# Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons

Hugo Vaysset, Chance Meers, Jean Cury, Aude Bernheim, Samuel Sternberg

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Hugo Vaysset, Chance Meers, Jean Cury, Aude Bernheim, Samuel Sternberg. Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons. Nature Microbiology, 2025, 10 (1), pp.20-27. 10.1038/s41564-024-01889-2. hal-05230530

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# Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS*110* transposons

Hugo Vaysset<fnref n="1" />,<fnref n="2" />,<fnref n="5" />, Chance Meers<fnref n="3" />,<fnref n="5" />, Jean Cury<fnref n="1" />, Aude Bernheim<fnref n="1" />,⁻<fnref n="3" />, Samuel H. Sternberg<fnref n="3" />,<fnref n="4" />,⁻<fnref n="3" />

## Abstract

Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS*110*-family transposases, which catalyse RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-*O*-methylation using C/D-box snoRNAs. On the basis of conservation of protein sequence, domain architecture, three-dimensional structure and non-coding RNA features, alongside phylogenetic analyses, we propose that programmable RNA modification emerged through the exaptation of components derived from IS*110*-like transposons. These findings underscore how recurrent domestication events of transposable elements have driven the evolution of RNA-guided mechanisms.

Transposons are present in all domains of life and increase genetic diversity via horizontal gene transfer and genome rearrangements. Genes encoded by transposons have also been repeatedly repurposed and exapted during the evolution of essential cellular functions, ranging from V(D)J recombination<fnref n="1" /> and programmed genome elimination<fnref n="2" />, to RNA-centric

Reprints and permissions information is available at www.nature.com/reprints.

ªCorrespondence and requests for materials should be addressed to Aude Bernheim or Samuel H. Sternberg.
aude.bernheim@pasteur.fr; shsternberg@gmail.com.

Author contributions

C.M. and S.H.S. conceived the project. C.M. and H.V. performed protein structural predictions and analyses. A.B., J.C. and H.V. designed and interpreted systematic homology searches and phylogenetic analyses, which were performed by H.V. All authors discussed the data and wrote the manuscript.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Competing interests

The authors declare no competing interests.

Extended data is available for this paper at https://doi.org/10.1038/s41564-024-01889-2.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41564-024-01889-2.

<footnote n="1">¹Molecular Diversity of Microbes Lab, CNRS UMR3525, Institut Pasteur, Paris, France.</footnote>

<footnote n="2">²AgroParisTech, Université Paris-Saclay, Paris, France.</footnote>

<footnote n="3">³Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.</footnote>

<footnote n="4">⁴Howard Hughes Medical Institute, Columbia University, New York, NY, USA.</footnote>

<footnote n="5">⁵These authors contributed equally: Hugo Vaysset, Chance Meers.</footnote>
processes such as pre-mRNA splicing³ and RNA-templated telomere maintenance⁴. In bacteria, RNA-guided DNA endonucleases encoded by CRISPR-Cas immune systems, including Cas9 and Cas12, were recently shown to have emerged from ancestral RNA-guided nucleases encoded by diverse transposons⁵⁻⁷. Interestingly, RNA-guided functions are also found in key biological maintenance pathways such as RNA-guided RNA modification⁸ and RNA-mediated splicing⁹, but their evolutionary origins are unknown.

In archaea and eukarya, C/D-box small nucleolar RNAs (snoRNAs) guide the sequence-specific 2'-*O*-methylation of ribosomal RNA (rRNA)¹⁰, a crucial step for accurate ribosomal processing and assembly¹¹. This methylation is enabled by Nop-family proteins —including archaeal Nop5 and its eukaryotic homologues, Nop56 and Nop58—which are integral components of the C/D-box ribonucleoprotein (RNP) complex. Together with snoRNAs, the RNA-binding protein L7Ae (or its eukaryotic counterpart, 15.5 kDa/Snu13p) and the methyltransferase fibrillarin, this complex meticulously directs site-specific RNA methylation¹⁰. Prp31, another member of the Nop5 family, facilitates essential RNA interactions in the U4/U6 di-snRNP spliceosomal complex, thereby supporting the assembly process crucial for RNA splicing¹². Remarkably, our findings suggest that the origin of RNA-guided activities for both C/D-box snoRNAs and U4/U6 small nuclear RNAs (snRNAs) can be traced back to prokaryotic IS *110*-like transposons, which similarly use guide RNA sequences, termed bridge RNAs, for programmable DNA recombination¹³,¹⁴.

IS *110*-family transposons are insertion sequence (IS) transposable elements found in bacterial and archaeal genomes that encode a unique transposase featuring a DEDD active site, making it more similar to RuvC-like Holliday junction resolvases (HJR)¹⁵ than to conventional DDE family transposases. While exploring homologues of IS *110* transposase (hereafter referred to as IS *110* Tnp for simplicity; Methods) using HHpred¹⁶ and Foldseek¹⁷, we noticed an unexpected structural similarity to Nop5 and Prp31 proteins, both of which share a Nop domain, suggesting a potential shared ancestral relationship (Extended Data Fig. 1). Surprisingly, we noticed similar secondary structures when comparing bridge RNAs (IS *110*) and snoRNAs (C/D-box), including large bulged-out regions that function as paired guide RNA-like sequences (Fig. 1a). On the basis of this structural similarity and the recent discovery that IS *110* and IS *1111*-family Tnp homologues use bridge guide RNAs during DNA transposition¹³,¹⁴, we hypothesized that the archaeal and eukaryotic mechanisms for RNA-guided RNA modification originated from primordial RNA-guided DNA recombination processes found in IS *110*-like elements, which later also became essential for RNA splicing.

We began by investigating these similarities using both computationally predicted and recently determined Tnp structures from IS*621* (ref. 18), an IS *110*-family transposon, to compare with experimental Nop5 (ref. 19) and Prp31 (ref. 12) structures. The IS *110* Tnp forms a tetrameric protein complex, as has been reported for archaeal snoRNP complexes²⁰, with each dimer component housing a guide RNA and its respective target or donor DNA sequence (Extended Data Fig. 2a,b)¹⁸. This examination revealed pronounced structural homology between IS *110* Tnp and Nop5, including an N-terminal RuvC-like domain, a coiled-coil region and a C-terminal α-helical domain; Prp31 also exhibits a similar fold, but lacks the N-terminal domain (Fig. 1b,d). The presence of a catalytically inactivated RNaseH/
RuvC-like fold²¹ in Nop5 (Fig. 1c), which has not been previously reported, together with structural rearrangements and an α-helical insertion that repositions this domain relative to the coiled-coil domain, highlights an evolutionary adaptation that favoured fibrillarin recruitment over nucleic acid cleavage, thereby supporting a newly exapted function within the larger RNP complex (Fig. 1c and Extended Data Fig. 2c,d). The coiled-coil domain facilitates dimerization for both *IS* *110* Tnp¹⁸ and Nop5 (ref. 22) (Fig. 1b and Extended Data Fig. 3a,b), followed by the C-terminal region that, in the case of *IS* *110*, is crucial for binding both DNA and bridge RNA¹⁸; in Nop5, this C-terminal region interacts with the RNA-binding protein L7Ae to facilitate C/D-box RNA binding¹⁰. Both proteins exhibit a prominent protrusion that separates the two guide strands (Extended Data Fig. 3c), and in *IS* *110*, this protrusion also contains the Tnp active site, positioning the catalytic serine residues adjacent to the RuvC domain to form a composite active site¹⁸,²³. Focusing on nucleic acid ligands, the overall structures of the bridge RNA and C/D-box RNA not only exhibit similarity but also appear to be positioned similarly within the complex, along with their cognate target nucleic acid molecule (Fig. 1e). In the case of Prp31, the overall positioning of the RNA within the complex is different¹², but each U4 snRNA binds similarly to the C-terminal domain (Extended Data Fig. 3d). Altogether, the pronounced structural homology, RNA binding similarities and shared mechanisms across these proteins underscore their evolutionary adaptability and illustrate how similar architectures have been repurposed for distinct yet related functions.

To systematically identify all Nop-family homologues, we used sensitive searches with either Foldseek¹⁷ or HHpred¹⁶. We identified a set of homologous proteins that includes archaeal Nop5 and eukaryotic Nop56/Nop58 homologues, eukaryotic Prp31 and prokaryotic *IS* *110* Tnp (Methods, and Supplementary Tables 1 and 2). More exhaustive homology searches against a database of 30,255 prokaryotic and eukaryotic genomes failed to reveal homologues outside these three families (Supplementary Table 3), suggesting an absence of additional proteins that exhibit this particular domain composition. When we analysed the N-terminal domain of the *IS* *110* Tnp in isolation, homologues within the family of RuvC-like HJRs were identified (Supplementary Table 3)²¹,²⁴,²⁵. Homology searches using HHpred starting from the Nop5- and Prp31-encoded Nop domain alone robustly retrieved the C-terminal region of the *IS* *110* Tnp (Pfam PF02371, probability = 0.96; Supplementary Table 4), and a reciprocal search starting from the ‘Nop’ domain of the *IS* *110* Tnp similarly returned the homologous domains from Nop5 and Prp31, with high confidence (Pfam PF01798, probability = 0.96; Supplementary Table 4). Collectively, this reciprocal retrieval provides strong support for a shared ancestry linking the *IS* *110* Tnp with Nop5- and Prp31-family proteins.

We next investigated whether the Nop5 family emerged from *IS* *110* Tnp. Testing this hypothesis required constructing a phylogeny to evaluate the evolutionary relationship between *IS* *110* Tnp and Nop5, relative to other protein families. Since sensitive full-length protein homology searches did not yield homologues beyond the *IS* *110* Tnp, Nop5 and Prp31 families, we focused on each individual protein domain (N-terminal, coiled-coil, C-terminal), retrieved all corresponding homologues, built domain-level alignments and concatenated them to construct a phylogeny (Fig. 2a). This method allowed us to capture the evolutionary history of bacterial/archaeal *IS* *110* Tnp, archaeal/eukaryotic Nop5 and all
their domain-level homologues within a single phylogenetic tree (Methods). The obtained alignment revealed broad conservation of key residues across the Nop domain, including a highly conserved glycine residue in the C-terminal α-helical domain (Extended Data Fig. 4a,b). Both IS*110* Tnp and Nop5 form monophyletic clades within the tree, and the relative position of both clades within the RuvC-like superfamily indicates that these protein families are close relatives, rather than Nop5 following a unique evolutionary trajectory to converge on the same domain architecture (Extended Data Fig. 5). Interestingly, the phylogenetic tree topology indicates that archaeal and eukaryotic Nop5 homologues did not descend from an archaeal IS*110* Tnp clade present in our database, instead suggesting that the Nop5 lineage originated either from an archaeal IS*110* Tnp clade that is extinct today, or from a more ancient IS*110* Tnp ancestor (Fig. 2c and Extended Data Fig. 5). This scenario is also supported by full-length protein and single-domain trees including IS*110* Tnp and Nop5 homologues (Extended Data Figs. 6 and 7). Given the widespread distribution of IS*110*-family transposons in bacteria and archaea and the diverse changes in Nop5 (notably loss of the RuvC active site), the IS*110* Tnp is likely to represent the ancestral state. Finally, we exploited the shared Nop domain to build a phylogenetic tree of archaeal Nop5, eukaryotic Nop56/Nop58 and Prp31, which revealed the likely emergence of Prp31 from a duplication event of Nop56 or Nop58 that must have occurred in early eukaryotes (Fig. 2b,c)²⁶.

