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PMID: 1333586 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Analysis of spliceosome assembly and the structure of a regulated intron in Drosophila in vitro splicing extracts.

Nucleic acids research ·Vol. 20 ·No. 21 ·1992-11-11 ·Pages 5719-27

Spikes D, Bingham PM

Abstract

We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.

MeSH Terms
Animals Base Sequence DNA Drosophila Exoribonucleases/metabolism In Vitro Techniques Introns Micrococcal Nuclease Molecular Sequence Data RNA Splicing RNA, Small Nuclear/metabolism Ribonuclease H/metabolism Spliceosomes/metabolism
Chemicals
RNA, Small Nuclear DNA Exoribonucleases exoribonuclease II Ribonuclease H Micrococcal Nuclease
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Spikes D
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794.
Bingham P M
References (36)
36 references, click to expand
  1. Developmental expression of a regulatory gene is programmed at the level of splicing.
    EMBO J. 1987 Dec 20;6(13):4095-104 PMID: 2832151
  2. A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity.
    Cell. 1984 Feb;36(2):357-69 PMID: 6537904
  3. 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
  4. Spliceosomal snRNAs.
    Annu Rev Genet. 1988;22:387-419 PMID: 2977088
  5. Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing.
    Cell. 1985 Nov;43(1):131-42 PMID: 3000599
  6. Structure, organization, and transcription of Drosophila U6 small nuclear RNA genes.
    J Biol Chem. 1987 Jan 25;262(3):1187-93 PMID: 3027083
  7. 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):1037-46 PMID: 3144435
  8. 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
  9. Evidence that a regulatory gene autoregulates splicing of its transcript.
    EMBO J. 1987 Dec 20;6(13):4105-11 PMID: 3443103
  10. Intron sequences involved in lariat formation during pre-mRNA splicing.
    Cell. 1985 May;41(1):95-105 PMID: 3888410
  11. Divergence of U2 snRNA sequences in the genome of D. melanogaster.
    Nucleic Acids Res. 1984 Dec 21;12(24):9543-50 PMID: 6083551
  12. The small nuclear RNAs of Drosophila.
    J Mol Biol. 1984 Dec 25;180(4):927-45 PMID: 6084723
  13. Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins.
    Nature. 1984 Jul 5-11;310(5972):25-31 PMID: 6330566
  14. 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
  15. Mass isolation of pole cells from Drosophila melanogaster.
    Dev Biol. 1977 APR;56(2):372-81 PMID: 403099
  16. Mechanisms of alternative pre-mRNA splicing.
    Science. 1991 Jan 4;251(4989):33-4 PMID: 1824726
  17. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators.
    Cell. 1991 Jul 26;66(2):383-94 PMID: 1830244
  18. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly.
    Cell. 1990 Oct 19;63(2):293-302 PMID: 2170025
  19. Sex in flies: the splice of life.
    Nature. 1989 Aug 17;340(6234):521-4 PMID: 2505080
  20. In vitro RNA synthesis with SP6 RNA polymerase.
    Methods Enzymol. 1987;155:397-415 PMID: 2828872
  21. On/off regulation of gene expression at the level of splicing.
    Trends Genet. 1988 May;4(5):134-8 PMID: 2853467
  22. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes.
    Cell. 1987 Jun 19;49(6):763-74 PMID: 2953438
  23. In vitro splicing of simian virus 40 early pre mRNA.
    Nucleic Acids Res. 1986 Feb 11;14(3):1219-35 PMID: 3005968
  24. The control of alternative splicing at genes regulating sexual differentiation in D. melanogaster.
    Cell. 1988 Apr 22;53(2):229-36 PMID: 3129196
  25. Positive control of pre-mRNA splicing in vitro.
    Science. 1992 Apr 10;256(5054):237-40 PMID: 1566072
  26. Arginine/serine-rich domains of the su(wa) and tra RNA processing regulators target proteins to a subnuclear compartment implicated in splicing.
    Cell. 1991 Oct 18;67(2):335-42 PMID: 1655279
  27. Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution.
    EMBO J. 1991 Jan;10(1):207-14 PMID: 1824937
  28. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators.
    Cell. 1991 Jul 26;66(2):373-82 PMID: 1855257
  29. Drosophila melanogaster genes for U1 snRNA variants and their expression during development.
    Nucleic Acids Res. 1990 Dec 11;18(23):6971-9 PMID: 2124674
  30. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene.
    Cell. 1987 Aug 28;50(5):739-47 PMID: 2441872
  31. Branch point selection in alternative splicing of tropomyosin pre-mRNAs.
    Nucleic Acids Res. 1989 Jul 25;17(14):5633-50 PMID: 2762151
  32. In vitro transcription of the Drosophila engrailed gene.
    Genes Dev. 1988 Jan;2(1):68-81 PMID: 3356339
  33. Isolation and characterization of two putative full-length Drosophila U4 small nuclear RNA genes.
    J Biol Chem. 1986 Jul 5;261(19):8750-3 PMID: 3722171
  34. GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast.
    Cell. 1985 Nov;43(1):177-88 PMID: 3907851
  35. Sequencing end-labeled DNA with base-specific chemical cleavages.
    Methods Enzymol. 1980;65(1):499-560 PMID: 6246368
  36. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.
    Mol Cell Biol. 1988 Feb;8(2):814-21 PMID: 2832738
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/20.21.5719
Published
1992-11-11
Pages
5719-27
Language
English
Region
England
NLM ID
0411011
PMCID
PMC334408
Subset
IM
Grants
NIGMS NIH HHS · GM32003 · United States
External Links
PubMed source DOI: 10.1093/nar/20.21.5719 PMC Full Text View journal details
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