Abstract
Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adam S. A., Nakagawa T., Swanson M. S., Woodruff T. K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol. 1986 Aug;6(8):2932–2943. doi: 10.1128/mcb.6.8.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Augenlicht L. H. Protected nucleotide sequences in nuclear ribonucleoprotein. Biochemistry. 1979 Aug 21;18(17):3780–3786. doi: 10.1021/bi00584a022. [DOI] [PubMed] [Google Scholar]
- Banerjee A. K. Transcription and replication of rhabdoviruses. Microbiol Rev. 1987 Mar;51(1):66–87. doi: 10.1128/mr.51.1.66-87.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barnett S. F., Friedman D. L., LeStourgeon W. M. The C proteins of HeLa 40S nuclear ribonucleoprotein particles exist as anisotropic tetramers of (C1)3 C2. Mol Cell Biol. 1989 Feb;9(2):492–498. doi: 10.1128/mcb.9.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beyer A. L., Bouton A. H., Miller O. L., Jr Correlation of hnRNP structure and nascent transcript cleavage. Cell. 1981 Oct;26(2 Pt 2):155–165. doi: 10.1016/0092-8674(81)90299-3. [DOI] [PubMed] [Google Scholar]
- Beyer A. L., Christensen M. E., Walker B. W., LeStourgeon W. M. Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell. 1977 May;11(1):127–138. doi: 10.1016/0092-8674(77)90323-3. [DOI] [PubMed] [Google Scholar]
- Beyer A. L., Miller O. L., Jr, McKnight S. L. Ribonucleoprotein structure in nascent hnRNA is nonrandom and sequence-dependent. Cell. 1980 May;20(1):75–84. doi: 10.1016/0092-8674(80)90236-6. [DOI] [PubMed] [Google Scholar]
- Billings P. B., Martin T. E. Proteins of nuclear ribonucleoprotein subcomplexes. Methods Cell Biol. 1978;17:349–376. doi: 10.1016/s0091-679x(08)61154-1. [DOI] [PubMed] [Google Scholar]
- Burd C. G., Swanson M. S., Görlach M., Dreyfuss G. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9788–9792. doi: 10.1073/pnas.86.24.9788. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Choi Y. D., Dreyfuss G. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7471–7475. doi: 10.1073/pnas.81.23.7471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Choi Y. D., Grabowski P. J., Sharp P. A., Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. doi: 10.1126/science.3952495. [DOI] [PubMed] [Google Scholar]
- Cobianchi F., Karpel R. L., Williams K. R., Notario V., Wilson S. H. Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids. J Biol Chem. 1988 Jan 15;263(2):1063–1071. [PubMed] [Google Scholar]
- Cobianchi F., SenGupta D. N., Zmudzka B. Z., Wilson S. H. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem. 1986 Mar 15;261(8):3536–3543. [PubMed] [Google Scholar]
- Cole C. N., Stacy T. P. Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation. Mol Cell Biol. 1985 Aug;5(8):2104–2113. doi: 10.1128/mcb.5.8.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Conway G., Wooley J., Bibring T., LeStourgeon W. M. Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles. Mol Cell Biol. 1988 Jul;8(7):2884–2895. doi: 10.1128/mcb.8.7.2884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Dreyfuss G., Choi Y. D., Adam S. A. Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol Cell Biol. 1984 Jun;4(6):1104–1114. doi: 10.1128/mcb.4.6.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dreyfuss G. Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu Rev Cell Biol. 1986;2:459–498. doi: 10.1146/annurev.cb.02.110186.002331. [DOI] [PubMed] [Google Scholar]
- Dreyfuss G., Swanson M. S., Piñol-Roma S. Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci. 1988 Mar;13(3):86–91. doi: 10.1016/0968-0004(88)90046-1. [DOI] [PubMed] [Google Scholar]