Collectively, our findings provide strong support for the emergence of both Nop5 and Prp31-family proteins from an ancestral IS*110* Tnp (Fig. 2c), unifying the seemingly disparate processes of RNA-guided DNA recombination, RNA-guided RNA modification and RNA-mediated RNA splicing. We propose that the Nop5 lineage emerged in early archaea from an ancestral protein that probably possessed similar structural and functional features as the IS*110* Tnp present in extant bacteria and archaea. Nop5 evolution was marked by notable functional changes, including loss of nuclease activity in the N-terminal domain and the acquisition of protein-interacting functions to engage other components of snoRNP/snRNP complexes for novel RNA-guided processes. When taken together with recent work demonstrating a transposon origin for RuvC nuclease domain-containing CRISPR effectors such as Cas9 and Cas12 (refs. 5,6,27,28), our work highlights the repeated emergence of complex RNA-guided functions in bacterial mobile genetic elements, showcasing their pervasive influence on complex cellular pathways. These diverse programmable features have been exapted by their host organisms to fulfill novel biological roles, and they present exciting opportunities to further advance genome engineering technologies.

# Methods

## Nomenclature

The *IS*110 family of transposons is divided into two subfamilies: *IS*110/*IS*621 and *IS*111, named after their representative sequences¹⁴ (Extended Data Figs. 6 and 7). All transposons in the *IS*110 family consist of a transposase (Tnp) and a non-coding 'bridge' RNA. In the main text, we refer to these transposons as 'IS*110* transposons' and their associated transposases as 'IS*110* Tnp' for simplicity, even if the Tnp protein derives from a more specific *IS*110-family element such as *IS*621. *IS*110 Tnp consists of three domains: an N-
terminal domain called RuvC, due to its homology with the RuvC nuclease of the RNaseH fold superfamily; a coiled-coil (CC) domain made up of two α-helices separated by a short variable region; and a C-terminal α-helical bundle (Fig. 1d).

We generally prefer the term ‘transposon’ over ‘insertion sequence (IS)’ throughout the text to capture the broadest category of mobile elements capable of transposition from one site in DNA to another. This term should not be confused with bacterial composite transposons that are flanked by two separate IS elements, or with other transposons such as Tn7-family elements, which mobilize additional cargo genes.

The Nop5 family of proteins uses C/D-box snoRNAs to direct RNA-guided RNA methylation. While these molecules are often called sRNAs in archaea, we consistently refer to them as snoRNAs throughout the text to maintain a universal nomenclature and avoid confusion with small RNAs. Archaea encode a single Nop5 homologue, while eukaryotic organisms possess two paralogues, known as Nop56 and Nop58. For clarity, we refer to this entire family of proteins, including both archaeal and eukaryotic homologues, as ‘Nop5’. In archaea, Nop5 and snoRNAs form a sno-like RNP (sRNP) complex, which consists of Nop5, an RNA-binding protein called L7Ae, and fibrillarin (an RNA methyltransferase). Similarly, in eukaryotes, this complex is referred to as the small nucleolar ribonucleoprotein particle (snoRNP) and includes Nop56 and Nop58 (homologues of Nop5), 15.5 kDa/Snu13p (homologue of L7Ae) and fibrillarin²⁹. In addition to Nop56 and Nop58, eukaryotes encode another Nop domain protein called Prp31, which functions in the spliceosome by binding U4 and U6 small nuclear RNAs (snRNA). Similar to Nop5 homologues, Prp31 also interacts with 15.5 kDa/Snu13p¹².

## Database of eukaryotic, archaeal and bacterial genomes

Eukaryotic genomes (4,616) from 2,407 species and 22,804 bacterial complete genomes were downloaded from Genbank in January 2022³⁰. Archaeal genomes (2,339) were downloaded from the Genome Taxonomy Database (GTDB) in January 2023 and concatenated with 496 in-house Asgard archaea genomes, resulting in a database of 2,835 archaeal genomes³¹. Altogether, our database comprised 30,255 genomes across the three domains of life.

## Remote homology between IS*110* Tnp and Nop5

Remote homology detection was assessed using the HHpred web-server and Foldseek (v.8-ef4e960)¹⁷,³². The initial observation suggesting a putative homology link between Nop5 and the *IS*110* Tnp was made by querying individual *IS*110* Tnp sequences from *K. pneumoniae* (A0A809T667) and *S. flexneri* (QKW72470) against the Pfam-A (v.36) and the PDB_mmCIF70 databases using HHpred with default parameters (Supplementary Table 2)³². This association was also assessed using Foldseek, querying (options -c 0.8 and -max-seqs 10000) representative sequences of *IS*110* Tnp, Nop5, Nop56 and Nop58 against the UniProt50 Foldseek database, as downloaded in February 2024¹⁷. The obtained hits were filtered (probability > 0.8) and further annotated using the Pfam-A (v.36) database to determine their identities (Supplementary Table 1)³³.
## Protein structure prediction

When experimentally solved protein structures were not available from the RCSB Protein Data Bank, protein structure predictions were retrieved from the AlphaFold Protein Structure database (AFDB)³⁴,³⁵. When neither experimental structures nor predicted protein structures were readily available in these databases, protein structure prediction was performed using AlphaFold2 through the ColabFold distribution (v.1.5.2) as implemented in the localcolabfold package³⁶,³⁷. When predicting multimeric structures, AlphaFold-Multimer v.3 was used³⁷. Protein structure prediction was performed using three independent models with 20 prediction recycles each. Template information was used and multiple sequence alignments were computed in mmseqs2_uniref_env mode using only paired sequences. Protein structures were visualized and drawn using ChimeraX (v.1.7.1)³⁸.

