylation of exogenous RNA substrate. Cell. 1985 Jul;41(3):845–855. doi: 10.1016/s0092-8674(85)80065-9. [DOI] [PubMed] [Google Scholar]
  43. Murphy J. T., Burgess R. R., Dahlberg J. E., Lund E. Transcription of a gene for human U1 small nuclear RNA. Cell. 1982 May;29(1):265–274. doi: 10.1016/0092-8674(82)90111-8. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Pederson T. Chromatin structure and the cell cycle. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2224–2228. doi: 10.1073/pnas.69.8.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Penman S., Scherrer K., Becker Y., Darnell J. E. POLYRIBOSOMES IN NORMAL AND POLIOVIRUS-INFECTED HELA CELLS AND THEIR RELATIONSHIP TO MESSENGER-RNA. Proc Natl Acad Sci U S A. 1963 May;49(5):654–662. doi: 10.1073/pnas.49.5.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Reddy R., Henning D., Das G., Harless M., Wright D. The capped U6 small nuclear RNA is transcribed by RNA polymerase III. J Biol Chem. 1987 Jan 5;262(1):75–81. [PubMed] [Google Scholar]
  48. Ro-Choi T. S., Raj N. B., Pike L. M., Busch H. Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA. Biochemistry. 1976 Aug 24;15(17):3823–3828. doi: 10.1021/bi00662a027. [DOI] [PubMed] [Google Scholar]
  49. Rohleder A., Wieben E. Solid-phase processing of U2 snRNA precursors. Biochemistry. 1986 Oct 7;25(20):5910–5914. doi: 10.1021/bi00368a011. [DOI] [PubMed] [Google Scholar]
  50. Roop D. R., Kristo P., Stumph W. E., Tsai M. J., O'Malley B. W. Structure and expression of a chicken gene coding for U1 RNA. Cell. 1981 Mar;23(3):671–680. doi: 10.1016/0092-8674(81)90430-x. [DOI] [PubMed] [Google Scholar]
  51. Skuzeski J. M., Lund E., Murphy J. T., Steinberg T. H., Burgess R. R., Dahlberg J. E. Synthesis of human U1 RNA. II. Identification of two regions of the promoter essential for transcription initiation at position +1. J Biol Chem. 1984 Jul 10;259(13):8345–8352. [PubMed] [Google Scholar]
  52. Tamm I., Kikuchi T., Darnell J. E., Jr, Salditt-Georgieff M. Short capped hnRNA precursor chains in HeLa cells: continued synthesis in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Biochemistry. 1980 Jun 10;19(12):2743–2748. doi: 10.1021/bi00553a032. [DOI] [PubMed] [Google Scholar]
  53. Tani T., Watanabe-Nagasu N., Okada N., Ohshima Y. Molecular cloning and characterization of a gene for rat U2 small nuclear RNA. J Mol Biol. 1983 Aug 15;168(3):579–594. doi: 10.1016/s0022-2836(83)80303-9. [DOI] [PubMed] [Google Scholar]
  54. Van Arsdell S. W., Weiner A. M. Human genes for U2 small nuclear RNA are tandemly repeated. Mol Cell Biol. 1984 Mar;4(3):492–499. doi: 10.1128/mcb.4.3.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Watanabe-Nagasu N., Itoh Y., Tani T., Okano K., Koga N., Okada N., Ohshima Y. Structural analysis of gene loci for rat U1 small nuclear RNA. Nucleic Acids Res. 1983 Mar 25;11(6):1791–1801. doi: 10.1093/nar/11.6.1791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weil P. A., Segall J., Harris B., Ng S. Y., Roeder R. G. Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. J Biol Chem. 1979 Jul 10;254(13):6163–6173. [PubMed] [Google Scholar]
  57. Westin G., Lund E., Murphy J. T., Pettersson U., Dahlberg J. E. Human U2 and U1 RNA genes use similar transcription signals. EMBO J. 1984 Dec 20;3(13):3295–3301. doi: 10.1002/j.1460-2075.1984.tb02293.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Westin G., Zabielski J., Hammarström K., Monstein H. J., Bark C., Pettersson U. Clustered genes for human U2 RNA. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3811–3815. doi: 10.1073/pnas.81.12.3811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wieben E. D., Nenninger J. M., Pederson T. Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles. J Mol Biol. 1985 May 5;183(1):69–78. doi: 10.1016/0022-2836(85)90281-5. [DOI] [PubMed] [Google Scholar]
  60. Yang V. W., Lerner M. R., Steitz J. A., Flint S. J. A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1371–1375. doi: 10.1073/pnas.78.3.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yuo C. Y., Ares M., Jr, Weiner A. M. Sequences required for 3' end formation of human U2 small nuclear RNA. Cell. 1985 Aug;42(1):193–202. doi: 10.1016/s0092-8674(85)80115-x. [DOI] [PubMed] [Google Scholar]
  62. Zeller R., Carri M. T., Mattaj I. W., De Robertis E. M. Xenopus laevis U1 snRNA genes: characterisation of transcriptionally active genes reveals major and minor repeated gene families. EMBO J. 1984 May;3(5):1075–1081. doi: 10.1002/j.1460-2075.1984.tb01931.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zieve G., Penman S. Small RNA species of the HeLa cell: metabolism and subcellular localization. Cell. 1976 May;8(1):19–31. doi: 10.1016/0092-8674(76)90181-1. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES