Antagonistic conflict between transposon-encoded introns and guide RNAs

Abstract

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life, presumably due to the critical roles they play in transposon proliferation. IS605-family TnpB homologs function in bacteria as programmable homing endonucleases by exploiting transposon-encoded guide RNAs to cleave vacant genomic sites, thereby driving transposon maintenance through DSB-stimulated homologous recombination. Whether this pathway is conserved in other genetic contexts, and in association with other transposases, is unknown. Here we uncover molecular mechanisms of transposition and RNA-guided DNA cleavage by IS607-family elements that, remarkably, also encode catalytic, self-splicing group I introns. After reconstituting and systematically investigating each of these biochemical activities for a candidate ‘IStron’ derived fromClostridium botulinum, we discovered sequence and structural features of the transposon-encoded RNA that satisfy molecular requirements of a group I intron and TnpB guide RNA, while still retaining the ability to be faithfully mobilized at the DNA level by the TnpA transposase. Strikingly, intron splicing was strongly repressed not only by TnpB, but also by the secondary structure of ωRNA alone, allowing the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work explains how diverse enzymatic activities emerged during the selfish spread of IS607-family elements and highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.

Emergence of RNA-guided transcription factors via domestication of transposon-encoded TnpB nucleases

“…Canonical tnpB genes in bacteria, alongside their ωRNA guides, are encoded within IS200/ IS605- or IS607-family transposons that can be straightforwardly identified using both comparative genomics and by defining the transposon left end (LE) and right end (RE) 1–4 ; in addition, a hallmark feature is their frequent association with tnpA transposase genes 29,37 ( Fig. 2a, left ).…”

“…4a ). Previous work has shown that TnpB proteins catalyze processing of their own guides through a RuvC-dependent mechanism 4,47 , however the absence of an intact catalytic triad in TldR proteins suggests that the mature gRNA may instead represent the sequence protected from cleavage by cellular ribonucleases.…”

“… AbstractTransposon-encoded tnpB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination 1–4 . This widespread gene family was repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12 5,6 .…”

“…The ‘universal’ cas1 gene encodes an integrase responsible for preserving memories of past infections by splicing viral DNA fragments into the CRISPR array, in a biochemical reaction reminiscent of transposon integration 16,17 , and ancestral CRISPR-less Cas1 homologs perform similar reactions within the context of transposable elements known as casposons 18,19 . Analogously, recent studies have demonstrated that the biochemical activities of Cas9 and Cas12, which perform RNA-guided DNA binding and cleavage during an immune response, can be traced back to ancestral transposon-encoded nucleases known as IscB and TnpB, respectively 1,2 , which perform similar reactions to promote transposon maintenance and spread 3,4 . In turn, nuclease-deficient CRISPR-Cas systems have been repurposed by transposons on at least four independent occasions, to facilitate a novel RNA-guided DNA integration pathway mediated by CRIS-PR-associated transposases (CASTs) 20–22 .…”

“…1a ). We recently demonstrated that TnpA-mediated transposition generates a scarless excision product at the donor site that is rapidly recognized and cleaved by TnpB-ωRNA complexes, in a reaction dependent on RNA-DNA complementarity and the presence of a cognate transposon/target-adjacent motif (TAM), leading to transposon reinstallation via DSB-mediated homologous recombination 3,4 . RNA-guided DNA cleavage thus first arose as a biochemical strategy to prevent permanent transposon loss and was only later adapted for a role in antiviral immunity.…”