RESUMEN
An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.
Asunto(s)
Apolipoproteínas B/genética , Edición de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Línea Celular , ADN/genética , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Luciferasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Papio , Mutación Puntual , ARN Mensajero/efectos de la radiación , Ratas , Transfección , Rayos UltravioletaRESUMEN
The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro. To expand our understanding of how this protein functions in vivo, we have created a null allele of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and employed it in a variety of genetic complementation assays. First, analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations. Second, despite extensive structural conservation, we find that metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59. Third, because the activity of U2AF65 in vitro involves binding to the 3' polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575). Our data indicate that all four introns tested, including two that lack extensive runs of pyrimidines between the branchpoint and 3' splice site, show splicing defects upon shifting to the nonpermissive condition. In all cases, splicing is blocked prior to the first transesterification reaction in the mutants, consistent with the role inferred for human U2AF65 based on in vitro experiments.
Asunto(s)
Intrones/genética , Mutación/genética , Proteínas Nucleares , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , División Celular/genética , ARN Helicasas DEAD-box , Proteínas Fúngicas/genética , Expresión Génica/genética , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Fenotipo , Precursores del ARN/genética , Empalme del ARN/genética , ARN de Hongos/genética , Alineación de Secuencia , Factor de Empalme U2AFRESUMEN
The signal recognition particle (SRP) is a ribonucleoprotein required for targeting a subset of nascent pre-secretory proteins to the endoplasmic reticulum membrane. Of the six SRP polypeptides, the most highly conserved is Srp54p, a modular protein consisting of an amino-terminal (N) domain of unknown function, a central GTPase (G) domain, and a carboxyl-terminal (M) domain implicated in the recognition of both signal sequences and SRP RNA. To identify regions of Srp54p that interact with other SRP subunits or regulatory proteins, we carried out systematic mutagenesis of the fission yeast homolog, principally using a "clustered charged-to-alanine" strategy. Of the 35 alleles examined, 13 are unable to support growth, two confer cold-sensitivity, five confer heat-sensitivity, and 15 produce no discernible phenotype. The lethal and conditional mutations map throughout the protein to several conserved regions, confirming that these motifs play critical roles in Srp54p function. The effects of the amino-acid substitutions are analyzed with reference to the recently determined tertiary structures of the N/G domain and the intact protein from a thermophilic bacterium.