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. 1993 Mar;12(3):1229–1238. doi: 10.1002/j.1460-2075.1993.tb05764.x

The low abundance of U7 snRNA is partly determined by its Sm binding site.

C Grimm 1, B Stefanovic 1, D Schümperli 1
PMCID: PMC413326  PMID: 8458335

Abstract

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4- to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Sm-mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

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Selected References

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  1. Ach R. A., Weiner A. M. The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation. Mol Cell Biol. 1987 Jun;7(6):2070–2079. doi: 10.1128/mcb.7.6.2070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birchmeier C., Grosschedl R., Birnstiel M. L. Generation of authentic 3' termini of an H2A mRNA in vivo is dependent on a short inverted DNA repeat and on spacer sequences. Cell. 1982 Apr;28(4):739–745. doi: 10.1016/0092-8674(82)90053-8. [DOI] [PubMed] [Google Scholar]
  3. Birchmeier C., Schümperli D., Sconzo G., Birnstiel M. L. 3' editing of mRNAs: sequence requirements and involvement of a 60-nucleotide RNA in maturation of histone mRNA precursors. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1057–1061. doi: 10.1073/pnas.81.4.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bond U. M., Yario T. A., Steitz J. A. Multiple processing-defective mutations in a mammalian histone pre-mRNA are suppressed by compensatory changes in U7 RNA both in vivo and in vitro. Genes Dev. 1991 Sep;5(9):1709–1722. doi: 10.1101/gad.5.9.1709. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Cotten M., Gick O., Vasserot A., Schaffner G., Birnstiel M. L. Specific contacts between mammalian U7 snRNA and histone precursor RNA are indispensable for the in vitro 3' RNA processing reaction. EMBO J. 1988 Mar;7(3):801–808. doi: 10.1002/j.1460-2075.1988.tb02878.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Lorenzi M., Rohrer U., Birnstiel M. L. Analysis of a sea urchin gene cluster coding for the small nuclear U7 RNA, a rare RNA species implicated in the 3' editing of histone precursor mRNAs. Proc Natl Acad Sci U S A. 1986 May;83(10):3243–3247. doi: 10.1073/pnas.83.10.3243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feeney R. J., Sauterer R. A., Feeney J. L., Zieve G. W. Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles. J Biol Chem. 1989 Apr 5;264(10):5776–5783. [PubMed] [Google Scholar]
  9. Feeney R. J., Zieve G. W. Nuclear exchange of the U1 and U2 snRNP-specific proteins. J Cell Biol. 1990 Apr;110(4):871–881. doi: 10.1083/jcb.110.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fresco L. D., Kurilla M. G., Keene J. D. Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus. Mol Cell Biol. 1987 Mar;7(3):1148–1155. doi: 10.1128/mcb.7.3.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Georgiev O., Mous J., Birnstiel M. L. Processing and nucleo-cytoplasmic transport of histone gene transcripts. Nucleic Acids Res. 1984 Nov 26;12(22):8539–8551. doi: 10.1093/nar/12.22.8539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gruber A., Soldati D., Burri M., Schümperli D. Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA. Biochim Biophys Acta. 1991 Jan 17;1088(1):151–154. doi: 10.1016/0167-4781(91)90167-k. [DOI] [PubMed] [Google Scholar]
  13. Gruber A., Streit A., Reist M., Benninger P., Böhni R., Schümperli D. Structure of a mouse histone-encoding gene cluster. Gene. 1990 Nov 15;95(2):303–304. doi: 10.1016/0378-1119(90)90377-4. [DOI] [PubMed] [Google Scholar]
  14. Gurdon J. B., Wickens M. P. The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 1983;101:370–386. doi: 10.1016/0076-6879(83)01028-9. [DOI] [PubMed] [Google Scholar]
  15. Hamm J., Darzynkiewicz E., Tahara S. M., Mattaj I. W. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
  16. Hamm J., van Santen V. L., Spritz R. A., Mattaj I. W. Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins. Mol Cell Biol. 1988 Nov;8(11):4787–4791. doi: 10.1128/mcb.8.11.4787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hernandez N., Lucito R. Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation. EMBO J. 1988 Oct;7(10):3125–3134. doi: 10.1002/j.1460-2075.1988.tb03179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Howard E. F., Michael S. K., Dahlberg J. E., Lund E. Functional, developmentally expressed genes for mouse U1a and U1b snRNAs contain both conserved and non-conserved transcription signals. Nucleic Acids Res. 1986 Dec 22;14(24):9811–9825. doi: 10.1093/nar/14.24.9811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones M. H., Guthrie C. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 1990 Aug;9(8):2555–2561. doi: 10.1002/j.1460-2075.1990.tb07436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kambach C., Mattaj I. W. Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA. J Cell Biol. 1992 Jul;118(1):11–21. doi: 10.1083/jcb.118.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  24. Lerner E. A., Lerner M. R., Janeway C. A., Jr, Steitz J. A. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc Natl Acad Sci U S A. 1981 May;78(5):2737–2741. doi: 10.1073/pnas.78.5.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lund E., Kahan B., Dahlberg J. E. Differential control of U1 small nuclear RNA expression during mouse development. Science. 1985 Sep 20;229(4719):1271–1274. doi: 10.1126/science.2412294. [DOI] [PubMed] [Google Scholar]
  26. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Melin L., Soldati D., Mital R., Streit A., Schümperli D. Biochemical demonstration of complex formation of histone pre-mRNA with U7 small nuclear ribonucleoprotein and hairpin binding factors. EMBO J. 1992 Feb;11(2):691–697. doi: 10.1002/j.1460-2075.1992.tb05101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Michaud N., Goldfarb D. Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways. J Cell Biol. 1992 Feb;116(4):851–861. doi: 10.1083/jcb.116.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mowry K. L., Steitz J. A. Identification of the human U7 snRNP as one of several factors involved in the 3' end maturation of histone premessenger RNA's. Science. 1987 Dec 18;238(4834):1682–1687. doi: 10.1126/science.2825355. [DOI] [PubMed] [Google Scholar]
  34. Neuman de Vegvar H. E., Dahlberg J. E. Nucleocytoplasmic transport and processing of small nuclear RNA precursors. Mol Cell Biol. 1990 Jul;10(7):3365–3375. doi: 10.1128/mcb.10.7.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nigg E. A., Baeuerle P. A., Lührmann R. Nuclear import-export: in search of signals and mechanisms. Cell. 1991 Jul 12;66(1):15–22. doi: 10.1016/0092-8674(91)90135-l. [DOI] [PubMed] [Google Scholar]
  36. Phillips S. C., Birnstiel M. L. Analysis of a gene cluster coding for the Xenopus laevis U7 snRNA. Biochim Biophys Acta. 1992 May 7;1131(1):95–98. doi: 10.1016/0167-4781(92)90104-8. [DOI] [PubMed] [Google Scholar]
  37. Phillips S. C., Turner P. C. A transcriptional analysis of the gene encoding mouse U7 small nuclear RNA. Gene. 1992 Jul 15;116(2):181–186. doi: 10.1016/0378-1119(92)90514-p. [DOI] [PubMed] [Google Scholar]
  38. Phillips S. C., Turner P. C. Nucleotide sequence of the mouse U7 snRNA gene. Nucleic Acids Res. 1991 Mar 25;19(6):1344–1344. doi: 10.1093/nar/19.6.1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reddy R., Busch H. Small nuclear RNAs and RNA processing. Prog Nucleic Acid Res Mol Biol. 1983;30:127–162. doi: 10.1016/s0079-6603(08)60685-6. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Smith H. O., Tabiti K., Schaffner G., Soldati D., Albrecht U., Birnstiel M. L. Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9784–9788. doi: 10.1073/pnas.88.21.9784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Soldati D., Schümperli D. Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA. Mol Cell Biol. 1988 Apr;8(4):1518–1524. doi: 10.1128/mcb.8.4.1518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Southgate C., Busslinger M. In vivo and in vitro expression of U7 snRNA genes: cis- and trans-acting elements required for RNA polymerase II-directed transcription. EMBO J. 1989 Feb;8(2):539–549. doi: 10.1002/j.1460-2075.1989.tb03408.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Strub K., Galli G., Busslinger M., Birnstiel M. L. The cDNA sequences of the sea urchin U7 small nuclear RNA suggest specific contacts between histone mRNA precursor and U7 RNA during RNA processing. EMBO J. 1984 Dec 1;3(12):2801–2807. doi: 10.1002/j.1460-2075.1984.tb02212.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Suter-Crazzolara C., Keller W. Organization and transient expression of the gene for human U11 snRNA. Gene Expr. 1991 May;1(2):91–102. [PMC free article] [PubMed] [Google Scholar]
  46. Watkins N. J., Phillips S. C., Turner P. C. The Xenopus U7 snRNA-encoding gene has an unusually compact structure. Gene. 1992 Oct 21;120(2):271–276. doi: 10.1016/0378-1119(92)90104-w. [DOI] [PubMed] [Google Scholar]
  47. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  48. Yang H., Moss M. L., Lund E., Dahlberg J. E. Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes. Mol Cell Biol. 1992 Apr;12(4):1553–1560. doi: 10.1128/mcb.12.4.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zieve G. W., Sauterer R. A. Cell biology of the snRNP particles. Crit Rev Biochem Mol Biol. 1990;25(1):1–46. doi: 10.3109/10409239009090604. [DOI] [PubMed] [Google Scholar]

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