RESUMO
We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4(+) gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73(ts) at the restrictive temperature (36 degrees ). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73(ts), indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73(ts) mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Precursores de RNA/genética , Splicing de RNA , Proteínas Repressoras/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Proteínas Fúngicas/metabolismo , Genótipo , Reação em Cadeia da Polimerase , Fatores de Processamento de RNA , RNA Fúngico/genética , RNA Mensageiro/genética , Ribonucleoproteína Nuclear Pequena U5 , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Supressão GenéticaRESUMO
Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the quantitative balance between DRADA, cofactor and substrate GLuR-B RNA.
