Transposon-encoded nucleases use guide RNAs to selfishly bias their inheritance

Abstract

Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605 elements undergo peel-and-paste transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB/IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related IS elements from Geobacillus stearothermophilus that encode diverse TnpB/IscB orthologs, and showed that a single TnpA transposase was active for transposon excision. The donor joints formed upon religation of IS-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB/IscB nucleases, and co-expression of TnpB together with TnpA led to significantly greater transposon retention, relative to conditions in which TnpA was expressed alone. Remarkably, TnpA and TnpB/IscB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between collaborating transposase and nuclease proteins. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.

The Transposon-Encoded Protein TnpB Processes Its Own mRNA into ωRNA for Guided Nuclease Activity

“… 51 The limited TAM diversity of TnpBs may be explained by the co-evolution of TnpA and TnpB, as TnpA also utilizes the TAM to recognize the transposon ssDNA for excision and insertion. 1 , 2 , 6 , 43 , 52 Although it is not yet clear how, if at all, TnpB interacts with TnpA and the transposon DNA, the fact that both proteins possess the same TAM sequence is a clear constraint on the TAM diversity.…”

“…According to recent evidence, TnpB improves retention of its transposon whereby an ωRNA expressed from one transposon locus targets transposon-lacking versions of that locus in the same cell, that is, loci where the transposon has not yet inserted or those which have undergone excision. 6 Therefore, it appears that TnpB DNA cleavage serves to fix the transposon-containing locus in the population by eliminating loci that have undergone excision 1 and/or homing to un-inserted loci. 2 , 54 Under this hypothesis, the TnpB mRNA could serve as a temporally-sensitive signal of active transcription of the transposon and its continued presence in the genome and, therefore, exert negative feedback on TnpB DNA cleavage to prevent unnecessary genome instability.…”

“… 4 TnpB is not known to be a transposase, but rather is thought to function akin to a homing endonuclease 5 that prevents loss of the transposon upon excision. 1 , 2 , 6 …”

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

“…52 Experimental reconstitution of IS200/IS605 elements from Geobacillus stearothermophilus showed that IscB targeted the donor joint for cleavage, allowing for retention of the transposable element in the donor strand following peel-out-paste-in transposition. 53 Type II systems have a nearly uniform gene composition and locus organization compared to the diversity of class 1 CRISPR. Nevertheless, there are three type II subtypes, of which two encompass distinct, signature genes.…”

“…Recently, it has been shown that IscB is a guide RNA-dependent endonuclease that is potentially involved in RNA-guided homing of the respective transposons, in a manner similar to that proposed for other IS200/IS605 transposons . Experimental reconstitution of IS200/IS605 elements from Geobacillus stearothermophilus showed that IscB targeted the donor joint for cleavage, allowing for retention of the transposable element in the donor strand following peel-out-paste-in transposition …”