Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 May;11(5):2432–2438. doi: 10.1128/mcb.11.5.2432

Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA.

G M Gilmartin 1, J R Nevins 1
PMCID: PMC360003  PMID: 2017162

Abstract

Poly(A) site processing of a pre-mRNA requires the participation of multiple nuclear factors. Two of these factors recognize specific sequences in the pre-mRNA and form a stable processing complex. Since these initial interactions are likely critical for the recognition of the poly(A) site and the efficiency of poly(A) site use, we have characterized these factors and the nature of their interaction with the pre-mRNA. The AAUAAA specificity factor PF2 is a large, multicomponent complex composed of at least five distinct polypeptides ranging in molecular size from 170 to 42 kDa. The 170-kDa polypeptide appears to mediate interaction with the pre-mRNA. Factor CF1, which provides specificity for the downstream G + U-rich element and stabilizes the PF2 interaction on the RNA, is also a multicomponent complex but is less complex than PF2. CF1 is composed of three polypeptides of molecular sizes 76, 64, and 48 kDa. UV cross-linking assays demonstrate that the 64-kDa polypeptide makes direct contact with the RNA, dependent on the G + U-rich downstream sequence element. Moreover, it is clear that these RNA-protein interactions are influenced by the apparent cooperative interaction involving PF2 and CF1, interactions that contribute to the efficiency of poly(A) site processing.

