Home LiteratureArticle Details
PMID: 4092689 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme.

The EMBO journal ·Vol. 4 ·No. 13A ·1985-12-16 ·Pages 3571-81

Krämer A, Keller W

Abstract

We have used a complementation assay to test for activities required for the splicing of pre-mRNA in vitro. During the hypotonic lysis of HeLa cells, two components are released from the nuclei that specifically stimulate splicing in an extract prepared from washed nuclei. The two activities separate during chromatography on DEAE-Sepharose. One of these activities [splicing factor (SF)2] co-purified through several steps with the lariat debranching enzyme and with a nuclease which degrades the linear portion of lariat RNAs. These enzymes could, however, be separated from SF2 by chromatography on heparin-Sepharose. SF2 fractionates as a single protein with an apparent mol. wt. of 50 000. SF2 is resistant to mild heat treatment and to treatment with micrococcal nuclease, but it is inactivated by N-ethylmaleimide, suggesting that it is a protein which is not associated with an essential RNA component. When SF2 is absent in a complementation assay, the generation of both intermediates and final products of the splicing reaction is completely abolished. Thus, SF2 functions in an early step of the splicing process.

MeSH Terms
Cell Nucleus/enzymology Endoribonucleases/isolation & purification HeLa Cells Humans Macromolecular Substances Molecular Weight Nuclear Proteins Nucleic Acid Precursors/metabolism Nucleoproteins/isolation & purification Proteins/isolation & purification RNA Nucleotidyltransferases/isolation & purification RNA Splicing RNA, Heterogeneous Nuclear/metabolism RNA, Messenger/biosynthesis RNA-Binding Proteins Serine-Arginine Splicing Factors
Chemicals
Macromolecular Substances Nuclear Proteins Nucleic Acid Precursors Nucleoproteins Proteins RNA, Heterogeneous Nuclear RNA, Messenger RNA-Binding Proteins Serine-Arginine Splicing Factors RNA Nucleotidyltransferases lariat debranching enzyme Endoribonucleases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Krämer A
Keller W
References (40)
40 references, click to expand
  1. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    Anal Biochem. 1976 May 7;72:248-54 PMID: 942051
  2. In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence.
    Cell. 1981 Dec;27(3 Pt 2):487-96 PMID: 6101203
  3. Are snRNPs involved in splicing?
    Nature. 1980 Jan 10;283(5743):220-4 PMID: 7350545
  4. A mechanism for RNA splicing.
    Proc Natl Acad Sci U S A. 1980 Apr;77(4):1877-9 PMID: 6246511
  5. Organization and expression of eucaryotic split genes coding for proteins.
    Annu Rev Biochem. 1981;50:349-83 PMID: 6791577
  6. Novel models for RNA splicing that involve a small nuclear RNA.
    Proc Natl Acad Sci U S A. 1981 Jul;78(7):4471-4 PMID: 6170064
  7. In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.
    Proc Natl Acad Sci U S A. 1981 Sep;78(9):5430-4 PMID: 6946482
  8. A catalogue of splice junction sequences.
    Nucleic Acids Res. 1982 Jan 22;10(2):459-72 PMID: 7063411
  9. Accurate in vitro splicing of human beta-globin RNA.
    Nucleic Acids Res. 1982 Sep 25;10(18):5429-45 PMID: 6292841
  10. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes.
    Nucleic Acids Res. 1982 Oct 25;10(20):6353-62 PMID: 6294601
  11. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
    Nucleic Acids Res. 1983 Mar 11;11(5):1475-89 PMID: 6828386
  12. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro.
    Cell. 1983 Jun;33(2):509-18 PMID: 6190573
  13. Splicing of adenovirus RNA in a cell-free transcription system.
    Proc Natl Acad Sci U S A. 1983 Sep;80(17):5230-4 PMID: 6577417
  14. Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein.
    Cell. 1983 Nov;35(1):101-7 PMID: 6194895
  15. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.
    Cell. 1983 Nov;35(1):89-99 PMID: 6194902
  16. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.
    Cell. 1984 Apr;36(4):993-1005 PMID: 6323033
  17. Cofactor requirements of splicing of purified messenger RNA precursors.
    Nature. 1984 Mar 22-28;308(5957):375-7 PMID: 6709044
  18. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate.
    Cell. 1984 Jun;37(2):415-27 PMID: 6722880
  19. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors.
    Cell. 1984 Aug;38(1):299-307 PMID: 6235919
  20. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro.
    Cell. 1984 Aug;38(1):317-31 PMID: 6088074
  21. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.
    Science. 1984 Aug 31;225(4665):898-903 PMID: 6206566
  22. 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):7035-56 PMID: 6091052
  23. Processing and nucleo-cytoplasmic transport of histone gene transcripts.
    Nucleic Acids Res. 1984 Nov 26;12(22):8539-51 PMID: 6095202
  24. Intron splicing: a conserved internal signal in introns of animal pre-mRNAs.
    Proc Natl Acad Sci U S A. 1984 Dec;81(23):7417-20 PMID: 6209716
  25. The RNA lariat: a new ring to the splicing of mRNA precursors.
    Cell. 1984 Dec;39(3 Pt 2):423-5 PMID: 6568879
  26. In vivo splicing products of the rabbit beta-globin pre-mRNA.
    Cell. 1984 Dec;39(3 Pt 2):589-602 PMID: 6096012
  27. In vivo characterization of yeast mRNA processing intermediates.
    Cell. 1984 Dec;39(3 Pt 2):603-10 PMID: 6096013
  28. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing.
    Cell. 1984 Dec;39(3 Pt 2):611-21 PMID: 6096014
  29. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors.
    Nature. 1985 Feb 14-20;313(6003):552-7 PMID: 2578627
  30. Intron sequences involved in lariat formation during pre-mRNA splicing.
    Cell. 1985 May;41(1):95-105 PMID: 3888410
  31. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction.
    Science. 1985 May 24;228(4702):963-7 PMID: 3890181
  32. Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro.
    Proc Natl Acad Sci U S A. 1985 Jul;82(13):4351-5 PMID: 3859867
  33. An RNA processing activity that debranches RNA lariats.
    Science. 1985 Jul 12;229(4709):135-40 PMID: 2990042
  34. A multicomponent complex is involved in the splicing of messenger RNA precursors.
    Cell. 1985 Aug;42(1):345-53 PMID: 3160482
  35. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences.
    Cell. 1985 Aug;42(1):355-67 PMID: 3160483
  36. Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.
    Nucleic Acids Res. 1985 Jul 11;13(13):4971-81 PMID: 2410858
  37. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro.
    Cell. 1985 Oct;42(3):725-36 PMID: 2996774
  38. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing.
    Cell. 1985 Oct;42(3):737-50 PMID: 2996775
  39. Role of the 3' splice site consensus sequence in mammalian pre-mRNA splicing.
    Nature. 1985 Oct 24-30;317(6039):732-4 PMID: 4058579
  40. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries.
    Proc Natl Acad Sci U S A. 1978 Oct;75(10):4853-7 PMID: 283395
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1985-12-16
Pages
3571-81
Language
English
Region
England
NLM ID
8208664
PMCID
PMC554699
Subset
IM
External Links
PubMed source PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2