In vitro reconstitution of functional yeast U2 snRNPs.

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Abstract
A system for the functional reconstitution of yeast U2 snRNPs using synthetic U2 RNAs is described. We use oligonucleotide-directed RNase H cleavage to specifically deplete yeast extracts of their endogenous full-length U2 snRNA and consequently inactivate pre-mRNA splicing activity. The subsequent addition of synthetic yeast U2 RNAs, derived by in vitro transcription (T7U2 RNAs), to these oligonucleotide-treated extracts efficiently reconstitutes their ability to splice pre-mRNA. The use of deletion derivatives of the T7U2 RNA has demonstrated that the region downstream from the conserved Sm-binding site sequence in the yeast U2 RNA is not absolutely required for pre-mRNA splicing activity in vitro. Furthermore, we found that both human and rat U2 RNAs can function in yeast extracts. We also show that point mutations in the yeast U2 RNA can be analyzed using the in vitro reconstitution system. Allele-specific suppression of mutations in pre-mRNA branch site sequence is observed when the appropriate compensatory mutations in the branch site recognition region of the T7U2 RNA are introduced. Finally, we present a model for the interaction of the U2 and U6 snRNAs during pre-mRNA splicing.