## Systematic identification of *IS*110 *Tnp* and *Nop*5 homologues

IS*110* Tnp homologues were identified using iterative profile HMM search on our database of 30,255 bacterial, archaeal and eukaryotic genomes. An initial BLAST (v.2.12.0) search was performed using a *K. pneumonia* IS*110* Tnp (A0A809T667) as a query to identify all closely related homologues (-max_target_seqs 1000000 -evalue 1e-80)³⁹. The retained hits (*n* = 6,407) were aligned using the MAFFT (v.7.520) L-INS-i algorithm (-localpair), and an initial profile HMM was built and used as a query to search for additional homologues using hmmsearch in the HMMER package (v.3.3.2), with default parameters⁴⁰,⁴¹. All hits (*n* = 55,007) with an *e*-value below 1 × 10⁻⁵ and a protein length between 200–1,000 amino acids were retained for another round of search. Hits were clustered at 80% identity and 80% coverage using mmseqs easy-cluster (v.13.45111)⁴². Representative sequences (*n* = 4,047) were aligned using MAFFT L-INS-i, and a new profile HMM was built from this alignment. All multiple sequence alignments (MSA) were visualized using the Unipro UGene software (v.49.1)⁴³.

The same procedure was repeated to obtain 58,533 hits satisfying the inclusion criteria (*e*-value < 1 × 10⁻⁵ and 200 < length < 1,000). Homology search was then terminated, as another round of search did not yield any new hits satisfying the inclusion criteria. The set of identified *IS*110 *Tnp* homologues was further filtered to include only protein hits covering at least 80% of the profile HMM, leading to a final set of 58,533 *IS*110 *Tnp* homologues among bacteria and archaea (Supplementary Table 3). As proteins retrieved using this procedure were mostly of bacterial origin, an extra round of search was performed using a profile HMM built from only the identified archaeal *IS*110 *Tnp* as a query (*n* = 662) against the database of archaeal genomes. This round of search did not yield any additional archaeal *IS*110 *Tnp* homologues⁴¹.

Nop56 and Nop58 homologues were identified using a similar iterative profile HMM search procedure⁴¹. Animal orthologues of Nop56 and Nop58 were retrieved from the NCBI Orthologs database and clustered at 98% identity and 98% coverage using mmseqs, leading to a set of 498 representative orthologues which were aligned using MAFFT L-INS-i to build an initial Nop56/Nop58 profile HMM⁴⁰⁻⁴². Homology search using this profile led to 15,253 hits satisfying the inclusion criteria (*e*-value < 1 × 10⁻⁵ and 300 < length < 700 amino acids), which included the archaeal Nop5 protein and the eukaryotic proteins Nop56,
Nop58 and Prp31. Those homologues were clustered at 80% identity and 80% coverage, and representative sequences (n = 2,397) were used for another round of search against the genome database. This round yielded 15,932 hits satisfying the inclusion criteria (e-value < 1 × 10⁻⁵ and 300 < length < 700 amino acids) (Supplementary Table 3). Hits were reannotated using Pfam-A (v.36) and all belonged to either the Nop5, Nop56/Nop58 or Prp31 family³³.

## Systematic identification of individual domain homologues

We sought to identify candidate homologues of each individual domain for IS*110* Tnp and Nop5. We decided to split the proteins into three parts on the basis of structural features: (1) the N-terminal domain (RuvC-like in the case of IS*110* Tnp), (2) the coiled-coil domain and (3) the C-terminal domain. Each domain was manually extracted from the whole protein alignments to build domain-level profile HMM and search for putative homologues in our database. First, single protein profile HMMs were built and used as queries in hmmsearch against our database of genomes⁴¹. As no significant hits belonged to new protein families using this approach (e-value < 1 × 10⁻⁵), ‘mixed’ profile HMMs were built by aligning sequences of a domain shared between distinct proteins (for example, both IS*110* Tnp and Nop5 N-terminal domains). Again, no novel significant hits could be identified using this approach.

To increase the sensitivity of the homology search for each individual domain, an MSA including the N-terminal domain of representative sequences (mmseqs easy-cluster at 60% identity and 60% coverage) of both IS*110* Tnp and Nop5, as well as of the compact RuvC nuclease domain, was queried in HHpred against the Protein Data Bank and the Pfam-A databases (5 iterations against UniRef30 for MSA generation, minimum probability of 20%)³²,³³,³⁵,⁴². Hits that satisfied the inclusion criteria were filtered (probability > 0.8) for further analysis. This approach uncovered new proteins from the family of the RuvC-like Holliday junction resolvase, in particular RuvC (PF02075), poxvirus A22 (PF04848), *Lactococcus* phage protein M3 (PF07066) and the YqgF/RuvX/Tex family of proteins (CL0580/PF14639, PF03652, PF16921). Proteins from these families were retrieved in our database by querying their respective Pfam-A profile HMM in hmmsearch (with option -cut_ga) against our database of genomes³³,⁴¹. These hits were added to the list of candidate homologues for the N-terminal domain of Nop5 and IS*110* Tnp for downstream phylogenetic inference. The same approach was applied to the other domains of Nop5 and IS*110* Tnp but did not provide any significant matches apart from Nop5 and IS*110* Tnp themselves. Foldseek searches were also performed using the individual domains of representative sequences for IS*110* Tnp, Nop5, Nop56, Nop58 and Prp31. Individual domains were manually extracted for these proteins and queried using Foldseek against the UniProt50 database (options -c 0.8 and -max_seqs 10000)¹⁷,³⁴. Hits were filtered (probability > 0.8) and annotated using profile HMM from the Pfam-A database³³,⁴¹ (Supplementary Table 1).

## Phylogenetic inference regarding IS*110* Tnp, Nop5, RuvC and RuvC-like homologues

We first built a phylogenetic tree of full-length IS*110* Tnp and Nop5 proteins (Extended Data Fig. 4). This tree was used to infer specific subfamilies within the IS*110* Tnp and
Nop5 families and to assess the relationship between the *IS*110Tnp and Nop5 proteins. All *IS*110Tnp and Nop5 proteins detected in the database of genomes were first clustered at 60% identity and 60% coverage using mmseqs easy-cluster⁴². Representative sequences (*n* = 2,898) were aligned using MAFFT L-INS-i and positions with more than 99% gaps were trimmed from the MSA using Clipkit (v.1.3.0, -m gappy -g 0.99), leading to a trimmed alignment of 1,237 positions⁴⁰,⁴⁴. IQTree was used to build a maximum likelihood phylogenetic tree that was optimized for 900 iterations using the Q.pfam+G4 substitution model which was selected using ModelFinder, and branch support was assessed using 1,000 UltraFast Bootstrap (UFBoot) trees⁴⁵⁻⁴⁷.

Individual protein domains (N-terminal, coiled-coil and C-terminal) were also separately
aligned using MAFFT L-INS-i, including representative sequences (mmseqs easy-cluster
at 60% identity and 60% coverage) of the proteins that possess each domain⁴⁰,⁴². These
alignments were first used to build domain-level phylogenetic trees for the N-terminal
and C-terminal domains, including all relevant homologues for each domain (Extended
Data Fig. 7). Single-domain phylogenies were built to assess putative evolutionary
convergence involving multiple independent domain assembly events which would translate
in incongruent domain-level trees. For the N-terminal domain, we included RuvC and
RuvC homologues (A22, M3 and YqgF/RuvX/Tex), in addition to *IS*110Tnp and the
Nop5 N-terminal domain²⁴,²⁵. Provided that *IS*110Tnp and Nop are homologous, under
the hypothesis that *IS*110Tnp are ‘not’ the closest relatives of Nop5 proteins within the set
of RuvC homologues, one would expect to observe Nop5 forming a clade with its closest
relative (one of the RuvC-like homologues identified through homology search) without
*IS*110Tnp in the clade. In line with this idea, we chose the YqgF/RuvX/Tex family of
proteins as an outgroup to root the tree. Since this family is the most distantly related of
the rest of the RuvC-like homologues identified through homology detection (as can be
seen in its catalytic residues, that is, DED instead of DEDD in all other RuvC homologues,
including *IS*110Tnp), this choice of an outgroup allows to assess the organization of
protein families within the clade of RuvC homologues, and thus to identify where the
Nop5 N-terminal domain would be located within the set of RuvC homologues²⁵. All
proteins were clustered at 60% identity and 60% coverage using mmseqs, and representative
sequences were aligned using MAFFT L-INS-i⁴⁰,⁴². The MSA was manually inspected
and trimmed using Clipkit⁴⁴. Maximum likelihood trees were searched for 3,000 iterations
using IQTree (v.2.2.0.3), with the Q.Pfam+G4 substitution model which was selected using
ModelFinder⁴⁵⁻⁴⁷. Branch support was assessed by generating 1,000 UFBoot trees⁴⁶. For
the C-terminal domain, only *IS*110Tnp and Nop5 proteins were included, as no other
candidate homologues were identified. Sequences were processed as described for the N-
terminal domain (clustering at 60% identity and 60% coverage using mmseqs easy-cluster,
alignment using MAFFT L-INS-i and trimming using Clipkit gappy to positions containing
more than 99% gaps), and a maximum likelihood tree was inferred using IQTree for 3,000
iterations with the LG+G4 substitution model selected using ModelFinder⁴⁰,⁴²,⁴⁴⁻⁴⁶. Branch
support was assessed using 1,000 UFBoot trees⁴⁶. The MSA was also visualized using
UGene⁴³, and conserved residues were mapped onto predicted or experimental structures of
*IS*110Tnp and Nop5, respectively, using ChimeraX³⁸.
The domain-level alignments were also used to build a phylogenetic tree at the full-length protein level for IS*110* Tnp and Nop5 (Fig. 2a). This strategy was adopted as (1) aligning at the single-domain level allows to obtain good-quality MSA including all the homologues possessing each individual domain in the absence of full protein homologues and (2) building a tree of the concatenated MSA allows to infer the evolutionary history of the full IS*110* Tnp and Nop5 proteins in a single tree. To do so, individual domain alignments were concatenated horizontally to obtain a unique MSA modelling the full-length protein while including representative sequences (clustered at 60% identity and 60% coverage using mmseqs) of each domain-level homologue identified during the search (n = 3,950 sequences in total)40,42. These domain-level homologues include the identified RuvC-family protein (RuvC, A22, M3 and YqgF/RuvX/Tex) for the N-terminal domain of IS*110* and Nop5 (ref. 25). As no homologue was identified for the C-terminal domain, only homologues of the N-terminal domain were included in the tree. The MSA was trimmed using clipkit gappy (removing positions with more than 99% gaps), leaving 762 positions satisfying the inclusion criteria44. A maximum likelihood phylogenetic tree of the full-length protein was then inferred from this concatenated MSA using IQTree for 4,000 iterations with the Q.Pfam+G4 substitution model which was selected using ModelFinder, and 1,000 UFBoot trees for assessing branch support40,45–47. Similar to the tree focusing on the N-terminal domain (Extended Data Fig. 7), YqgF/RuvX/Tex was chosen as an outgroup to root the tree because of its more important divergence from other RuvC homologues among all the included families of proteins (RuvC, YqgF/RuvX/Tex, A22, M3, IS*110* Tnp and Nop5)21,24,25. This choice of outgroup allowed us to assess the organization of protein families in the clade of RuvC homologues to test where the Nop5/Nop56/Nop58 family would branch in the diversity of RuvC close homologues.

To assess the phylogenetic relationship between the Nop5 and Prp31 lineages, we built a phylogenetic tree of the Nop domain from Nop5 and Prp31 (Extended Data Fig. 5). The Nop domain refers to the section of the protein spanning from the variable region separating the two α-helices of the coiled-coil region14,23 to the C-terminal orthogonal α-helical bundle, including the second helix of the coiled-coil region. This region was chosen to build the phylogenetic tree of the proteins, as it contains conserved residues among Nop5/Nop56/ Nop58 and Prp31; in contrast, the N-terminal RuvC-like domain of Nop5 proteins is not present in Prp31. The Nop domain region was manually extracted from all identified Nop5 and Prp31 homologues, and proteins were clustered at 80% identity and 80% coverage using mmseqs easy-cluster42. Representative sequences (n = 1,703) were aligned using MAFFT L-INS-i and a maximum likelihood tree was inferred using IQTree (LG+G4 substitution model, 5,000 iterations, 1,000 UFBoot trees)40,45,46. A small set of n = 23 representative IS*110* Tnp was randomly sampled to serve as an outgroup to root the tree, as we previously showed that (1) conserved residues are shared in the C-terminal region and (2) no other homologue outside of the IS*110* Tnp, Nop5/Nop56/Nop58 and Prp31 families of proteins could be identified. Changing the set of IS*110* Tnp rooting the tree did not impact the overall topology of the tree.
Extended Data

<figure><img src="image_2.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>IS110 Tnp
(S. flexneri
AF: QKW72470.1)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Nop5
(S. solfataricus
PDB: 5GIN)</figcaption></figure>

Nop Domain

Extended Data Fig. 1 | Profile HMM-based homology detection and secondary structure prediction of IS110 transposase.

a, Homology detection and secondary structure prediction for IS *110* Tnp were performed using HHpred. The left panel displays the alignment of *IS 110* Tnp with various protein domains identified through HHpred, indicating structural and functional similarities based on hits from the Protein Data Bank (PDB). The right panel provides a detailed view of the alignment with C/D-box snoRNP subunit Nop5 from *S. solfataricus* (PDB: 7XPL_B), showing the probability of homology, E-value, and secondary structure elements. Predicted secondary structures (α-helices and β-strands) are indicated above the amino acid sequence.

b, Homology detection and secondary structure prediction for IS *110* Tnp were performed using Foldseek. The left panel displays the AlphaFold-predicted structure of *IS 110* Tnp (Genbank: QKW72470.1) and the empirically determined structure of Nop5 from *S. solfataricus* (PDB: 5GIN). The Nop domain is outlined in blue. 3Di+AA FoldSeek alignment is shown on the right, with the Nop domain outlined in blue.
<figure><img src="image_5.png" /><figcaption>Extended Data Fig. 2 | Key structural differences in the N-terminal RuvC-like regions of IS110 Tnp and Nop5.</figcaption></figure>

a, Active tetrameric IS 110 Tnp structure¹⁸, with each dimer shown in light green or blue, bridge RNA in red, donor DNA in dark green, and target DNA in dark blue. Note that this particular IS 110-family element is known as IS621 but is referred to as IS 110 Tnp throughout, for simplicity. b, Each IS 110 Tnp dimer from a, with similar coloring. c, Structural changes that presumably accompanied the emergence of Nop5-family proteins. Comparison of the IS 110 Tnp (left) and Nop5 (center) highlights a large α-helix insertion (purple) that repositions the RuvC-like domain relative to the target nucleic acid (RNA for Nop5) and facilitates recruitment of the RNA methyltransferase Fibrillarin (yellow, at right). d, Hypothetical changes between the RuvC domain of IS 110 Tnp (PDB:8WT7) (left) and the one of Nop5 (center and right) for Fibrillarin recruitment. In Nop5 (PDB:7XPL), α1 (green) and α4 (orange) are repositioned to facilitate interactions for recruiting Fibrillarin (yellow). α3, which contains part of the active site motif, is partially degraded, possibly
due to the lack of need for nucleic acid cleavage. An inserted alpha-helix (gray) allows the restructured domain to extend and maintain the characteristic feature of the RNaseH fold: a five-stranded β-sheet composed of three anti-parallel strands and two parallel strands²¹.

<figure><img src="image_6.png" /><figcaption>**IS110 Tnp**
(E. coli, PDB: 8WT7)

**Nop5**
(A. fulgidus, PDB: 1NT2)

**IS110 Tnp**
(S. flexneri, AF: QKW72470.1)

**Nop5**
(S. solfataricus, PDB: 7XPL)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**IS110 Tnp**
(S. flexneri, AF: QKW72470.1)

**Nop5**
(E. coli, PDB: 8WT7)

**IS110 Tnp**
(S. solfataricus, PDB: 7XPL)

**Nop5**
(S. solfataricus, PDB: 7XPL)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**IS110 Tnp**
(E. coli, PDB: 8WT7)

**Nop5**
(S. solfataricus, PDB: 3PLA)

**Prp31**
(H. sapiens, PDB: 2OZB)</figcaption></figure>

**Extended Data Fig. 3** | **Key structural differences in the coiled coil and C-terminal regions of IS110 Tnp and Nop5.**

a, Comparison of the *E. coli* IS621 Tnp (left, PDB: 8WT7) and *A. fulgidus* Nop5 (right, PDB: 1NT2), which both lack α-helical bundle insertions between the anti-parallel α-helices that form the coiled-coil domain. In contrast, the predicted structure of an *IS110*-family transposase homolog from *Shigella flexneri* (protein accession: QKW72470.1) features an α-helical bundle insertion (teal), referred to as the ‘variable region’²³, that closely resembles a similar ‘tip insertion’ domain¹⁴ found in an *S. solfataricus* Nop5 homolog (PDB: 7XPL).

b, Structure-guided alignment of the *IS110* Tnp variable region (AlphaFold prediction) and Nop tip insertion domain (crystal structure) from *a*. The RMSD between 17 pruned atom
pairs is 1.199 Å; the RMSD across all 52 pairs is 3.862 Å. c, C-terminal protrusions of IS 110 Tnp (left, PDB: 8WT7) and Nop5 (right, PDB: 7XPL) separate the two strands of the non-coding guide RNA (bridge RNA for IS 110 Tnp, and C/D-box RNA for Nop5). d, Magnified view showing how the C-terminal region of IS 110 Tnp (left), Nop5 (center) and Prp31 (right) bind the non-coding guide RNA (red). Two specific ribonucleotides (green) exhibit flipped out bases that straddle the C-terminal region, shown in blue. PDB IDs are listed.

<figure><img src="image_9.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_10.png" /></figure>

E. coli IS621 Tnp
(PDB: 8WT7)

S. solfataricus Nop5
(PDB: 7XPL)

<figure><img src="image_11.png" /><figcaption>c</figcaption></figure>

Extended Data Fig. 4 | Alignment and phylogenetic tree built on the Nop domain of IS110 Tnp, Nop5, Nop56/Nop58, and Prp31 homologs.

a, Multiple sequence alignment (MSA) of the Nop domain from representative Nop5 (green), Nop56/Nop58 (pink), Prp31 (purple) and IS 110 Tnp (blue) homologs. A region exhibiting strong amino acid conservation is highlighted (yellow box), and the green triangle indicates a strictly conserved glycine. Protein names colored in yellow are highlighted in panel. b, Structure of IS 110 Tnp (PDB: 8WT7) and Nop5 proteins (PDB: 7XPL), with the boxed region from a colored in yellow. The conserved glycine is indicated with a green arrow, and the bridge RNA and snRNA are colored red. c, Phylogenetic tree built on the Nop domain of Nop5 (green), Nop56/Nop58 (pink), and Prp31 (purple), with a set of representative IS 110 Tnp homologs chosen as an outgroup to root the tree (blue). UFBoot
support values for each branch are indicated in green and are explicitly specified for key branches. A subsampled version of this tree is presented in Fig. 2b.

<figure><img src="image_12.png" /><figcaption>**Extended Data Fig. 5** | Three potential evolutionary scenarios to explain the relationship between *IS110* Tnp and *Nop5* proteins.</figcaption></figure>

Each diagram describes a potential evolutionary trajectory of *IS110* Tnp (blue lines) and *Nop5* (green lines) proteins among bacteria, archaea and eukaryotes (dark blue, gray and green colours) over time. The three scenarios explore distinct events, namely speciation (empty circle), duplication (filled circle), and extinction (cross). Note that all scenarios depict multiple lineages of *IS110* Tnp emerging in archaea via independent horizontal gene transfer (HGT) events, as supported by our phylogenetic analyses (Extended Data Fig. 6). **a**, The “Evolutionary Convergence” scenario posits that the observed structural similarity between *IS110* Tnp and *Nop5* are not the consequence of shared ancestry but instead due to evolutionary convergence, leading to the association of similar domains. This scenario implies that the *Nop5* lineage (green lines) emerged from an ancestor that was wholly independent of *IS110* Tnp (blue line), and emerged relatively early, given the widespread presence of *Nop5* among archaea. We reject the “Convergence” scenario based on the following observations: (i) We were unable to detect *IS110* Tnp-independent homologs of *Nop5* in our database, suggesting that if this ancestor once existed, it must have gone extinct; (ii) The conservation of key amino acid residues within the *Nop* domain of *IS110* Tnp and *Nop5* proteins renders a convergence scenario unlikely (Extended Data Fig. 4a, b); and (iii) *IS110* Tnp and *Nop5* exist as sister clades within the broader family of RuvC-like proteins (Fig. 2a), indicating that the closest relative of *Nop5* is likely to be *IS110* Tnp. **b**, The “Archaeal *IS110* origin” scenario posits that the *Nop5* lineage originated from an archaeal *IS110* Tnp through a duplication event (black circle). Under this scenario, one would expect to observe the whole clade of archaeal *Nop5* and eukaryotic *Nop56*/*Nop58* homologs to branch within a clade of archaeal *IS110* Tnp, but full-length and domain-specific phylogenetic trees do not exhibit such topologies. However, we cannot reject the possibility that the clade of archaeal *IS110* Tnp that gave rise to the *Nop5*
lineage have gone extinct in the past, nor can we exclude that the clade of archaeal IS*110* Tnp homologs ancestral to Nop5 are not present in the archaeal genomes included in our database. Thus, we cannot completely reject this evolutionary scenario. c, The “Ancient IS*110* origin” scenario posits that Nop5-family proteins and IS*110* Tnp are homologs and share a more ancient common ancestor. In the last common ancestor of bacteria and archaea, an ancestral protein would have given rise to both the IS*110* Tnp lineages in bacteria, and to Nop5 lineages in archaea (empty circle). This scenario is supported by the observed taxonomic distribution of proteins, the detection of IS*110* Tnp and Nop5 homologs across domains of life, and the topology of our phylogenetic trees showing that IS*110* Tnp and Nop5 proteins are sister clades within the family of RuvC-like proteins. We thus favor this evolutionary scenario for the emergence of the Nop5 lineage from its last common ancestor with the IS*110* Tnp lineage. The presence of ancestral features in IS*110* Tnp such as the DEDD catalytic motif, also found in the ancient RuvC nuclease family of proteins, makes it more likely that the last common ancestor of Nop5 and IS*110* Tnp resembled IS*110* Tnp found in extant organisms.
<figure><img src="image_13.png" /><figcaption>Extended Data Fig. 6 | Phylogenetic tree based on full-length IS*110* Tnp, Nop5 and Nop56/Nop58 proteins.</figcaption></figure>

