Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 May;16(5):2204–2213. doi: 10.1128/mcb.16.5.2204

Pre-mRNA topology is important for 3'-end formation in Saccharomyces cerevisiae and mammals.

G Stumpf 1, A Goppelt 1, H Domdey 1
PMCID: PMC231208  PMID: 8628287

Abstract

Various signal motifs that are required for efficient pre-mRNA 3'-end formation in the yeast Saccharomyces cerevisiae have been reported. None of these known signal sequences appears to be of the same general importance as is the mammalian AAUAAA motif. To establish the importance of yeast pre-mRNA termini in 3'-end formation, the ends of a pre-mRNA transcript synthesized in vitro were ligated before incubation in a yeast whole-cell extract. Such covalently closed circular RNAs were not cleaved at their poly(A) sites. Interestingly, pseudocircular RNAs with complementary 3'- and 5'-terminal sequences allowing the formation of panhandle structures were also resistant to cleavage. However, 3'-end processing was impeded neither by terminal hairpins at either or at both ends nor by RNA oligonucleotides complementary to either or both ends of a linear pre-mRNA. Intriguingly mammalian pseudocircular pre-mRNAs also were not cleaved at their poly(A) sites when incubated in a HeLa cell nuclear extract. These results provide evidence for the general importance of RNA topology in the formation of an active 3'-end processing complex in S. cerevisiae and higher eukaryotes. The possibility of a torus-shaped factor involved in 3'-end formation is discussed.

Full Text

The Full Text of this article is available as a PDF (490.3 KB).

Selected References

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

  1. Abe A., Hiraoka Y., Fukasawa T. Signal sequence for generation of mRNA 3' end in the Saccharomyces cerevisiae GAL7 gene. EMBO J. 1990 Nov;9(11):3691–3697. doi: 10.1002/j.1460-2075.1990.tb07581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahmed Y. F., Gilmartin G. M., Hanly S. M., Nevins J. R., Greene W. C. The HTLV-I Rex response element mediates a novel form of mRNA polyadenylation. Cell. 1991 Feb 22;64(4):727–737. doi: 10.1016/0092-8674(91)90502-p. [DOI] [PubMed] [Google Scholar]
  3. Brown P. H., Tiley L. S., Cullen B. R. Effect of RNA secondary structure on polyadenylation site selection. Genes Dev. 1991 Jul;5(7):1277–1284. doi: 10.1101/gad.5.7.1277. [DOI] [PubMed] [Google Scholar]
  4. Burgers P. M., Yoder B. L. ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends. J Biol Chem. 1993 Sep 25;268(27):19923–19926. [PubMed] [Google Scholar]
  5. Butler J. S., Platt T. RNA processing generates the mature 3' end of yeast CYC1 messenger RNA in vitro. Science. 1988 Dec 2;242(4883):1270–1274. doi: 10.1126/science.2848317. [DOI] [PubMed] [Google Scholar]
  6. Butler J. S., Sadhale P. P., Platt T. RNA processing in vitro produces mature 3' ends of a variety of Saccharomyces cerevisiae mRNAs. Mol Cell Biol. 1990 Jun;10(6):2599–2605. doi: 10.1128/mcb.10.6.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C. Y., Sarnow P. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science. 1995 Apr 21;268(5209):415–417. doi: 10.1126/science.7536344. [DOI] [PubMed] [Google Scholar]
  8. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  9. Chen J., Moore C. Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA. Mol Cell Biol. 1992 Aug;12(8):3470–3481. doi: 10.1128/mcb.12.8.3470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dandekar T., Hentze M. W. Finding the hairpin in the haystack: searching for RNA motifs. Trends Genet. 1995 Feb;11(2):45–50. doi: 10.1016/s0168-9525(00)88996-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
  13. Dominski Z., Kole R. Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing. Mol Cell Biol. 1994 Nov;14(11):7445–7454. doi: 10.1128/mcb.14.11.7445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gil A., Proudfoot N. J. A sequence downstream of AAUAAA is required for rabbit beta-globin mRNA 3'-end formation. 1984 Nov 29-Dec 5Nature. 312(5993):473–474. doi: 10.1038/312473a0. [DOI] [PubMed] [Google Scholar]
  15. Gil A., Proudfoot N. J. Position-dependent sequence elements downstream of AAUAAA are required for efficient rabbit beta-globin mRNA 3' end formation. Cell. 1987 May 8;49(3):399–406. doi: 10.1016/0092-8674(87)90292-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Goodall G. J., Wiebauer K., Filipowicz W. Analysis of pre-mRNA processing in transfected plant protoplasts. Methods Enzymol. 1990;181:148–161. doi: 10.1016/0076-6879(90)81117-d. [DOI] [PubMed] [Google Scholar]
  18. Gunderson S. I., Beyer K., Martin G., Keller W., Boelens W. C., Mattaj L. W. The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase. Cell. 1994 Feb 11;76(3):531–541. doi: 10.1016/0092-8674(94)90116-3. [DOI] [PubMed] [Google Scholar]
  19. Guo Z., Sherman F. 3'-end-forming signals of yeast mRNA. Mol Cell Biol. 1995 Nov;15(11):5983–5990. doi: 10.1128/mcb.15.11.5983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heidmann S., Obermaier B., Vogel K., Domdey H. Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Sep;12(9):4215–4229. doi: 10.1128/mcb.12.9.4215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heidmann S., Schindewolf C., Stumpf G., Domdey H. Flexibility and interchangeability of polyadenylation signals in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Jul;14(7):4633–4642. doi: 10.1128/mcb.14.7.4633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Henikoff S., Cohen E. H. Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1515–1520. doi: 10.1128/mcb.4.8.1515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Herendeen D. R., Kelly T. J. DNA polymerase III: running rings around the fork. Cell. 1996 Jan 12;84(1):5–8. doi: 10.1016/s0092-8674(00)80069-0. [DOI] [PubMed] [Google Scholar]
  24. Humphrey T., Christofori G., Lucijanic V., Keller W. Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes. EMBO J. 1987 Dec 20;6(13):4159–4168. doi: 10.1002/j.1460-2075.1987.tb02762.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hyman L. E., Seiler S. H., Whoriskey J., Moore C. L. Point mutations upstream of the yeast ADH2 poly(A) site significantly reduce the efficiency of 3'-end formation. Mol Cell Biol. 1991 Apr;11(4):2004–2012. doi: 10.1128/mcb.11.4.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Irniger S., Egli C. M., Braus G. H. Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3060–3069. doi: 10.1128/mcb.11.6.3060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Irniger S., Sanfaçon H., Egli C. M., Braus G. H. Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants. Mol Cell Biol. 1992 May;12(5):2322–2330. doi: 10.1128/mcb.12.5.2322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Keller W. No end yet to messenger RNA 3' processing! Cell. 1995 Jun 16;81(6):829–832. doi: 10.1016/0092-8674(95)90001-2. [DOI] [PubMed] [Google Scholar]
