Regulation of splicing at an intermediate step in the formation of the spliceosome

In vivo experiments have demonstrated that the ribosomal protein L32 of Saccharomyces cerevisiae brings about the inhibition of splicing of the transcript of its own gene through an RNA structure comprised largely of the first exon. We now show that L32, itself, binds specifically to this RNA. Splicing of the RPL32 transcript in vitro is blocked by the presence of L32. Furthermore, addition of the 75-nucleotide RNA representing the 5' end of the RPL32 transcript stimulates specifically the splicing of the RPL32 substrate, presumably by competing for L32 present in the extract. Use of RNAs carrying mutations shown to abolish the regulation of splicing, either as substrates or as competitors, confirmed that the in vitro reaction is a faithful representation of the situation in vivo. We conclude that the regulation of splicing occurs through the specific binding of L32 to an RNA structure within the first 75 nucleotides of the RPL32 transcript. The RPL32 substrate, bound to L32, forms a complex with U1 snRNP, the first step in spliceosome assembly. The presence of L32 prevents the ATP-dependent association of the U2 snRNP necessary to form a complete spliceosome.