Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Feb 15;89(4):1403–1407. doi: 10.1073/pnas.89.4.1403

The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs.

Y Takagaki 1, C C MacDonald 1, T Shenk 1, J L Manley 1
PMCID: PMC48459  PMID: 1741396

Abstract

Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted. An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs.

Full text

PDF
1403

Images in this article

1404Image
on p.1404
1404Image
on p.1404
1404Image
on p.1404
1404Image
on p.1404
1407Image
on p.1407

Selected References

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

  1. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  2. Bardwell V. J., Zarkower D., Edmonds M., Wickens M. The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase. Mol Cell Biol. 1990 Feb;10(2):846–849. doi: 10.1128/mcb.10.2.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackwood E. M., Eisenman R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991 Mar 8;251(4998):1211–1217. doi: 10.1126/science.2006410. [DOI] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [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. 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]
  9. Gentz R., Chen C. H., Rosen C. A. Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis. Proc Natl Acad Sci U S A. 1989 Feb;86(3):821–824. doi: 10.1073/pnas.86.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Gilmartin G. M., Nevins J. R. Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA. Mol Cell Biol. 1991 May;11(5):2432–2438. doi: 10.1128/mcb.11.5.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  13. Higgs D. R., Goodbourn S. E., Lamb J., Clegg J. B., Weatherall D. J., Proudfoot N. J. Alpha-thalassaemia caused by a polyadenylation signal mutation. Nature. 1983 Nov 24;306(5941):398–400. doi: 10.1038/306398a0. [DOI] [PubMed] [Google Scholar]
  14. Horikoshi M., Wang C. K., Fujii H., Cromlish J. A., Weil P. A., Roeder R. G. Cloning and structure of a yeast gene encoding a general transcription initiation factor TFIID that binds to the TATA box. Nature. 1989 Sep 28;341(6240):299–303. doi: 10.1038/341299a0. [DOI] [PubMed] [Google Scholar]
  15. Humphrey T., Proudfoot N. J. A beginning to the biochemistry of polyadenylation. Trends Genet. 1988 Sep;4(9):243–245. doi: 10.1016/0168-9525(88)90028-5. [DOI] [PubMed] [Google Scholar]
  16. Jagus R. Hybrid selection of mRNA and hybrid arrest of translation. Methods Enzymol. 1987;152:567–572. doi: 10.1016/0076-6879(87)52063-8. [DOI] [PubMed] [Google Scholar]
  17. Kemp B. E., Pearson R. B. Protein kinase recognition sequence motifs. Trends Biochem Sci. 1990 Sep;15(9):342–346. doi: 10.1016/0968-0004(90)90073-k. [DOI] [PubMed] [Google Scholar]
  18. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  23. Manley J. L. Polyadenylation of mRNA precursors. Biochim Biophys Acta. 1988 May 6;950(1):1–12. doi: 10.1016/0167-4781(88)90067-x. [DOI] [PubMed] [Google Scholar]
  24. Manley J. L., Sharp P. A., Gefter M. L. Rna synthesis in isolated nuclei processing of adenovirus serotype 2 late messenger rna precursors. J Mol Biol. 1982 Aug 25;159(4):581–599. doi: 10.1016/0022-2836(82)90102-4. [DOI] [PubMed] [Google Scholar]
  25. Marqusee S., Baldwin R. L. Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8898–8902. doi: 10.1073/pnas.84.24.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Moore C. L., Skolnik-David H., Sharp P. A. Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site. EMBO J. 1986 Aug;5(8):1929–1938. doi: 10.1002/j.1460-2075.1986.tb04446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  29. Nevins J. R., Darnell J. E., Jr Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing. Cell. 1978 Dec;15(4):1477–1493. doi: 10.1016/0092-8674(78)90071-5. [DOI] [PubMed] [Google Scholar]
  30. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prendergast G. C., Ziff E. B. DNA-binding motif. Nature. 1989 Oct 5;341(6241):392–392. doi: 10.1038/341392a0. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Ptashne M. How eukaryotic transcriptional activators work. Nature. 1988 Oct 20;335(6192):683–689. doi: 10.1038/335683a0. [DOI] [PubMed] [Google Scholar]
  35. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  36. Ryner L. C., Takagaki Y., Manley J. L. Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation. Mol Cell Biol. 1989 Oct;9(10):4229–4238. doi: 10.1128/mcb.9.10.4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Sheets M. D., Stephenson P., Wickens M. P. Products of in vitro cleavage and polyadenylation of simian virus 40 late pre-mRNAs. Mol Cell Biol. 1987 Apr;7(4):1518–1529. doi: 10.1128/mcb.7.4.1518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
  40. Sundaralingam M., Drendel W., Greaser M. Stabilization of the long central helix of troponin C by intrahelical salt bridges between charged amino acid side chains. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7944–7947. doi: 10.1073/pnas.82.23.7944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Takagaki Y., Ryner L. C., Manley J. L. Four factors are required for 3'-end cleavage of pre-mRNAs. Genes Dev. 1989 Nov;3(11):1711–1724. doi: 10.1101/gad.3.11.1711. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Terns M. P., Jacob S. T. Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor. Mol Cell Biol. 1989 Apr;9(4):1435–1444. doi: 10.1128/mcb.9.4.1435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vuorio E., de Crombrugghe B. The family of collagen genes. Annu Rev Biochem. 1990;59:837–872. doi: 10.1146/annurev.bi.59.070190.004201. [DOI] [PubMed] [Google Scholar]
  46. Weiss E. A., Gilmartin G. M., Nevins J. R. Poly(A) site efficiency reflects the stability of complex formation involving the downstream element. EMBO J. 1991 Jan;10(1):215–219. doi: 10.1002/j.1460-2075.1991.tb07938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Williams T., Tjian R. Characterization of a dimerization motif in AP-2 and its function in heterologous DNA-binding proteins. Science. 1991 Mar 1;251(4997):1067–1071. doi: 10.1126/science.1998122. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES