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PMID: 1824937 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution.

The EMBO journal ·Vol. 10 ·No. 1 ·1991-01-00 ·Pages 207-14

Zamore PD, Green MR

Abstract

U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. We have performed biochemical complementation and immunological assays to characterize U2AF in greater detail. First, we use an extract lacking only U2AF activity to show that U2AF is an essential splicing factor. Second, we show that all U2AF activity in vitro resides in the 65 kd U2AF polypeptide. Third, based upon both immunological and functional criteria, we show that U2AF is evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenically related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, we show that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers.' This localization differs strikingly from that reported for snRNP antigens and splicing factors. Our data, in conjunction with those in the accompanying paper [Carmo-Fonseca et al. (1991) EMBO J., 10, 195-206.], suggest that these centers represent novel aspects of nuclear organization.

MeSH Terms
Amino Acid Sequence Animals Biological Evolution Cell Line Cell Nucleus/metabolism Fluorescent Antibody Technique HeLa Cells/cytology,metabolism Humans Immunoblotting Molecular Sequence Data RNA Precursors/genetics RNA Splicing Ribonucleoproteins/genetics,isolation & purification,metabolism Ribonucleoproteins, Small Nuclear Species Specificity
Chemicals
RNA Precursors Ribonucleoproteins Ribonucleoproteins, Small Nuclear
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Zamore P D
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
Green M R
References (36)
36 references, click to expand
  1. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites.
    Cell. 1990 Jul 13;62(1):35-42 PMID: 2364434
  2. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly.
    Cell. 1990 Oct 19;63(2):293-302 PMID: 2170025
  3. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro.
    Cell. 1990 Jul 13;62(1):25-34 PMID: 2163768
  4. A mammalian protein of 220 kDa binds pre-mRNAs in the spliceosome: a potential homologue of the yeast PRP8 protein.
    Proc Natl Acad Sci U S A. 1990 Apr;87(8):3082-6 PMID: 2139226
  5. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus.
    Nature. 1990 Feb 1;343(6257):437-41 PMID: 2137203
  6. Higher order nuclear organization: three-dimensional distribution of small nuclear ribonucleoprotein particles.
    Proc Natl Acad Sci U S A. 1990 Jan;87(1):147-51 PMID: 2136950
  7. 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
  8. Nonhistone nuclear antigens reactive with autoantibodies. Immunofluorescence studies on distribution in synchronized cells.
    J Cell Biol. 1981 Dec;91(3 Pt 1):654-60 PMID: 7035465
  9. A simple method of reducing the fading of immunofluorescence during microscopy.
    J Immunol Methods. 1981;43(3):349-50 PMID: 7019347
  10. Characterization of a soluble nuclear ribonucleoprotein antigen reactive with SLE sera.
    J Immunol. 1971 Nov;107(5):1281-90 PMID: 4330162
  11. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.
    EMBO J. 1991 Jan;10(1):195-206 PMID: 1824936
  12. 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):755-66 PMID: 2946417
  13. Sex in flies: the splice of life.
    Nature. 1989 Aug 17;340(6234):521-4 PMID: 2505080
  14. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.
    Mol Cell Biol. 1988 Feb;8(2):814-21 PMID: 2832738
  15. Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA.
    Proc Natl Acad Sci U S A. 1988 Aug;85(15):5459-62 PMID: 2969592
  16. Spliceosomal snRNAs.
    Annu Rev Genet. 1988;22:387-419 PMID: 2977088
  17. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly.
    Cell. 1988 Jan 29;52(2):207-19 PMID: 2963698
  18. Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107.
    J Cell Biol. 1985 Aug;101(2):560-7 PMID: 2991302
  19. Intranuclear localization of snRNP antigens.
    J Cell Biol. 1986 Jan;102(1):137-44 PMID: 2934400
  20. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing.
    Nature. 1987 Feb 19-25;325(6106):673-8 PMID: 2950324
  21. A protein associated with small nuclear ribonucleoprotein particles recognizes the 3' splice site of premessenger RNA.
    Cell. 1986 Dec 26;47(6):973-84 PMID: 2946421
  22. Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.
    EMBO J. 1986 May;5(5):997-1002 PMID: 2941274
  23. Distribution of the 70K U1 RNA-associated protein during interphase and mitosis. Correlation with other U RNP particles and proteins of the nuclear matrix.
    J Cell Sci. 1986 Dec;86:173-90 PMID: 2958489
  24. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.
    EMBO J. 1987 Aug;6(8):2415-24 PMID: 2959470
  25. The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome.
    Proc Natl Acad Sci U S A. 1989 Nov;86(22):8742-6 PMID: 2479028
  26. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.
    Genes Dev. 1989 Dec;3(12A):1874-86 PMID: 2533575
  27. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor.
    Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243-7 PMID: 2531895
  28. Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing.
    Cell. 1989 Mar 10;56(5):749-58 PMID: 2924347
  29. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells.
    Nucleic Acids Res. 1989 Aug 11;17(15):6419 PMID: 2771659
  30. Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein.
    Nature. 1989 Dec 14;342(6251):819-21 PMID: 2532307
  31. Presplicing complex formation requires two proteins and U2 snRNP.
    Genes Dev. 1988 Sep;2(9):1155-67 PMID: 3192077
  32. Accurate and efficient pre-mRNA splicing in Drosophila cell-free extracts.
    Proc Natl Acad Sci U S A. 1988 May;85(9):2904-8 PMID: 3362856
  33. Colocalization of U1 and U2 small nuclear RNPs by immunocytochemistry.
    Biol Cell. 1984;51(1):109-12 PMID: 6207877
  34. Splicing of messenger RNA precursors.
    Science. 1987 Feb 13;235(4790):766-71 PMID: 3544217
  35. Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme.
    EMBO J. 1985 Dec 16;4(13A):3571-81 PMID: 4092689
  36. Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript.
    Nature. 1990 Mar 29;344(6265):461-3 PMID: 1690860
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1991-01-00
Pages
207-14
Language
English
Region
England
NLM ID
8208664
PMCID
PMC452631
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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