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Unlike the majority of genes encoding small nuclear RNAs, which are transcribed by RNA polymerase B, the U6 gene contains features found in both class B and class C genes, indicating the involvement of a combination of transcription factors normally specific to each class of genes. We present direct genetic and biochemical evidence that the U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C in vivo as well as in vitro. A mutant strain with a temperature‐sensitive defect in the large subunit of RNA polymerase C that results in defective transcription of tRNA and 5S RNA genes shows a corresponding defect in U6 RNA levels. Also, purified RNA polymerase C transcribes the U6 gene when supplemented with partially purified TFIIIB. The other class C transcription factors, TFIIIA and Tau (TFIIIC), are not required in this system.

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The process of nuclear pre-messenger RNA splicing is similar in Saccharomyces cerevisiae and metazoan cells in that the two-step mechanism is identical and the reaction occurs in a large ribonucleoprotein complex, the spliceosome. Little is known, however, about the degree of conservation of splicing factors other than of the small nuclear RNAs (snRNAs). Yeast counterparts of the metazoan spliceosomal snRNAs (U1, U2, U4, U5 and U6) have been identified but, with the exception of U6, the yeast snRNAs are larger and sequence similarity is limited to short regions. By using antibodies against the yeast PRP8 protein, a pre-mRNA splicing factor of relative molecular mass 280,000 (Mr280K) stably associated with U5 small nuclear ribonucleoproteins (snRNPs), we have now identified an immunologically related protein in HeLa cell nuclear extracts. The HeLa cell protein has an Mr greater than 200K and is associated with purified 20S U5 snRNPs. This is the first report of phylogenetic conservation between yeast and man of a protein splicing factor.

A mammalian protein of approximately 220 kIa (p220) was UV-crosslinked to precursor mRNAs (premRNAs) under splicing conditions. The kinetics and biochemical requirements of the UV-crosslinking of p220 corresponded to the kinetics and biochemical requirements of spliceosome formation. On Western blots, antibodies against the yeast splicing factor PRP8 recognized a doublet ofproteins, the faster migrating of which comigrated with p220. Furthermore, UVcrosslinked p220 was immunoprecipitated with anti-PRP8 antisera. These results suggest structural conservation of the splicing factor PRP8 from yeast to mammals and show that this protein is in close proximity to the pre-mRNA in the spliceosome.Splicing of precursor mRNAs (pre-mRNAs) requires the U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs) and several protein factors (1-8). These assemble to form a splicing body, the spliceosome (9, 10). The U-small nuclear RNAs (7) and the spliceosome formation pathway (11)(12)(13)(14) have been conserved from yeast to man. In yeast and mammalian systems, this pathway can be divided into distinct steps by the identification of intermediates in the formation of the spliceosome (3,9,(12)(13)(14). Ul and U2 snRNPs bind pre-mRNAs to form pre-spliceosome complexes (4,9,12,13). Subsequently, U4, U5, and U6 snRNPs associate in the absence of exogenously added pre-mRNA to form a U4,5,6 multi-snRNP complex (12), and this complex binds the prespliceosome complexes to form spliceosomes (12)(13)(14).Genetic and biochemical analyses predict that a large number of gene products are required for pre-mRNA splicing. The analysis of pre-mRNA splicing conditional mutants in Saccharomyces cerevisiae has led to the identification and characterization of more than 20 gene products essential for splicing (15)(16)(17)(18)(19). One of these, PRP8, is a U5 snRNPassociated 280-kDa protein, shown to be required for premRNA splicing in vivo and in vitro (20). Under splicing conditions PRP8 associates with the U4,5,6 multi-snRNP complex and with pre-mRNA (19). Fractionation of in vitro splicing systems has revealed the existence of several proteins that collaborate with snRNPs in splicing pre-mRNAs (3,5). It remains to be determined whether some of these act directly on the pre-mRNA or have a role in the assembly of the spliceosome.To identify proteins that interact directly with pre-mRNAs during splicing reactions, we have used UV-crosslinking of RNA to protein (21). We have identified a 220-kDa protein (p220) in HeLa cell nuclear extracts that binds pre-mRNAs under splicing conditions. p220 binds pre-mRNAs with specificity; point mutants in the splice sites that are impaired in splicing do not bind to p220. We investigated the relationship between HeLa p220 and the yeast splicing factor PRP8. Rabbit antibodies against yeast PRP8 protein (19) detect a protein that comigrates in SDS/PAGE with p220 and immunoprecipitate UV-crosslinked p220, strongly suggesting these two proteins are related. MATERIALS AND METHODSRNAs, Nuclear...

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