Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site.
Human autoantibodies of the Sm specificity recognize a conserved set of proteins found in the U class small nuclear ribonucleoproteins (U snRNPs), key trans-acting factors involved in the splicing of mRNA precursors. The Sm protein binding site in U snRNAs is unusual because of its single-stranded nature and its simple sequence motif (AU5-6GPu). Here we use genetics to probe this specific protein-RNA interaction by saturation mutagenesis of the Sm binding site of the Saccharomyces cerevisiae U5 snRNA. The assay system used to analyze these mutations takes advantage of a conditionally expressed U5 gene which does not support growth under non-permissive conditions; U5 genes containing Sm site mutations were tested for their ability to complement this lethal phenotype. Our results indicate that the Sm binding site is remarkably tolerant to mutation despite its high degree of conservation, suggesting that relatively few or redundant specific contacts can determine recognition of single-stranded RNA by protein. A complementary biochemical analysis of these mutants demonstrates that integrity of the Sm site is necessary for snRNP stability in vivo and in vitro.
Jones
M H
MH
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Guthrie
C
C
D001324:Autoantigens; D001483:Base Sequence; D001665:Binding Sites; D005075:Biological Evolution; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D010957:Plasmids; D012322:RNA Precursors / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics*; D015183:Restriction Mapping; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D055517:snRNP Core Proteins
D001324:Autoantigens; D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins