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
. 1986 Jun;83(11):3718–3722. doi: 10.1073/pnas.83.11.3718

Identification of proteins that bind tightly to pre-mRNA during in vitro splicing.

S H Mayrand, N Pedersen, T Pederson
PMCID: PMC323594  PMID: 3459150

Abstract

Incubation of a human beta-globin pre-mRNA in a HeLa cell nuclear extract under conditions permissive for efficient splicing resulted in the assembly of the RNA into ribonucleoprotein (RNP) complexes. This RNP formation occurred largely within the characteristic lag period that precedes splicing. Two classes of RNP were detected by the criterion of their stability in Cs2SO4 gradients. One was unstable and contained mainly aberrant RNA cleavage products. The other class of RNP complexes comprised 50-85% of the beta-globin RNA, formed only under splicing-permissive conditions, was stable in Cs2SO4 gradients, and contained both unspliced pre-mRNA molecules and the lariat intron 1-exon 2 splicing intermediate. This latter class of RNP complexes banded at approximately equal to 1.30 g/cm3, a density very similar to that of native heterogeneous nuclear RNP particles that contain pre-mRNA. RNA-protein crosslinking revealed major proteins of Mr approximately equal to 38,000 and 41,000 in the stable class of RNP. The use of antibodies specific for heterogeneous nuclear RNP core proteins and for small nuclear RNA-associated proteins, in conjunction with [32P]RNA-protein crosslinking, revealed polypeptides having the molecular weights of both sets of antigens. These results show that both heterogeneous nuclear RNP particle core proteins and small nuclear RNA-associated proteins bind tightly to pre-mRNA during splicing in vitro.

Full text

PDF
3718

Images in this article

3719Image
on p.3719
3721Image
on p.3721
3721Image
on p.3721
3721Image
on p.3721
3721Image
on p.3721

