Construction of an in vivo-regulated U6 snRNA transcription unit as a tool to study U6 function.
U6 snRNA is the only spliceosomal snRNA transcribed by RNA polymerase III in yeast. We have constructed a regulated U6 snRNA transcription unit by introducing the binding site for the Escherichia coli lacI repressor protein in the U6 snRNA promoter. GAL-induced expression of lacI protein led to a decrease in U6 snRNA levels and blocked cell growth. lacI dissociation from the promoter, and consequent U6 snRNA transcription, could be induced by addition of IPTG and repression of lacI transcription. To test the usefulness of this system in studying spliceosomal U6 snRNA function, we conditionally expressed U6 snRNAs with a single base substitution in position A51. We demonstrate that expression of the U6-A51 mutations confers a strong dominant negative phenotype as shown by severe reductions in growth rate. In these strains, splicing of endogenous pre-mRNAs was blocked before the second step.
Luukkonen
B G
BG
European Molecular Biology Laboratory, Heidelberg, Germany.
Séraphin
B
B
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005800:Genes, Fungal; D056968:Lac Repressors; D009154:Mutation; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012342:RNA, Small Nuclear; D012097:Repressor Proteins