- Economidis I. V., Pederson T. Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1599–1602. doi: 10.1073/pnas.80.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gerke V., Steitz J. A. A protein associated with small nuclear ribonucleoprotein particles recognizes the 3' splice site of premessenger RNA. Cell. 1986 Dec 26;47(6):973–984. doi: 10.1016/0092-8674(86)90812-3. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Hart R. P., McDevitt M. A., Ali H., Nevins J. R. Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites. Mol Cell Biol. 1985 Nov;5(11):2975–2983. doi: 10.1128/mcb.5.11.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hart R. P., McDevitt M. A., Nevins J. R. Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences. Cell. 1985 Dec;43(3 Pt 2):677–683. doi: 10.1016/0092-8674(85)90240-5. [DOI] [PubMed] [Google Scholar]
- Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
- Holcomb E. R., Friedman D. L. Phosphorylation of the C-proteins of HeLa cell hnRNP particles. Involvement of a casein kinase II-type enzyme. J Biol Chem. 1984 Jan 10;259(1):31–40. [PubMed] [Google Scholar]
- Kumar A., Williams K. R., Szer W. Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins. J Biol Chem. 1986 Aug 25;261(24):11266–11273. [PubMed] [Google Scholar]
- Lothstein L., Arenstorf H. P., Chung S. Y., Walker B. W., Wooley J. C., LeStourgeon W. M. General organization of protein in HeLa 40S nuclear ribonucleoprotein particles. J Cell Biol. 1985 May;100(5):1570–1581. doi: 10.1083/jcb.100.5.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Malcolm D. B., Sommerville J. The structure of nuclear ribonucleoprotein of amphibian oocytes. J Cell Sci. 1977 Apr;24:143–165. doi: 10.1242/jcs.24.1.143. [DOI] [PubMed] [Google Scholar]
- Mason P. J., Elkington J. A., Lloyd M. M., Jones M. B., Williams J. G. Mutations downstream of the polyadenylation site of a Xenopus beta-globin mRNA affect the position but not the efficiency of 3' processing. Cell. 1986 Jul 18;46(2):263–270. doi: 10.1016/0092-8674(86)90743-9. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- McKnight S. L., Miller O. L., Jr Ultrastructural patterns of RNA synthesis during early embryogenesis of Drosophila melanogaster. Cell. 1976 Jun;8(2):305–319. doi: 10.1016/0092-8674(76)90014-3. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Merrill B. M., Barnett S. F., LeStourgeon W. M., Williams K. R. Primary structure differences between proteins C1 and C2 of HeLa 40S nuclear ribonucleoprotein particles. Nucleic Acids Res. 1989 Nov 11;17(21):8441–8449. doi: 10.1093/nar/17.21.8441. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Merrill B. M., LoPresti M. B., Stone K. L., Williams K. R. High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus. J Biol Chem. 1986 Jan 15;261(2):878–883. [PubMed] [Google Scholar]
- 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]
- 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]
- Ohlsson R. I., van Eekelen C., Philipson L. Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor. Nucleic Acids Res. 1982 May 25;10(10):3053–3068. doi: 10.1093/nar/10.10.3053. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osheim Y. N., Miller O. L., Jr, Beyer A. L. RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell. 1985 Nov;43(1):143–151. doi: 10.1016/0092-8674(85)90019-4. [DOI] [PubMed] [Google Scholar]
- Patton J. R., Chae C. B. Specific regions of the intervening sequences of beta-globin RNA are resistant to nuclease in 50S heterogeneous nuclear RNA-protein complexes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8414–8418. doi: 10.1073/pnas.82.24.8414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piñol-Roma S., Choi Y. D., Matunis M. J., Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 1988 Feb;2(2):215–227. doi: 10.1101/gad.2.2.215. [DOI] [PubMed] [Google Scholar]
