Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA

Abstract

The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers. Reversal of the group I intron self-splicing reaction, termed reverse splicing, coupled with reverse transcription and genomic integration potentially mediate an RNA-based intron mobility pathway. Compared to the well characterized endonuclease-mediated intron homing, reverse splicing is less specific and represents a likely explanation for many intron transpositions into new genomic sites. However, the frequency and general role of an RNA-based mobility pathway in the spread of natural group I introns is still unclear. We have used the twin-ribozyme intron (Dir.S956-1) from the myxomycete Didymium iridis to test how a mobile group I intron containing a homing endonuclease gene (HEG) selects between potential insertion sites in the small subunit (SSU) rRNA in vitro, in Escherichia coli and in yeast. Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site. Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species.

The recent transfer of a homing endonuclease gene

“…Target 2 was the intron-containing rDNA allele. For Intron 1, Target 2 was the SphI-linearized pYGAL-DirS956-1 plasmid (12) that contains the Intron 1 (i.e. Dir.S956-1) and some flanking rDNA exons.…”

Reverse splicing of a mobile twin-ribozyme group I intron into the natural small subunit rRNA insertion site

“…Analysis of the entire E. coli SSU rRNA confirms that intron integration was detected exclusively at site S956 (natural integration site), and contrasts the more relaxed stringency obtained from reverse splicing of the Tetrahymena intron in E. coli [12][13][14]. Finally, reverse splicing was found to be dependent on the intron splicing ribozyme DiGIR2, but independent of the processing ribozyme (DiGIR1) and the I-DirI HEG [24].…”

“…rRNAs of E. coli and yeast, was subsequently expressed in these cells and, finally, the generated recombined RNAs were evaluated by Northern-blot analysis and reverse transcriptase-PCR sequencing [24]. Furthermore, we also investigated the roles of intron domains (ribozymes and HEG) in reverse splicing by including various deletion constructs of the intron in the analysis ( Figure 2B).…”

Nuclear group I introns in self-splicing and beyond

“…Here, an excised intron attacks the ligated exons at the intron-lacking cognate insertion site and integrates into the precursor RNA. Reverse splicing has been reported in vitro , in yeast and in Escherichia coli for both the Tetrahymena intron [44-46] and the Didymium intron [47]. Interestingly, in vitro integration of full-length circular intron RNA has also been noted, suggesting a biological role for the circularization pathway in propagation and intron spread [47].…”

“…Reverse splicing has been reported in vitro , in yeast and in Escherichia coli for both the Tetrahymena intron [44-46] and the Didymium intron [47]. Interestingly, in vitro integration of full-length circular intron RNA has also been noted, suggesting a biological role for the circularization pathway in propagation and intron spread [47]. Less frequently, reverse splicing may lead to intron spread at novel rRNA sites, and may explain the low frequency transposition features of nuclear group I introns observed in phylogenetic studies [5,22,48].…”