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  5. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  6. Calvet J. P., Pederson T. Nucleoprotein organization of inverted repeat DNA transcripts in heterogeneous nuclear RNA-ribonucleoprotein particles from HeLa cells. J Mol Biol. 1978 Jul 5;122(3):361–378. doi: 10.1016/0022-2836(78)90195-x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Economidis I. V., Pederson T. In vitro assembly of a pre-messenger ribonucleoprotein. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4296–4300. doi: 10.1073/pnas.80.14.4296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  12. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  13. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  14. Greenberg J. R. Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res. 1979 Feb;6(2):715–732. doi: 10.1093/nar/6.2.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones R. E., Okamura C. S., Martin T. E. Immunofluorescent localization of the proteins of nuclear ribonucleoprotein complexes. J Cell Biol. 1980 Jul;86(1):235–243. doi: 10.1083/jcb.86.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karn J., Vidali G., Boffa L. C., Allfrey V. G. Characterization of the non-histone nuclear proteins associated with rapidly labeled heterogeneous nuclear RNA. J Biol Chem. 1977 Oct 25;252(20):7307–7322. [PubMed] [Google Scholar]
  17. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  18. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  19. Lerner M. R., Steitz J. A. Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5495–5499. doi: 10.1073/pnas.76.11.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Lin W. L., Pederson T. Ribonucleoprotein organization of eukaryotic RNA. XXXI. Structure of the U1 small nuclear ribonucleoprotein. J Mol Biol. 1984 Dec 25;180(4):947–960. doi: 10.1016/0022-2836(84)90265-1. [DOI] [PubMed] [Google Scholar]
  22. Martin T., Billings P., Levey A., Ozarslan S., Quinlan T., Swift H., Urbas L. Some properties of RNA:protein complexes from the nucleus of eukaryotic cells. Cold Spring Harb Symp Quant Biol. 1974;38:921–932. doi: 10.1101/sqb.1974.038.01.094. [DOI] [PubMed] [Google Scholar]
  23. Mayrand S., Pederson T. Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells. Mol Cell Biol. 1983 Feb;3(2):161–171. doi: 10.1128/mcb.3.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mayrand S., Pederson T. Nuclear ribonucleoprotein particles probed in living cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2208–2212. doi: 10.1073/pnas.78.4.2208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miller O. L., Jr, Bakken A. H. Morphological studies of transcription. Acta Endocrinol Suppl (Copenh) 1972;168:155–177. doi: 10.1530/acta.0.071s155. [DOI] [PubMed] [Google Scholar]
  26. Miller O. L., Jr The nucleolus, chromosomes, and visualization of genetic activity. J Cell Biol. 1981 Dec;91(3 Pt 2):15s–27s. doi: 10.1083/jcb.91.3.15s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  28. Munroe S. H. Secondary structure of splice sites in adenovirus mRNA precursors. Nucleic Acids Res. 1984 Nov 26;12(22):8437–8456. doi: 10.1093/nar/12.22.8437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Pederson T., Davis N. G. Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors. J Cell Biol. 1980 Oct;87(1):47–54. doi: 10.1083/jcb.87.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pederson T. Nuclear RNA-protein interactions and messenger RNA processing. J Cell Biol. 1983 Nov;97(5 Pt 1):1321–1326. doi: 10.1083/jcb.97.5.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pederson T. Proteins associated with heterogeneous nuclear RNA in eukaryotic cells. J Mol Biol. 1974 Feb 25;83(2):163–183. doi: 10.1016/0022-2836(74)90386-6. [DOI] [PubMed] [Google Scholar]
  35. Pettersson I., Hinterberger M., Mimori T., Gottlieb E., Steitz J. A. The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies. J Biol Chem. 1984 May 10;259(9):5907–5914. [PubMed] [Google Scholar]
  36. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  37. Samarina O. P., Lukanidin E. M., Molnar J., Georgiev G. P. Structural organization of nuclear complexes containing DNA-like RNA. J Mol Biol. 1968 Apr 14;33(1):251–263. doi: 10.1016/0022-2836(68)90292-1. [DOI] [PubMed] [Google Scholar]
  38. Sass H., Pederson T. Transcription-dependent localization of U1 and U2 small nuclear ribonucleoproteins at major sites of gene activity in polytene chromosomes. J Mol Biol. 1984 Dec 25;180(4):911–926. doi: 10.1016/0022-2836(84)90263-8. [DOI] [PubMed] [Google Scholar]
  39. Setyono B., Pederson T. Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo. J Mol Biol. 1984 Apr 5;174(2):285–295. doi: 10.1016/0022-2836(84)90339-5. [DOI] [PubMed] [Google Scholar]
  40. Treisman R., Proudfoot N. J., Shander M., Maniatis T. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing. Cell. 1982 Jul;29(3):903–911. doi: 10.1016/0092-8674(82)90452-4. [DOI] [PubMed] [Google Scholar]
  41. Wieben E. D., Pederson T. Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock. Mol Cell Biol. 1982 Aug;2(8):914–920. doi: 10.1128/mcb.2.8.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wieben E. D., Rohleder A. M., Nenninger J. M., Pederson T. cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7914–7918. doi: 10.1073/pnas.82.23.7914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wilt F. H., Anderson M., Ekenberg E. Centrifugation of nuclear ribonucleoprotein particles of sea urchin embryos in cesium sulfate. Biochemistry. 1973 Feb 27;12(5):959–966. doi: 10.1021/bi00729a027. [DOI] [PubMed] [Google Scholar]
  44. Wollenzien P. L., Cantor C. R., Grant D. M., Lambowitz A. M. RNA splicing in neurospora mitochondria: structure of the unspliced 35S precursor ribosomal RNA detected by psoralen cross-linking. Cell. 1983 Feb;32(2):397–407. doi: 10.1016/0092-8674(83)90459-2. [DOI] [PubMed] [Google Scholar]
  45. Zieve G., Penman S. Subnuclear particles containing a small nuclear RNA and heterogeneous nuclear RNA. J Mol Biol. 1981 Jan 25;145(3):501–523. doi: 10.1016/0022-2836(81)90542-8. [DOI] [PubMed] [Google Scholar]
  46. 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]

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