Natural diversity of group II introns

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Mobile DNAs are selfish genetic elements that proliferate without immediate benefits to their hosts; however, over time they have been the source of many useful evolutionary inventions such as telomerase. The dynamics by which mobile DNAs are agents of mutagenesis, yet also enablers of adaptation, are not well understood. Group II introns are a type of mobile DNA in bacteria that possesses properties of both catalytic RNAs and retroelements. The sequence of the introns encodes two components: 1) a catalytic RNA component that carries out a self-splicing reaction; and 2) an intron-encoded reverse transcriptase that facilitates the splicing reaction and allows the introns to integrate into new genomic locations. Group II introns are exceptionally versatile, having evolved into many variant forms, with the most important arguably being eukaryotic spliceosomal introns and non-LTR retrotransposons, which make up ~25% and ~20% of the human genome, respectively. Past research in the Zimmerly lab has helped to establish the breadth of group II introns that exist in nature. Here we will further investigate how group II introns have differentiated and evolved to acquire new characteristics and biological impacts, with three aims. 1) We will use bioinformatic tools to curate group II introns in GenBank. In addition to generating an updated census, this is expected to identify new intron variants. Phylogenetic analysis of the accumulated sequence data will allow reconstruction of the evolution of a major lineage of the introns. 2) We will investigate the phenomenon of binary splicing, which is carried out by a subset of group II introns. While most group II introns recognize their 5' exon by a single exon-binding motif, a subset can alternately use either of two exon-binding motifs. We will dissect the mechanism for binary splicing and determine its effect on intron mobility, which we hypothesize to be the biological function of the unusual splicing property. 3) We will examine the unprecedented reaction of alternative splicing of the C.te.I1 intron in Clostridium tetani. Here an intron has taken on a presumably beneficial role, allowing a single 5' exon to be ligated to one of four downstream exons, producing five protein variants from a single genetic locus. To address the biological consequences of this reaction, we will investigate the intron in three patient isolates of C. tetani and compare data to a reference strain. The genomes of the three strains will be sequenced, and RNA-Seq will be used to detect splicing and transcription properties. Together these three approaches will help illuminate the functional variants and adaptations of group II introns, and contribute to our understanding of how mobile DNAs and ribozymes adapt and diversify over evolutionary time, sometimes becoming domesticated to take on useful roles within their bacterial hosts.