RESUMEN
Group II introns are usually removed from precursor RNAs as lariats comprised of a circular component and a short 3' tail. We find that group II introns can also be excised as complete circles. Circle formation requires release of the 3' exon of a splicing substrate, apparently by a trans splicing mechanism. After 3' exon release, the terminal uridine of the intron attacks the 5' splice site, releasing the 5' exon and joining the first and last intron residues by a 2'-5' phosphodiester bond. RNA isolated from yeast mitochondria also contains circles, indicating that at least one group II intron (aI2) forms circles in vivo. Furthermore, analysis of RNA and DNA from certain mutant yeast strains shows that circular DNA introns exist and are produced by reverse transcription of RNA, rather than by ectopic homing.
Asunto(s)
Intrones/genética , Precursores del ARN/metabolismo , Empalme del ARN , ADN Ligasas/metabolismo , ADN Mitocondrial/química , Células HeLa , Humanos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , ARN/química , Precursores del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Levaduras/genéticaRESUMEN
Few point mutations have been described that specifically inhibit the second step of group II intron splicing. Furthermore, the effects of such mutations are typically not apparent unless the mutations are studied in the context of a substrate that harbors a very short 5' exon. Truncation of the 5' exon slows the second step of splicing. Once the second step has been slowed, the effects of point mutations can be seen. We report the unexpected observation that the deletion of a conserved GA dinucleotide dramatically inhibits the second step of splicing, even when the mutation is studied in the context of a full-length substrate. In contrast, we find that this mutation does not significantly affect the first step of splicing, unless the mutation is studied in combination with a second point mutation that is known to inhibit the first step. Even in that context, the effect of the GA deletion mutation on the first step is modest. These observations, together with the inferred location of the GA dinucleotide in the three-dimensional structure of the intron, suggest that this dinucleotide plays a particularly important role in the second step of splicing.
Asunto(s)
Empalme Alternativo , Intrones , Precursores del ARN/genética , ARN/genética , Transcripción Genética , Clonación Molecular , Secuencia Conservada , Fosfatos de Dinucleósidos , Exones , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Mutación Puntual , ARN/química , Eliminación de SecuenciaRESUMEN
Here we describe two methods for generating DNA fragments with single-stranded overhangs, like those generated by the activity of many restriction enzymes, by simple methods that do not involve DNA digestion. The methods, RNA-overhang cloning (ROC) and DNA-overhang cloning (DOC), generate polymerase chain reaction (PCR) products composed of double-stranded (ds) DNA flanked by single-stranded (ss) RNA or DNA overhangs. The overhangs can be used to recombine DNA fragments at any sequence location, creating "perfect" chimeric genes composed of DNA fragments that have been joined without the insertion, deletion, or alteration of even a single base pair. The ROC method entails using PCR primers that contain regions of RNA sequence that cannot be copied by certain thermostable DNA polymerases. Using such a chimeric primer in PCR would yield a product with a 5' overhang identical to the sequence of the RNA component of the primer, which can be used for directional ligation of the amplified product to other preselected DNA molecules. This method provides complete control over both the length and sequence of the overhangs, and eliminates the need for restriction enzymes as tools for gene engineering.
Asunto(s)
Clonación Molecular/métodos , ADN , Ingeniería Genética/métodos , ARN , Cartilla de ADN/genética , Exones , Globinas/genética , Humanos , Intrones , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores AMPA/genéticaRESUMEN
We report the use of engineered ribozymes to shuffle exon cassettes in vitro. Specifically, we have designed derivatives of a group II intron that insert into selected sites in the human tissue plasminogen activator (t-PA) mRNA. The insertion reaction links t-PA sequences to the group II intron sequences so that trans-splicing reactions catalyzed by the intron can be employed to shuffle the t-PA sequences. We expect these results to be generalizable, so that similar ribozymes can be designed to target any desired 13 nucleotide sequence. In principle, the reactions we describe here should be able to link any RNA molecule to any other RNA molecule at any selected point.
Asunto(s)
ARN Catalítico/metabolismo , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Activador de Tejido Plasminógeno/biosíntesis , Secuencia de Bases , Cartilla de ADN , Factor de Crecimiento Epidérmico/biosíntesis , Exones , Fibronectinas/biosíntesis , Ingeniería Genética/métodos , Humanos , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Empalme del ARN , ARN Catalítico/biosíntesis , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
I describe the self-splicing of an RNA that consists of exon sequences flanked by group II intron sequences. I find that this RNA undergoes accurate splicing in vitro, yielding an excised exon circle. This splicing reaction involves the joining of the 5' splice site at the end of an exon to the 3' splice site at the beginning of the same exon; thus, I term it inverse splicing. Inverse splicing provides a potential mechanism for exon scrambling, for exon deletion in alternative splicing pathways, and for exon shuffling in gene evolution.
Asunto(s)
Empalme Alternativo , Intrones , Precursores del ARN/metabolismo , Secuencia de Bases , Exones , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Especificidad por Sustrato , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
We describe a 98-base-pair region (-38 to +60) in the long terminal repeat of the Drosophila gypsy retrotransposon that is sufficient for accurate normal-level transcription. We find that, unlike most RNA polymerase II (pol II) promoters, the gypsy promoter includes downstream sequences that are required for full activity. Also unlike most pol II promoters, the gypsy promoter, which lacks a TATA motif, was found to have an essential sequence at the transcription initiation site, mutation of which abolishes transcription. These three uncommon features of the gypsy promoter may be characteristic of a subset of pol II promoters, exemplified by certain retrotransposons and developmental genes of Drosophila and by Tdt, the mouse terminal deoxynucleotidyl-transferase (TdT) gene.
Asunto(s)
Elementos Transponibles de ADN , Drosophila/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Plásmidos , Poli A/genética , Poli A/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificación , ARN Polimerasa II/genética , ARN Mensajero , TransfecciónRESUMEN
A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.
Asunto(s)
Intrones , Mitocondrias/metabolismo , Empalme del ARN , Levaduras/genética , Electroforesis en Gel de Poliacrilamida , Levaduras/ultraestructuraRESUMEN
Reaction parameters were modified to enhance the in vitro reaction rate and to reveal partial and novel reactions of the group II intron 5g of the mitochondrial gene from Saccharomyces cerevisiae encoding cytochrome c oxidase subunit I. One alteration yields separate 5'- and 3'-exons plus linear excised intron as the main products. A linear reaction intermediate, containing intron and 3'-exon, and products resulting from cleavages at two unexpected sites were identified. Spliced exon "reopening," a novel reaction between excised intron and spliced exons, appears responsible for separate 5'- and 3'-exon products.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Genes Fúngicos , Intrones , Empalme del ARN , Saccharomyces cerevisiae/genética , Sulfato de Amonio/farmacología , Exones , Magnesio/farmacología , Cloruro de Magnesio , Cloruro de Potasio/farmacología , ARN de Hongos/genética , Saccharomyces cerevisiae/enzimología , Cloruro de Sodio/farmacologíaRESUMEN
We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.