Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein (original) (raw)

Abstract

The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one.

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  1. 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]
  2. Amrein H., Gorman M., Nöthiger R. The sex-determining gene tra-2 of Drosophila encodes a putative RNA binding protein. Cell. 1988 Dec 23;55(6):1025–1035. doi: 10.1016/0092-8674(88)90247-4. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bell L. R., Maine E. M., Schedl P., Cline T. W. Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell. 1988 Dec 23;55(6):1037–1046. doi: 10.1016/0092-8674(88)90248-6. [DOI] [PubMed] [Google Scholar]
  5. Bestagno M., Cerino A., Riva S., Astaldi Ricotti G. C. Improvements of Western blotting to detect monoclonal antibodies. Biochem Biophys Res Commun. 1987 Aug 14;146(3):1509–1514. doi: 10.1016/0006-291x(87)90820-5. [DOI] [PubMed] [Google Scholar]
  6. Biamonti G., Buvoli M., Bassi M. T., Morandi C., Cobianchi F., Riva S. Isolation of an active gene encoding human hnRNP protein A1. Evidence for alternative splicing. J Mol Biol. 1989 Jun 5;207(3):491–503. doi: 10.1016/0022-2836(89)90459-2. [DOI] [PubMed] [Google Scholar]
  7. Buvoli M., Biamonti G., Tsoulfas P., Bassi M. T., Ghetti A., Riva S., Morandi C. cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms. Nucleic Acids Res. 1988 May 11;16(9):3751–3770. doi: 10.1093/nar/16.9.3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Celis J. E., Bravo R., Arenstorf H. P., LeStourgeon W. M. Identification of proliferation-sensitive human proteins amongst components of the 40 S hnRNP particles. Identity of hnRNP core proteins in the HeLa protein catalogue. FEBS Lett. 1986 Jan 1;194(1):101–109. doi: 10.1016/0014-5793(86)80059-x. [DOI] [PubMed] [Google Scholar]
  9. Celis J. E., Ratz G. P., Celis A., Madsen P., Gesser B., Kwee S., Madsen P. S., Nielsen H. V., Yde H., Lauridsen J. B. Towards establishing comprehensive databases of cellular proteins from transformed human epithelial amnion cells (AMA) and normal peripheral blood mononuclear cells. Leukemia. 1988 Sep;2(9):561–601. [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Crowl R., Seamans C., Lomedico P., McAndrew S. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene. 1985;38(1-3):31–38. doi: 10.1016/0378-1119(85)90200-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. LeStourgeon W. M., Beyer A. L., Christensen M. E., Walker B. W., Poupore S. M., Daniels L. P. The packaging proteins of core hnRNP particles and the maintenance of proliferative cell states. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):885–898. doi: 10.1101/sqb.1978.042.01.090. [DOI] [PubMed] [Google Scholar]
  17. Leser G. P., Escara-Wilke J., Martin T. E. Monoclonal antibodies to heterogeneous nuclear RNA-protein complexes. The core proteins comprise a conserved group of related polypeptides. J Biol Chem. 1984 Feb 10;259(3):1827–1833. [PubMed] [Google Scholar]
  18. 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]
  19. Mayrand S., Setyono B., Greenberg J. R., Pederson T. Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells. J Cell Biol. 1981 Aug;90(2):380–384. doi: 10.1083/jcb.90.2.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Merrill B. M., Stone K. L., Cobianchi F., Wilson S. H., Williams K. R. Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein. J Biol Chem. 1988 Mar 5;263(7):3307–3313. [PubMed] [Google Scholar]
  21. Michel B., Zinder N. D. In vitro binding of the bacteriophage f1 gene V protein to the gene II RNA-operator and its DNA analog. Nucleic Acids Res. 1989 Sep 25;17(18):7333–7344. doi: 10.1093/nar/17.18.7333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  23. Nielsen D. A., Shapiro D. J. Preparation of capped RNA transcripts using T7 RNA polymerase. Nucleic Acids Res. 1986 Jul 25;14(14):5936–5936. doi: 10.1093/nar/14.14.5936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  25. Pandolfo M., Valentini O., Biamonti G., Rossi P., Riva S. Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose. Eur J Biochem. 1987 Jan 2;162(1):213–220. doi: 10.1111/j.1432-1033.1987.tb10563.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  34. 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]