  29. Konarska M., Filipowicz W., Domdey H., Gross H. J. Binding of ribosomes to linear and circular forms of the 5'-terminal leader fragment of tobacco-mosaic-virus RNA. Eur J Biochem. 1981 Feb;114(2):221–227. doi: 10.1111/j.1432-1033.1981.tb05139.x. [DOI] [PubMed] [Google Scholar]
  30. Kondrakhin YuV, Shamin V. V., Kolchanov N. A. Construction of a generalized consensus matrix for recognition of vertebrate pre-mRNA 3'-terminal processing sites. Comput Appl Biosci. 1994 Dec;10(6):597–603. doi: 10.1093/bioinformatics/10.6.597. [DOI] [PubMed] [Google Scholar]
  31. Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
  32. Kozak M. Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989 Nov;9(11):5134–5142. doi: 10.1128/mcb.9.11.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kozak M. Inability of circular mRNA to attach to eukaryotic ribosomes. Nature. 1979 Jul 5;280(5717):82–85. doi: 10.1038/280082a0. [DOI] [PubMed] [Google Scholar]
  34. Kuriyan J., O'Donnell M. Sliding clamps of DNA polymerases. J Mol Biol. 1993 Dec 20;234(4):915–925. doi: 10.1006/jmbi.1993.1644. [DOI] [PubMed] [Google Scholar]
  35. McDevitt M. A., Hart R. P., Wong W. W., Nevins J. R. Sequences capable of restoring poly(A) site function define two distinct downstream elements. EMBO J. 1986 Nov;5(11):2907–2913. doi: 10.1002/j.1460-2075.1986.tb04586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McDevitt M. A., Imperiale M. J., Ali H., Nevins J. R. Requirement of a downstream sequence for generation of a poly(A) addition site. Cell. 1984 Jul;37(3):993–999. doi: 10.1016/0092-8674(84)90433-1. [DOI] [PubMed] [Google Scholar]
  37. McLauchlan J., Gaffney D., Whitton J. L., Clements J. B. The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini. Nucleic Acids Res. 1985 Feb 25;13(4):1347–1368. doi: 10.1093/nar/13.4.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Minvielle-Sebastia L., Preker P. J., Keller W. RNA14 and RNA15 proteins as components of a yeast pre-mRNA 3'-end processing factor. Science. 1994 Dec 9;266(5191):1702–1705. doi: 10.1126/science.7992054. [DOI] [PubMed] [Google Scholar]
  40. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  41. Orkin S. H., Cheng T. C., Antonarakis S. E., Kazazian H. H., Jr Thalassemia due to a mutation in the cleavage-polyadenylation signal of the human beta-globin gene. EMBO J. 1985 Feb;4(2):453–456. doi: 10.1002/j.1460-2075.1985.tb03650.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  43. Proudfoot N. J., Brownlee G. G. Sequence at the 3' end of globin mRNA shows homology with immunoglobulin light chain mRNA. Nature. 1974 Nov 29;252(5482):359–362. doi: 10.1038/252359a0. [DOI] [PubMed] [Google Scholar]
  44. Proudfoot N. Poly(A) signals. Cell. 1991 Feb 22;64(4):671–674. doi: 10.1016/0092-8674(91)90495-k. [DOI] [PubMed] [Google Scholar]
  45. Russo P., Li W. Z., Hampsey D. M., Zaret K. S., Sherman F. Distinct cis-acting signals enhance 3' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae. EMBO J. 1991 Mar;10(3):563–571. doi: 10.1002/j.1460-2075.1991.tb07983.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sadhale P. P., Platt T. Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4262–4270. doi: 10.1128/mcb.12.10.4262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schindewolf C. A., Domdey H. Splicing of a circular yeast pre-mRNA in vitro. Nucleic Acids Res. 1995 Apr 11;23(7):1133–1139. doi: 10.1093/nar/23.7.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stukenberg P. T., Studwell-Vaughan P. S., O'Donnell M. Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem. 1991 Jun 15;266(17):11328–11334. [PubMed] [Google Scholar]
  49. Wahle E., Keller W. RNA-protein interactions in mRNA 3'-end formation. Mol Biol Rep. 1993 Aug;18(2):157–161. doi: 10.1007/BF00986771. [DOI] [PubMed] [Google Scholar]
  50. Wahle E., Keller W. The biochemistry of 3'-end cleavage and polyadenylation of messenger RNA precursors. Annu Rev Biochem. 1992;61:419–440. doi: 10.1146/annurev.bi.61.070192.002223. [DOI] [PubMed] [Google Scholar]
  51. Yisraeli J. K., Melton D. A. Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol. 1989;180:42–50. doi: 10.1016/0076-6879(89)80090-4. [DOI] [PubMed] [Google Scholar]
  52. Zaret K. S., Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. doi: 10.1016/0092-8674(82)90211-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES