Antagonistic conflict between transposon-encoded introns and guide RNAs.

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. IS605family 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 from Clostridium 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.

Transposon-encoded nucleases use guide RNAs to promote their selfish spread.

Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity 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 defence.

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

Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS 200 /IS 605 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.

ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants.

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin-mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin-associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N-terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME-deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony-sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)-dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin-independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild-type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1-dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT-dependent trafficking on endocytic recycling and the PM.