Purification of a protein required for the splicing of pre-mRNA and its separation from the lariat debranching enzyme

Abstract

We have used a complementation assay to test for activities required for the splicing of pre-mRNA in vitro. During the hypotonic lysis of HeLa cells, two components are released from the nuclei that specifically stimulate splicing in an extract prepared from washed nuclei. The two activities separate during chromatography on DEAE-Sepharose. One of these activities [splicing factor (SF)2] co-purified through several steps with the lariat debranching enzyme and with a nuclease which degrades the linear portion of lariat RNAs. These enzymes could, however, be separated from SF2 by chromatography on heparin-Sepharose. SF2 fractionates as a single protein with an apparent mol. wt. of 50 000. SF2 is resistant to mild heat treatment and to treatment with micrococcal nuclease, but it is inactivated by N-ethylmaleimide, suggesting that it is a protein which is not associated with an essential RNA component. When SF2 is absent in a complementation assay, the generation of both intermediates and final products of the splicing reaction is completely abolished. Thus, SF2 functions in an early step of the splicing process.