Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing

Conway, G. C., Krainer, A. R., Spector, D. L., Roberts, R. J. (1989) Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing. Molecular & Cellular Biology, 9 (12). pp. 5273-80. ISSN 0270-7306

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URL: http://www.ncbi.nlm.nih.gov/pubmed/2531283
DOI: 10.1128/​MCB.9.12.5273

Abstract

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

Item Type: Paper
Uncontrolled Keywords: Cell Fractionation Cell Nucleus metabolism ultrastructure Centrifugation Density Gradient Globins genetics Hela Cells metabolism Humans RNA Precursors genetics isolation and purification RNA Splicing RNA Small Nuclear isolation and purification Research Support Non US Govt Research Support US Govt PHS Ribonucleoproteins isolation and purification Ribonucleoproteins Small Nuclear
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA splicing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > splicing factor
CSHL Authors:
Communities: CSHL labs > Krainer lab
CSHL labs > Spector lab
Depositing User: Brian Soldo
Date Deposited: 04 Apr 2012 20:06
Last Modified: 19 Mar 2013 21:07
PMCID: PMC363691
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25651

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