Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome.
Putative components of the spliceosomal active site include a bulged helix between U2 and U6 snRNAs (U2-U6 helix I) and the adjacent ACAGAG hexanucleotide in U6. We have developed an in vivo, bimolecular randomization-selection method to functionally dissect these elements. Although a portion of U2-U6 helix I resembles the G-binding site of group I introns, the data are inconsistent with an analogous functional role for this structure in the spliceosome. Instead, analysis of several novel covariants supports the existence of a structure in which the helix I bulge engages in a tertiary interaction with the terminal residue of the U6 hexanucleotide (ACAGAG). Such a higher order structure, together with other known interactions, would juxtapose the two clusters of residues of the U2-U6 complex that are specifically required for the second chemical step of pre-mRNA splicing with the 3' splice site. Indeed, mutations in the residues that participate in the tertiary interaction affect both the efficiency and fidelity of 3' splice site usage.
Madhani
H D
HD
Department of Biochemistry and Biophysics, University of California at San Francisco 94143-0448.
Guthrie
C
C
D000483:Alleles; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D015723:Gene Library; D014644:Genetic Variation; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry; D010641:Phenotype; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017381:Spliceosomes / Q000378:metabolism*; D013489:Suppression, Genetic
D009843:Oligoribonucleotides; D012331:RNA, Fungal; D012342:RNA, Small Nuclear