Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Feb;8(2):539–549. doi: 10.1002/j.1460-2075.1989.tb03408.x

In vivo and in vitro expression of U7 snRNA genes: cis- and trans-acting elements required for RNA polymerase II-directed transcription.

C Southgate 1, M Busslinger 1
PMCID: PMC400839  PMID: 2721491

Abstract

Three of five genes coding for U7 small nuclear (sn) RNA of the sea urchin Psammechinus miliaris were shown to be expressed during early embryogenesis by microinjection into sea urchin eggs followed by fertilization. Both in vivo and in nuclear extracts of blastula embryos, a minimal promoter of 80 bp of 5' flanking sequence is essential for their expression. Sequences upstream of position -80 enhance transcription in vivo, but not in vitro, approximately 5-fold. In vitro, transcription initiates at nucleotide +1 of the U7 snRNA and is directed by RNA polymerase II. Protein-DNA binding studies and site-directed mutagenesis demonstrate the presence of multiple proteins interacting with sequences between -57 and -26, which are essential for selection of the correct initiation site and for efficient in vitro transcription. Three of these factors recognize a TATA-like regulatory element between positions -53 and -45, suggesting a role for TATA-binding proteins in the initiation of sea urchin U7 snRNA transcription.

Full text

PDF
539

Images in this article

540Image
on p.540
541Image
on p.541
542Image
on p.542
542Image
on p.542
543Image
on p.543
544Image
on p.544
545Image
on p.545
546Image
on p.546

