Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes

Abstract

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Graphical Abstract

Figure 1.

Fanzors and a unique clade of TnpBs share unusual active site signatures. (A) Depiction of loci architecture of Fanzor and TnpB containing transposons. LE and RE correspond to left-end and right-end transposon boundaries. The transposon associated motif (TAM) and guide region are highlighted in orange and red, respectively. (B) Illustration of Fanzor/TnpB complexed with their right-end RNAs (reRNAs) restricting DNA. Fanzors/TnpBs recognize DNA containing both a TAM and sequence complementarity to the guide region of the reRNA. (C) Fanzors and TnpBs with RuvC (blue) and Zinc-Finger (grey) domain annotations. Red ticks indicate locations of D-E-D RuvC triad residues. Fanzors and TnpBs are differentiated by the DPG and DΦG, and E_alt and E_can signatures in the RuvC domain, respectively. Multiple Sequence Alignment encompassing RuvC and Zinc-Finger domains of Fanzors and TnpBs is shown. (D) Maximum likelihood tree of TnpBs and Fanzors annotated by the RuvC features. See legend for the annotations depicted in the track indicated below. (E) Pie charts detailing phyla and IS family classification of the prokaryotic group of TnpBs closely related to Fanzor2s (pro-Fanzors) in (D).

Figure 2.

Fanzor2s have been captured by non-IS607 transposons on multiple occasions. (A) Depiction of pre-integration and post-integration loci architecture of various TnpB and Fanzor encoding transposons. (B) Maximum likelihood phylogenetic tree of TnpBs, Fanzor2s, and Fanzor1s (gray, green and blue in the outer track) annotated based on their transposon associations (inner track). The numbers correspond to transposons and their loci architecture in (A).

Figure 3.

IS607 reRNAs evolved to overcome transposase imposed restrictions. (A) Depiction of loci architecture of the ISXfa1 transposon. Inlaid graph shows mapping of small-RNAs to the locus. (B) Overview of IS607 transposon life-cycle. LE and RE correspond to left-end and right-end transposon boundaries. LF and RF correspond to left and right-flanking nucleotides encoded in the insertion site. IS607 transposons mobilize as a double-stranded circular DNA intermediate and integrate into the acceptor site, both events happening via crossover between GG dinucleotides at the LE and RE of the transposon, and the donor or acceptor sites. (C) Results of TAM depletion assays with variable reRNA boundaries. Wild-type ISXfa1 shows depletion of GGG motif, whereas RF deletion mutant loses activity, demonstrating that the RF nucleotide is part of the reRNA scaffold. This is in contrast to the reRNA architecture of IS200/605-associated TnpBs, whose scaffold ends with the transposon boundaries.

Figure 4.

reRNA secondary structures are conserved between TnpBs and Fanzor2s across diverse transposon families. (A) Predicted secondary structures of reRNAs associated with various transposons. Right-flanking nucleotides (RF) and target site duplications (TSD) are colored in red. Putative pseudoknot (PK) interactions are highlighted in black dashed boxes. Transposase recognized bases required for transposition are circled in black. (B) Experimental design of PK interaction assays. Plasmids containing ISXfa1 TnpB and reRNAs with PK mutations were transformed into E. coli harboring an ampicillin resistance (AmpR) plasmid. As ISXfa1 TnpB restricts the AmpR plasmid and leads to ampicillin sensitization, effect of PK mutations was assayed via ampicillin selection. (C) Results of the E. coli plasmid interference assay confirming the PK interactions. ISXfa1 TnpB activity was disrupted by the U-130A mutation and subsequently rescued by the U-130A/A-3U mutation.

Figure 5.

reRNAs of TnpBs and Fanzors use similar strategies to co-function with their transposases. (A) Locus organization of Fanzor1 encoding transposon in S. frugiperda ascovirus 1a (SfAV). Red box indicates target-site duplication (TSD) which appears as a result of transposition. Boxed triangle indicates terminal inverted repeats (TIR). Inlaid graph shows mapping of small RNA-seq reads onto the reference locus encoding the Fanzor. (B) Workflow of in vivo Fanzor1 reRNA expression assays using SfAV and S. frugiperda larvae. (C) Putative secondary structure of SfAV Fanzor1 reRNA. TSD is color coded in red. Region where pseudoknot (PK) was expected is highlighted in black dashed box. Bases corresponding to TIR recognized by the transposase are circled. (D) Graphical representation of how boundaries are redefined for reRNAs occurring in different transposon families. Right-flanking nucleotide (RF) in IS607 transposons and TSD in eukaryotic transposons are both incorporated into the reRNA scaffold. (E) Schematic depicting MBP-GFP fused SfAV Fanzor1 construct. Picture of S. frugiperda larvae infected with recombinant baculovirus encoding his-tagged Fanzor (left) and MBP and GFP tag fused Fanzor is shown (right). (F) Western blot confirms expression of SfAV protein in vivo using anti-his and anti-GFP primary antibodies. Lane 1: Chameleon ladder. Lanes 2–5: his-tagged Fanzor containing lysate dilutions. Lanes 6–9: MBP and GFP tag fused Fanzor containing lysate dilutions (the full blot is shown in Supplementary Figure S6).

Figure 6.

Structural comparisons of TnpBs and Fanzors highlight key conserved features of the Fanzor lineage. (A) Example structures of IS200/605 and IS607-associated TnpBs and Fanzors. All models are AlphaFold2 models, except for that of ISDra2 (PDB-ID: 8EXA). Species information: ISDra2, Deinococcus radiodurans. ISXfa1, Xylella fastidiosa. ISSoc2, Synechococcus sp. ISvMimi1, Acanthamoeba polyphaga mimivirus. SfAV, Spodoptera frugiperda ascovirus 1a. Pca, Phytophthora cactorum. (B) Schematic representation of the structural organization of TnpBs and Fanzor2s, and regions of novel insertions in Fanzor1s. (C) AlphaFold2 structures and protein topology diagram of the two types of RuvC active site organization specific to proteins in (A). Dashed circles in the left, and colored circles in the right panels, indicate the RuvC2 glutamate residue in the canonical E_can and the alternative E_can locations. (D) CLANS analysis for TnpBs and Fanzors sequences. Note that the lepidopteran and viral Fanzors bridge Fanzor2s and the rest of Fanzor1s. (E) The proposed evolutionary of Fanzors inferred using extant sequences. Prokaryotic TnpBs with DPG and E_alt motifs, or pro-Fanzors, evolved into eukaryotic Fanzor2s via horizontal transfer. Acquisition of Fanzor2s with novel non-IS607 eukaryotic transposons gave rise to Fanzor2*s, one instance of which gave rise to Fanzor1s.