Full text

PDF
2432

Images in this article

2434Image
on p.2434
2435Image
on p.2435
2436Image
on p.2436

Selected References

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

  1. 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]
  2. Carswell S., Alwine J. C. Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences. Mol Cell Biol. 1989 Oct;9(10):4248–4258. doi: 10.1128/mcb.9.10.4248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Christofori G., Keller W. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell. 1988 Sep 9;54(6):875–889. doi: 10.1016/s0092-8674(88)91263-9. [DOI] [PubMed] [Google Scholar]
  4. Conway L., Wickens M. A sequence downstream of A-A-U-A-A-A is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3949–3953. doi: 10.1073/pnas.82.12.3949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeZazzo J. D., Imperiale M. J. Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit. Mol Cell Biol. 1989 Nov;9(11):4951–4961. doi: 10.1128/mcb.9.11.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeZazzo J. D., Kilpatrick J. E., Imperiale M. J. Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation. Mol Cell Biol. 1991 Mar;11(3):1624–1630. doi: 10.1128/mcb.11.3.1624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Falck-Pedersen E., Logan J. Regulation of poly(A) site selection in adenovirus. J Virol. 1989 Feb;63(2):532–541. doi: 10.1128/jvi.63.2.532-541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fitzgerald M., Shenk T. The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs. Cell. 1981 Apr;24(1):251–260. doi: 10.1016/0092-8674(81)90521-3. [DOI] [PubMed] [Google Scholar]
  10. Galli G., Guise J. W., McDevitt M. A., Tucker P. W., Nevins J. R. Relative position and strengths of poly(A) sites as well as transcription termination are critical to membrane versus secreted mu-chain expression during B-cell development. Genes Dev. 1987 Jul;1(5):471–481. doi: 10.1101/gad.1.5.471. [DOI] [PubMed] [Google Scholar]
  11. Galli G., Guise J., Tucker P. W., Nevins J. R. Poly(A) site choice rather than splice site choice governs the regulated production of IgM heavy-chain RNAs. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2439–2443. doi: 10.1073/pnas.85.8.2439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gilmartin G. M., McDevitt M. A., Nevins J. R. Multiple factors are required for specific RNA cleavage at a poly(A) addition site. Genes Dev. 1988 May;2(5):578–587. doi: 10.1101/gad.2.5.578. [DOI] [PubMed] [Google Scholar]
  15. Gilmartin G. M., Nevins J. R. An ordered pathway of assembly of components required for polyadenylation site recognition and processing. Genes Dev. 1989 Dec;3(12B):2180–2190. doi: 10.1101/gad.3.12b.2180. [DOI] [PubMed] [Google Scholar]
  16. Heath C. V., Denome R. M., Cole C. N. Spatial constraints on polyadenylation signal function. J Biol Chem. 1990 Jun 5;265(16):9098–9104. [PubMed] [Google Scholar]
  17. Leff S. E., Rosenfeld M. G., Evans R. M. Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem. 1986;55:1091–1117. doi: 10.1146/annurev.bi.55.070186.005303. [DOI] [PubMed] [Google Scholar]
  18. Lutz-Freyermuth C., Keene J. D., Lutz-Reyermuth C. The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs. Mol Cell Biol. 1989 Jul;9(7):2975–2982. doi: 10.1128/mcb.9.7.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McDevitt M. A., Gilmartin G. M., Reeves W. H., Nevins J. R. Multiple factors are required for poly(A) addition to a mRNA 3' end. Genes Dev. 1988 May;2(5):588–597. doi: 10.1101/gad.2.5.588. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Inhibition of RNA cleavage but not polyadenylation by a point mutation in mRNA 3' consensus sequence AAUAAA. Nature. 1983 Oct 13;305(5935):600–605. doi: 10.1038/305600a0. [DOI] [PubMed] [Google Scholar]
  24. Moore C. L., Chen J., Whoriskey J. Two proteins crosslinked to RNA containing the adenovirus L3 poly(A) site require the AAUAAA sequence for binding. EMBO J. 1988 Oct;7(10):3159–3169. doi: 10.1002/j.1460-2075.1988.tb03183.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Nevins J. R. The pathway of eukaryotic mRNA formation. Annu Rev Biochem. 1983;52:441–466. doi: 10.1146/annurev.bi.52.070183.002301. [DOI] [PubMed] [Google Scholar]
  27. Nevins J. R., Wilson M. C. Regulation of adenovirus-2 gene expression at the level of transcriptional termination and RNA processing. Nature. 1981 Mar 12;290(5802):113–118. doi: 10.1038/290113a0. [DOI] [PubMed] [Google Scholar]
  28. Peterson M. L., Perry R. P. The regulated production of mu m and mu s mRNA is dependent on the relative efficiencies of mu s poly(A) site usage and the c mu 4-to-M1 splice. Mol Cell Biol. 1989 Feb;9(2):726–738. doi: 10.1128/mcb.9.2.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  30. Russnak R., Ganem D. Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses. Genes Dev. 1990 May;4(5):764–776. doi: 10.1101/gad.4.5.764. [DOI] [PubMed] [Google Scholar]
  31. Sadofsky M., Alwine J. C. Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site. Mol Cell Biol. 1984 Aug;4(8):1460–1468. doi: 10.1128/mcb.4.8.1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simonsen C. C., Levinson A. D. Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids. Mol Cell Biol. 1983 Dec;3(12):2250–2258. doi: 10.1128/mcb.3.12.2250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  34. Takagaki Y., Manley J. L., MacDonald C. C., Wilusz J., Shenk T. A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev. 1990 Dec;4(12A):2112–2120. doi: 10.1101/gad.4.12a.2112. [DOI] [PubMed] [Google Scholar]
  35. Takagaki Y., Ryner L. C., Manley J. L. Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell. 1988 Mar 11;52(5):731–742. doi: 10.1016/0092-8674(88)90411-4. [DOI] [PubMed] [Google Scholar]
  36. Wilusz J., Feig D. I., Shenk T. The C proteins of heterogeneous nuclear ribonucleoprotein complexes interact with RNA sequences downstream of polyadenylation cleavage sites. Mol Cell Biol. 1988 Oct;8(10):4477–4483. doi: 10.1128/mcb.8.10.4477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wilusz J., Shenk T. A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell. 1988 Jan 29;52(2):221–228. doi: 10.1016/0092-8674(88)90510-7. [DOI] [PubMed] [Google Scholar]
  38. Wilusz J., Shenk T. A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal. Mol Cell Biol. 1990 Dec;10(12):6397–6407. doi: 10.1128/mcb.10.12.6397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilusz J., Shenk T., Takagaki Y., Manley J. L. A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates. Mol Cell Biol. 1990 Mar;10(3):1244–1248. doi: 10.1128/mcb.10.3.1244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zarkower D., Stephenson P., Sheets M., Wickens M. The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro. Mol Cell Biol. 1986 Jul;6(7):2317–2323. doi: 10.1128/mcb.6.7.2317. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES