Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.
U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs.
Miraglia
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Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Seiwert
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Igel
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Ares
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D001483:Base Sequence; D005799:Genes, Dominant; D005800:Genes, Fungal; D005804:Genes, Lethal; D014644:Genetic Variation; D006801:Humans; D006860:Hydrogen Bonding; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D010641:Phenotype; D010802:Phylogeny; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D015345:Oligonucleotide Probes; D012342:RNA, Small Nuclear