9603550 Patton It is becoming increasingly clear that modified nucleotides in RNA have important functional roles. Of the 93 different modified nucleotides currently known to occur in RNA molecules, pseudouridine is the most abundant. Pseudouridine is important in tRNA function and is thought to participate in the peptidyl transfer reaction of rRNA. Pseudouridine also occurs in highly conserved positions in snRNAs (small nuclear RNAs), essential co-factors in pre-mRNA splicing. Splicing is accomplished by the splicosome, an assembly of co-factors which includes snRNPs (small nuclear ribonucleoprotein particles) composed of snRNAs and proteins. Conserved nucleotide modifications in splicosomal snRNAs are concentrated near the catalytic center of the splicosome and nearly all are found where snRNAs interact with the pre-mRNA or other snRNAs. Pseudouridines occur in regions of U2 snRNA sequence know to be essential for its function as a spicing co-factor. This research will test the hypothesis that pseudouridine modification of U2 snRNA is required for the assembly and/or function of the U2 snRNP. U2 snRNPs will be assembled in vitro using either U2 RNA synthesized in vitro or fully modified U2 snRNA isolated from HeLa cells. Post-transcriptional inhibitors of pseudouridine formation which do not prevent other U2 RNA modifications such as methylation will be used to control the presence or absence the pseudouridine in the U2 RNA synthesized in vitro. U2 snRNPs reconstituted in vitro will be used in a complementation assay of pre-mRNA splicing to determine if pseudouridine modification of U2 snRNA affects splicosome function. It is becoming increasingly clear that modified nucleotides in RNA have important functional roles. Of the 93 different modified nucleotides currently known to occur in RNA molecules, pseudouridine is the most abundant and has been implicated in both tRNA and rRNA function. Pseudouridine also occurs in snRNAs (small nuclear RNAs), essential co-factors for the "sp licing" of pre-mRNAs into functional mRNAs in the cell nucleus. Splicing is accomplished by the splicosome, an assembly of snRNPs (small nuclear ribonucleoprotein particles) composed of snRNAs and proteins. Conserved nucleotide modifications in splicosomal snRNAs are concentrated near the catalytic center of the splicosome and nearly all are found where snRNAs interact with the pre-mRNA or other snRNAs. Pseudouridines occur in regions of U2 snRNA sequence known to be essential for its function as a spicing co-factor. This research will test the hypothesis that pseudouridine modification of U2 snRNA is required for the assembly and/or function of the U2 snRNP.
