RESUMO
RmInt1 is a group II intron encoding a reverse transcriptase protein (IEP) lacking the C-terminal endonuclease domain. RmInt1 is an efficient mobile retroelement that predominantly reverse splices into the transient single-stranded DNA at the template for lagging strand DNA synthesis during host replication, a process facilitated by the interaction of the RmInt1 IEP with DnaN at the replication fork. It has been suggested that group II intron ribonucleoprotein particles bind DNA nonspecifically, and then scan for their correct target site. In this study, we investigated RmInt1 binding sites throughout the Sinorhizobium meliloti genome, by chromatin-immunoprecipitation coupled with next-generation sequencing. We found that RmInt1 binding sites cluster around the bidirectional replication origin of each of the three replicons comprising the S. meliloti genome. Our results provide new evidence linking group II intron mobility to host DNA replication.
RESUMO
Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.
Assuntos
Bactérias/genética , Genoma Bacteriano , Genômica , Íntrons , Elementos de DNA Transponíveis , DNA Bacteriano , Genômica/métodos , Fases de Leitura Aberta , Splicing de RNA , RNA BacterianoRESUMO
Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.
Assuntos
Íntrons/fisiologia , Proteínas Nucleares/biossíntese , Splicing de RNA/fisiologia , Ribonucleoproteínas/biossíntese , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Ribonucleoproteínas/genéticaRESUMO
Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3' exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3' splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3-IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.
Assuntos
Íntrons/genética , Precursores de RNA/genética , Splicing de RNA/genética , RNA Catalítico/genética , Elementos de DNA Transponíveis/genética , Éxons/genética , Mutação , Conformação de Ácido Nucleico , Precursores de RNA/química , Sinorhizobium meliloti/genéticaRESUMO
Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.
