Multiple Splicing Factors Are Released from Endogenous Complexes during In Vitro Pre-mRNA Splicing
Year: 1989
Type: article
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.
Source: Molecular and Cellular Biology
Authors: G C Conway, A R Krainer, D L Spector, R J Roberts
Institution: Cold Spring Harbor Laboratory
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: National Cancer Institute
Award: G1200689522
OpenAlex ID: W1857528879