Inactivation of splicing factors in HeLa cells subjected to heat shock.

Abstract	The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.
Authors	R R Shukla, Z Dominski, T Zwierzynski, R Kole
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 265 Issue 33 Pg. 20377-83 (Nov 25 1990) ISSN: 0021-9258 [Print] United States
PMID	2147023 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References	RNA Precursors Ribonucleoproteins Ribonucleoproteins, Small Nuclear Micrococcal Nuclease
Topics	Cell Nucleus (metabolism) HeLa Cells (metabolism) Hot Temperature Humans Kinetics Micrococcal Nuclease RNA Precursors (genetics, metabolism) RNA Splicing Ribonucleoproteins (isolation & purification, metabolism) Ribonucleoproteins, Small Nuclear Templates, Genetic Transcription, Genetic

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