- Preugschat F., Wold B. Isolation and characterization of a Xenopus laevis C protein cDNA: structure and expression of a heterogeneous nuclear ribonucleoprotein core protein. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9669–9673. doi: 10.1073/pnas.85.24.9669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pullman J. M., Martin T. E. Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins. J Cell Biol. 1983 Jul;97(1):99–111. doi: 10.1083/jcb.97.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
- Ryner L. C., Takagaki Y., Manley J. L. Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly. Mol Cell Biol. 1989 Apr;9(4):1759–1771. doi: 10.1128/mcb.9.4.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Sadofsky M., Connelly S., Manley J. L., Alwine J. C. Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing. Mol Cell Biol. 1985 Oct;5(10):2713–2719. doi: 10.1128/mcb.5.10.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sierakowska H., Szer W., Furdon P. J., Kole R. Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA. Nucleic Acids Res. 1986 Jul 11;14(13):5241–5254. doi: 10.1093/nar/14.13.5241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sosnowski B. A., Belote J. M., McKeown M. Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell. 1989 Aug 11;58(3):449–459. doi: 10.1016/0092-8674(89)90426-1. [DOI] [PubMed] [Google Scholar]
- Steitz J. A., Kamen R. Arrangement of 30S heterogeneous nuclear ribonucleoprotein on polyoma virus late nuclear transcripts. Mol Cell Biol. 1981 Jan;1(1):21–34. doi: 10.1128/mcb.1.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stévenin J., Gattoni R., Keohavong P., Jacob M. Mild nuclease treatment as a probe for a non-random distribution of adenovirus-specific RNA sequences and of cellular RNA in nuclear ribonucleoprotein fibrils. J Mol Biol. 1982 Mar 5;155(3):185–205. doi: 10.1016/0022-2836(82)90001-8. [DOI] [PubMed] [Google Scholar]
- Swanson M. S., Dreyfuss G. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol. 1988 May;8(5):2237–2241. doi: 10.1128/mcb.8.5.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Swanson M. S., Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Swanson M. S., Nakagawa T. Y., LeVan K., Dreyfuss G. Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. Mol Cell Biol. 1987 May;7(5):1731–1739. doi: 10.1128/mcb.7.5.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tazi J., Alibert C., Temsamani J., Reveillaud I., Cathala G., Brunel C., Jeanteur P. A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein. Cell. 1986 Dec 5;47(5):755–766. doi: 10.1016/0092-8674(86)90518-0. [DOI] [PubMed] [Google Scholar]
- Thomas J. O., Glowacka S. K., Szer W. Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides. J Mol Biol. 1983 Dec 25;171(4):439–455. doi: 10.1016/0022-2836(83)90039-6. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsanev R. G., Djondjurov L. P. Ultrastructure of free ribonucleoprotein complexes in spread mammalian nuclei. J Cell Biol. 1982 Sep;94(3):662–666. doi: 10.1083/jcb.94.3.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilk H. E., Angeli G., Schäfer K. P. In vitro reconstitution of 35S ribonucleoprotein complexes. Biochemistry. 1983 Sep 13;22(19):4592–4600. doi: 10.1021/bi00288a038. [DOI] [PubMed] [Google Scholar]
- Wilk H. E., Werr H., Friedrich D., Kiltz H. H., Schäfer K. P. The core proteins of 35S hnRNP complexes. Characterization of nine different species. Eur J Biochem. 1985 Jan 2;146(1):71–81. doi: 10.1111/j.1432-1033.1985.tb08621.x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Zarkower D., Wickens M. A functionally redundant downstream sequence in SV40 late pre-mRNA is required for mRNA 3'-end formation and for assembly of a precleavage complex in vitro. J Biol Chem. 1988 Apr 25;263(12):5780–5788. [PubMed] [Google Scholar]
- van Eekelen C., Ohlsson R., Philipson L., Mariman E., van Beek R., van Venrooij W. Sequence dependent interaction of hnRNP proteins with late adenoviral transcripts. Nucleic Acids Res. 1982 Nov 25;10(22):7115–7131. doi: 10.1093/nar/10.22.7115. [DOI] [PMC free article] [PubMed] [Google Scholar]