Selected References

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

  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. Ares M., Jr, Chung J. S., Giglio L., Weiner A. M. Distinct factors with Sp1 and NF-A specificities bind to adjacent functional elements of the human U2 snRNA gene enhancer. Genes Dev. 1987 Oct;1(8):808–817. doi: 10.1101/gad.1.8.808. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Barberis A., Superti-Furga G., Busslinger M. Mutually exclusive interaction of the CCAAT-binding factor and of a displacement protein with overlapping sequences of a histone gene promoter. Cell. 1987 Jul 31;50(3):347–359. doi: 10.1016/0092-8674(87)90489-2. [DOI] [PubMed] [Google Scholar]
  5. Bark C., Weller P., Zabielski J., Janson L., Pettersson U. A distant enhancer element is required for polymerase III transcription of a U6 RNA gene. Nature. 1987 Jul 23;328(6128):356–359. doi: 10.1038/328356a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Bohmann D., Keller W., Dale T., Schöler H. R., Tebb G., Mattaj I. W. A transcription factor which binds to the enhancers of SV40, immunoglobulin heavy chain and U2 snRNA genes. Nature. 1987 Jan 15;325(6101):268–272. doi: 10.1038/325268a0. [DOI] [PubMed] [Google Scholar]
  8. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  9. Brown D. T., Morris G. F., Chodchoy N., Sprecher C., Marzluff W. F. Structure of the sea urchin U1 RNA repeat. Nucleic Acids Res. 1985 Jan 25;13(2):537–556. doi: 10.1093/nar/13.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carbon P., Murgo S., Ebel J. P., Krol A., Tebb G., Mattaj L. W. A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II. Cell. 1987 Oct 9;51(1):71–79. doi: 10.1016/0092-8674(87)90011-0. [DOI] [PubMed] [Google Scholar]
  11. Ciliberto G., Buckland R., Cortese R., Philipson L. Transcription signals in embryonic Xenopus laevis U1 RNA genes. EMBO J. 1985 Jun;4(6):1537–1543. doi: 10.1002/j.1460-2075.1985.tb03814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Das G., Henning D., Wright D., Reddy R. Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. EMBO J. 1988 Feb;7(2):503–512. doi: 10.1002/j.1460-2075.1988.tb02838.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Forbes D. J., Kirschner M. W., Caput D., Dahlberg J. E., Lund E. Differential expression of multiple U1 small nuclear RNAs in oocytes and embryos of Xenopus laevis. Cell. 1984 Oct;38(3):681–689. doi: 10.1016/0092-8674(84)90263-0. [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. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gilmartin G. M., Schaufele F., Schaffner G., Birnstiel M. L. Functional analysis of the sea urchin U7 small nuclear RNA. Mol Cell Biol. 1988 Mar;8(3):1076–1084. doi: 10.1128/mcb.8.3.1076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Janson L., Bark C., Pettersson U. Identification of proteins interacting with the enhancer of human U2 small nuclear RNA genes. Nucleic Acids Res. 1987 Jul 10;15(13):4997–5016. doi: 10.1093/nar/15.13.4997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kazmaier M., Tebb G., Mattaj I. W. Functional characterization of X. laevis U5 snRNA genes. EMBO J. 1987 Oct;6(10):3071–3078. doi: 10.1002/j.1460-2075.1987.tb02614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kemler I., Busslinger M. Characterization of two nonallelic pairs of late histone H2A and H2B genes of the sea urchin: differential regulation in the embryo and tissue-specific expression in the adult. Mol Cell Biol. 1986 Nov;6(11):3746–3754. doi: 10.1128/mcb.6.11.3746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krol A., Carbon P., Ebel J. P., Appel B. Xenopus tropicalis U6 snRNA genes transcribed by Pol III contain the upstream promoter elements used by Pol II dependent U snRNA genes. Nucleic Acids Res. 1987 Mar 25;15(6):2463–2478. doi: 10.1093/nar/15.6.2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Lund E., Bostock C. J., Dahlberg J. E. The transcription of Xenopus laevis embryonic U1 snRNA genes changes when oocytes mature into eggs. Genes Dev. 1987 Mar;1(1):47–56. doi: 10.1101/gad.1.1.47. [DOI] [PubMed] [Google Scholar]
  28. Lund E., Dahlberg J. E. Differential accumulation of U1 and U4 small nuclear RNAs during Xenopus development. Genes Dev. 1987 Mar;1(1):39–46. doi: 10.1101/gad.1.1.39. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Mattaj I. W., Lienhard S., Jiricny J., De Robertis E. M. An enhancer-like sequence within the Xenopus U2 gene promoter facilitates the formation of stable transcription complexes. Nature. 1985 Jul 11;316(6024):163–167. doi: 10.1038/316163a0. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. McMahon A. P., Flytzanis C. N., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. doi: 10.1016/0012-1606(85)90045-4. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Morris G. F., Marzluff W. F. Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA. Mol Cell Biol. 1985 May;5(5):1143–1150. doi: 10.1128/mcb.5.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Morris G. F., Price D. H., Marzluff W. F. Synthesis of U1 RNA in a DNA-dependent system from sea urchin embryos. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3674–3678. doi: 10.1073/pnas.83.11.3674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Murphy J. T., Skuzeski J. T., Lund E., Steinberg T. H., Burgess R. R., Dahlberg J. E. Functional elements of the human U1 RNA promoter. Identification of five separate regions required for efficient transcription and template competition. J Biol Chem. 1987 Feb 5;262(4):1795–1803. [PubMed] [Google Scholar]
  41. Müller M. M., Gerster T., Schaffner W. Enhancer sequences and the regulation of gene transcription. Eur J Biochem. 1988 Oct 1;176(3):485–495. doi: 10.1111/j.1432-1033.1988.tb14306.x. [DOI] [PubMed] [Google Scholar]
  42. Nijhawan P., Marzluff W. F. Metabolism of low molecular weight ribonucleic acids in early sea urchin embryos. Biochemistry. 1979 Apr 3;18(7):1353–1360. doi: 10.1021/bi00574a035. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Roebuck K. A., Walker R. J., Stumph W. E. Multiple functional motifs in the chicken U1 RNA gene enhancer. Mol Cell Biol. 1987 Dec;7(12):4185–4193. doi: 10.1128/mcb.7.12.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Saba J. A., Busch H., Wright D., Reddy R. Isolation and characterization of two putative full-length Drosophila U4 small nuclear RNA genes. J Biol Chem. 1986 Jul 5;261(19):8750–8753. [PubMed] [Google Scholar]
  47. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  48. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sive H. L., Roeder R. G. Interaction of a common factor with conserved promoter and enhancer sequences in histone H2B, immunoglobulin, and U2 small nuclear RNA (snRNA) genes. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6382–6386. doi: 10.1073/pnas.83.17.6382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Strub K., Birnstiel M. L. Genetic complementation in the Xenopus oocyte: co-expression of sea urchin histone and U7 RNAs restores 3' processing of H3 pre-mRNA in the oocyte. EMBO J. 1986 Jul;5(7):1675–1682. doi: 10.1002/j.1460-2075.1986.tb04411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Tebb G., Bohmann D., Mattaj I. W. Only two of the four sites of interaction with nuclear factors within the Xenopus U2 gene promoter are necessary for efficient transcription. Nucleic Acids Res. 1987 Aug 25;15(16):6437–6453. doi: 10.1093/nar/15.16.6437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vitelli L., Kemler I., Lauber B., Birnstiel M. L., Busslinger M. Developmental regulation of micro-injected histone genes in sea urchin embryos. Dev Biol. 1988 May;127(1):54–63. doi: 10.1016/0012-1606(88)90188-1. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Yu J. C., Nash M. A., Santiago C., Marzluff W. F. Structure and expression of a second sea urchin U1 RNA gene repeat. Nucleic Acids Res. 1986 Dec 22;14(24):9977–9988. doi: 10.1093/nar/14.24.9977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES