Characterization of the splicing reaction of the sinorhizobium meliloti rmint1 group ii intron

Resumen

Group II introns are both auto-catalytic RNAs and retrotransposable elements. They were initially found in organellar genomes, and later in bacterial and archaeal genomes. Several data suggest that they are the ancestors of eukaryotic spliceosomal introns. They consist of a catalytically active intron RNA (ribozyme) and an optional intron-encoded protein (IEP), which assists the intron in the splicing and mobility processes. The secondary structure of the ribozyme consists of six helical domains radiating from a central wheel, which in turn interact to each other creating a complex tertiary networking. Group II introns are spliced via a lariat o linear intermediate, and can retrohome in cognate intronless target sites they recognized through base pairing. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. This group II intron is excised, in vivo and in vitro, as both intron lariats and putative intron circles. However, until date, the complete splicing reaction in vivo remains to be elucidated. A lacZ reporter gene system, northern blotting and real-time reverse transcription were carried out to investigate RmInt1 splicing activity (Chapter 1 and 2). Splicing efficiency of 0.07 ± 0.02 % was recorded. These findings suggest that bacterial group II introns function more like retroelements than spliceosomal introns. Previous studies on the in vitro self-splicing of RmInt1 intron showed an inefficient reaction characterized by the formation of unconventional side products. By testing different RmInt1-transcritp contexts and reaction conditions, we obtained a RmInt1-construction by eliminating an IBS1-like sequence present in the first 10 or 11 nucleotides of the 3' exon from the natural target IS2011-2 insertion sequence (Chapter 4). The new construct prevented the formation of unusual truncated side products previously observed, producing an increment of the amplitude of the reaction. Finally, we analyzed the effect of point mutations in conserved positions of the ribozyme of RmInt1 on the efficiency of first and second splicing steps in vivo and in vitro (Chapter 3 and 4). We identified the occurrence of tertiary interactions common to other group IIB introns, such as the ¿-¿', EBS3-IBS3 and the non Watson-Crick interaction between first and penultimate nucleotides of the intron. In addition, we proved the existence of structural motifs conserved in group II introns as the triplex between domain V and the nucleotides within the linker region J2/3. Mutations disrupting interaction between components of this motif resulted in the alteration of both steps of splicing in vivo and in vitro, which supports the occurrence of a unique active site catalyzing both steps of the splicing reaction.