RESUMEN
Three introns whose splicing is activated during meiosis in S. cerevisiae contain a Mer1p-dependent splicing enhancer. The enhancer can impose Mer1p-activated splicing upon the constitutively spliced actin intron provided the basal splicing efficiency of actin is first reduced. Of several nonessential splicing factors tested, only the U1 snRNP protein Nam8p is indispensable for Mer1 p-activated splicing. We show that Mer1p associates with the U1 snRNP even in the absence of Nam8p or pre-mRNA. This work defines a yeast splicing enhancer and shows that constitutively expressed and cell type-specific factors combine to regulate splicing of a specific subset of pre-mRNAs including SPO70, MER2, and MER3.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Intrones , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Bases , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Precursores del ARN/genética , Precursores del ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome. The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence. We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome. Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80%. Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/ MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/ MTR2 produces at least five different spliced mRNAs. One gene (YGR225W/ SPO70 ) has an intron whose removal is activated during meiosis under control of the MER1 gene. We found eight new introns, suggesting that numerous introns still remain to be discovered. The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast.
Asunto(s)
Empalme Alternativo , Regulación Fúngica de la Expresión Génica , Intrones , Meiosis/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/citologíaRESUMEN
Introns have typically been discovered in an ad hoc fashion: introns are found as a gene is characterized for other reasons. As complete eukaryotic genome sequences become available, better methods for predicting RNA processing signals in raw sequence will be necessary in order to discover genes and predict their expression. Here we present a catalog of 228 yeast introns, arrived at through a combination of bioinformatic and molecular analysis. Introns annotated in the Saccharomyces Genome Database (SGD) were evaluated, questionable introns were removed after failing a test for splicing in vivo, and known introns absent from the SGD annotation were added. A novel branchpoint sequence, AAUUAAC, was identified within an annotated intron that lacks a six-of-seven match to the highly conserved branchpoint consensus UACUAAC. Analysis of the database corroborates many conclusions about pre-mRNA substrate requirements for splicing derived from experimental studies, but indicates that splicing in yeast may not be as rigidly determined by splice-site conservation as had previously been thought. Using this database and a molecular technique that directly displays the lariat intron products of spliced transcripts (intron display), we suggest that the current set of 228 introns is still not complete, and that additional intron-containing genes remain to be discovered in yeast. The database can be accessed at http://www.cse.ucsc.edu/research/compbi o/yeast_introns.html.
Asunto(s)
Biología Computacional , Genoma Fúngico , Intrones/genética , Saccharomyces cerevisiae/genética , Bases de Datos como Asunto , Cadenas de Markov , Precursores del ARN/genética , Empalme del ARN/genética , ARN Nuclear Pequeño/genética , Proteínas Ribosómicas/genética , Empalmosomas/genéticaRESUMEN
It has been hypothesized that selections for aptamers with high affinity for a given target molecule will of necessity identify aptamers that have high specificity for that target. We have attempted to assess this hypothesis by selecting aptamers that can bind to MS2 coat protein or to single- or double-substitution variants of the coat protein. Some aptamers selected to bind MS2 coat protein or its variants were mildly specific for their cognate targets, discriminating by two- to fourfold against closely related proteins. Specificity determinants on both the coat proteins and the aptamers could be identified. However, many aptamers could readily bind to each of the different coat proteins. The identification of such aptamer 'generalists' belies the proposed relationship between the affinities and specificities of selected RNA ligands. These results imply that, while aptamers may not finely discriminate between closely related targets, neither will their binding be negated by mutations in targets. Aptamer pharmaceuticals may therefore better resist the evolution of resistance.
Asunto(s)
Allolevivirus/genética , Proteínas de la Cápside , Cápside/genética , Variación Genética , Oligorribonucleótidos/química , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Unión Competitiva , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Oligorribonucleótidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
The coat proteins of the RNA phages MS2 and Qbetaare structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qbeta. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qbetatranslational operator led to the isolation of several MS2 mutants that acquired binding activity for QbetaRNA. Some of these also had reduced abilities to repress translation from the MS2 translational operator. These changes in RNA binding specificity were the results of substitutions of amino acid residues 87 and 89. Additional codon- directed mutagenesis experiments confirmed earlier results showing that the identity of Asn87 is important for specific binding of MS2 RNA. Glu89, on the other hand, is not required for recognition of MS2 RNA, but prevents binding of QbetaRNA.
Asunto(s)
Allolevivirus/genética , Proteínas de la Cápside , Cápside/metabolismo , Levivirus/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Cápside/genética , Codón , Evolución Molecular Dirigida , Biblioteca de Genes , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Regiones Operadoras Genéticas , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la EspecieRESUMEN
The coat proteins of the RNA bacteriophages Qbeta and MS2 are specific RNA binding proteins. Although they possess common tertiary structures, they bind different RNA stem loops and thus provide useful models of specific protein-RNA recognition. Although the RNA-binding site of MS2 coat protein has been extensively characterized previously, little is known about Qbeta. Here we describe the isolation of mutants that define the RNA-binding site of Qbeta coat protein, showing that, as with MS2, it resides on the surface of a large beta-sheet. Mutations are also described that convert Qbeta coat protein to the RNA binding specificity of MS2. The results of these and other studies indicate that, although they bind different RNAs, the binding sites of the two coat proteins are sufficiently similar that each is easily converted by mutation to the RNA binding specificity of the other.
Asunto(s)
Proteínas de la Cápside , Cápside/metabolismo , Colifagos/metabolismo , ARN Viral/metabolismo , Sitios de Unión , Levivirus/metabolismo , Mutagénesis , Conformación de Ácido Nucleico , Regiones Operadoras GenéticasRESUMEN
The coat proteins of RNA phages MS2 and GA are specific RNA-binding proteins which function to encapsidate viral RNA and to translationally repress synthesis of the viral replicase. The two proteins have highly homologous amino acid sequences, yet they show different RNA binding specificities, recognizing RNA stem-loop structures which differ primarily in the nucleotide sequences of their loops. We sought to convert MS2 coat protein to the RNA binding specificity of GA through the introduction of GA-like amino acid substitutions into the MS2 coat protein RNA-binding site. The effects of the mutations were determined by measuring the affinity of the coat protein variants for RNA in vitro and by measuring translational repression in vivo. We found five substitutions that affect RNA binding. One dramatically reduces binding of MS2 coat protein to both operators. Three others compensate for this defect by nonspecifically strengthening the interaction. Another substitution accounts for the ability to recognize the differences in the RNA loop sequence.