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A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple
U Utans1, S E Behrens, R Lührmann
1Abteilung Zellbiologie, Biozentrum der Universität Basel, Switzerland.
Abstract:
One of the consequences of the heat shock response is a shutdown of pre-mRNA splicing, a phenomenon that can be reproduced in extracts prepared from heat-shocked cells. The block in splicing occurs before the covalent modifications that generate spliced mRNA at the level of spliceosome formation. We have used extracts prepared from heat-shocked cells as a complementation system to characterize and partially purify a protein factor that is inactivated during the in vivo heat shock. The activity functions in the formation of the active spliceosome by assembling U4/U6 and U5 snRNPs into a triple snRNP particle. The factor appears to be different from previously isolated splicing factors and is functionally equivalent to several polypeptides that are specifically associated with the purified triple snRNP but not with individual U4/U6 or U5 snRNPs. Our data confirm the hypothesis that U4/U6 and U5 snRNPs enter the spliceosome as a triple snRNP complex and show for the first time a function of specific snRNP-associated polypeptides in the mammalian splicing pathway.
Insights
Heat shock response halts pre-messenger RNA (mRNA) splicing by inactivating a protein factor essential for spliceosome assembly. This study identifies and partially purifies this crucial factor, revealing its role in forming the triple small nuclear ribonucleoprotein (snRNP) complex.
Area of Science:
- Molecular Biology
- Cellular Stress Response
- RNA Processing
Background:
- Heat shock response globally impacts cellular processes, including pre-mRNA splicing.
- Splicing inhibition during heat shock occurs early in spliceosome formation.
- Understanding splicing regulation under stress is critical for cell survival.
Purpose of the Study:
- To characterize and partially purify a protein factor inactivated by heat shock that affects pre-mRNA splicing.
- To elucidate the role of this factor in spliceosome assembly.
- To investigate the function of snRNP-associated polypeptides in mammalian splicing.
Main Methods:
- Utilized extracts from heat-shocked cells as a complementation system.
- Partially purified the heat-inactivated protein factor.
- Assayed for the factor's activity in spliceosome formation, specifically the assembly of U4/U6 and U5 snRNPs.
Main Results:
- Identified and partially purified a protein factor inactivated during in vivo heat shock.
- Demonstrated this factor's essential role in assembling U4/U6 and U5 snRNPs into a triple snRNP particle.
- Showed functional equivalence between the purified factor and polypeptides associated with the triple snRNP.
Conclusions:
- Confirmed the hypothesis that U4/U6 and U5 snRNPs enter the spliceosome as a pre-assembled triple snRNP complex.
- Provided the first evidence for the function of specific snRNP-associated polypeptides in mammalian splicing.
- Highlighted a key regulatory mechanism of splicing during cellular stress.