ABSTRACT
U6 small nuclear RNA (snRNA) is an essential component of the spliceosome, the ribonucleoprotein complex that carries out the splicing of pre-mRNAs. The precise function of U6 RNA is unknown, but it has been proposed to participate directly in catalysis of the splicing reaction. We present biochemical and genetic evidence for an intramolecular stem/loop structure in the 3' half of U6 RNA of the yeast Saccharomyces cerevisiae that is mutually exclusive with the intermolecular base-pairing between U6 RNA and U4 snRNA. Strains with mutations that stabilize the U6 RNA 3'-intramolecular stem exhibit cold-sensitive growth and accumulate free U4 RNA, indicative of a block in U4/U6 snRNP assembly. The cold sensitivity can be partially suppressed by overexpression of U4 RNA. Mutations that disrupt base-pairing in the intramolecular 3' stem and mutations elsewhere in U6 RNA also suppress the growth defect. We conclude that a large conformational switch, involving melting of the U6 RNA 3' stem, is required for U4/U6 snRNP assembly. We hypothesize that formation of the U6 RNA intramolecular 3' stem after U4 RNA leaves the assembled spliceosome serves to activate U6 RNA for splicing by juxtaposing regions in U6 RNA that interact with U2 small nuclear RNA.
