Detection and characterization of a factor which rescues spliceosome assembly from a heat-inactivated HeLa cell nuclear extract.
Mild heat treatment of HeLa cell nuclear extracts (NE) selectively inhibits pre-mRNA splicing. Heat-inactivated extracts can be complemented by a small amount of untreated NE. Utilizing this complementation assay and a combination of ion-exchange, affinity, and hydrophobic chromatography, a heat reversal factor (HRF) was purified from NE that is required to rescue pre-mRNA splicing from a heat-inactivated extract. This activity in its most purified form consistently copurified in a fraction containing two 70-kDa proteins and a minor polypeptide of approximately 100 kDa. It was free of the major small nuclear RNAs, sensitive to protease, and required to rescue spliceosome formation from a heat-inactivated nuclear extract. These results suggest that this factor is a protein that may be an important component in pre-mRNA splicing, or alternatively, it may be involved in renaturation of a heat-sensitive splicing factor.
Delannoy
P
P
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Caruthers
M H
MH
D002467:Cell Nucleus / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000502:physiology; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000502:physiology*; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D012322:RNA Precursors