a, Phylogenetic tree of Nop5 and Nop56/Nop58 homologs built on full-length protein sequences. Archaeal Nop5 homologs were chosen as an outgroup to root the tree, and the outer ring indicates the domain of life that each protein belongs to. UltraFast Bootstrap support values are represented in shades of green on the tree branches, and explicit values are indicated for key branches. b, Phylogenetic tree of IS*110* Tnp homologs, built on full-length protein sequences, colored as in a. The tree is rooted on the branch between the two IS*110* subfamilies indicated by the outer ring (light blue, IS*1111* subfamily; dark blue, IS*110/IS621* subfamily). Representative Tnp homologs from the IS*110* family are labeled. c, Unrooted phylogenetic tree of IS*110* Tnp and Nop5 protein homologs aligned on their full-length amino acid sequence. Inner ring colors indicate the protein identity, and central ring colors indicate which domain of life each protein belongs to (blue, bacteria; gray, archaea; green, eukaryotes). For IS*110* Tnp, an outer ring indicates to which IS*110* subfamily
each protein belongs (light blue, IS *111* subfamily; dark blue, IS *110*/*IS621* subfamily).

Representative Tnp homologs from the IS *110* family are also indicated. UltraFast Bootstrap support values are indicated in shades of green on the tree branches. Gray arrows indicate major archaeal clades in the tree. IS *110* Tnp homologs found in archaeal organisms are distributed across several distinct clades and are mixed with bacterial homologs. This observation strongly supports the idea that archaeal IS *110* transposable elements were acquired in archaea through multiple independent horizontal gene transfer (HGT) events.

<figure><img src="image_14.png" /><figcaption>**Extended Data Fig. 7** | **Phylogenetic tree built on the N-terminal domain of IS*110* Tnp, Nop5, and Nop56/Nop58.**</figcaption></figure>

Candidate homologs were identified by querying a multiple sequence alignment of the N-terminal domain of IS *110* Tnp, Nop5, and Nop56/Nop58, alongside RuvC Holliday Junction Resolvase proteins, in HHpred against PfamA-v36 (see Methods). The YqgF/RuvX/Tex clade (orange) was chosen as an outgroup to root the tree. As in Fig. 2a, this choice of outgroup allows to assess the organization of RuvC close homologs compared with IS *110* Tnp and Nop5 in the tree. UltraFast Bootstrap support values for each branch are indicated in green and are explicitly specified for key branches.

**Supplementary Material**

Refer to Web version on PubMed Central for supplementary material.
Acknowledgements

We thank E. Rocha, H. Ghalei and members of the Sternberg lab for carefully reading the manuscript, providing constructive feedback and engaging in fruitful discussions. Several bioinformatic analyses were performed on the Core Cluster of the Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013). C.M. was supported by an NIH F32 Postdoctoral Fellowship (GM143924) and an ASGCT Career Development Award. A.B., J.C. and H.V. were supported by an ERC Starting Grant (PECAN 101040529). S.H.S. was supported by NSF Faculty Early Career Development Program (CAREER) Award 2239685, a Pew Biomedical Scholarship, an Irma T. Hirschl Career Scientist Award, and a generous startup package from the Columbia University Irving Medical Center Dean's Office and the Vagelos Precision Medicine Fund.

Data availability

All code and raw data are available on GitHub at https://github.com/mdmparis/is110_nop5 (ref. 48). Source data are provided with this paper.

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<figure><img src="image_15.png" /><figcaption>**Fig. 1 |** Functional and structural similarity between bacterial *IS110* Tnp, archaeal *Nop5* and eukaryotic *Prp31* proteins.</figcaption></figure>

a, Schematic illustrating RNA-guided mechanisms involving the *IS* 110 *Tnp* (left), C/D-box *snoRNP* (centre) and U4/U6 di-snRNP (right). *IS* 110 *bridge RNAs* (black) direct site-specific transposition between donor (green) and target (blue) DNA molecules by the transposase via RNA–DNA base-pairing interactions. C/D-box *snoRNPs* use similar structured RNAs to direct 2'-*O*-methylation via RNA–RNA base-pairing interactions, with *Nop5* playing a key structural role. An RNP complex formed at the 5'-stem loop of U4/U6 di-snRNAs exploits *Prp31* to stabilize the U4/U6 duplex, required for spliceosome assembly.

b, Comparison of *IS* 110 Tnp, *Nop5*, *Nop56*, *Nop58* and *Prp31* protein domain architectures. Both *IS* 110 Tnp and *Nop5*-family proteins exhibit a similar domain arrangement, including an N-terminal *RuvC*-like domain, a coiled-coil domain and a C-terminal region rich in α-helices. A red box highlights the *RuvC* and *RuvC*-like domains compared in c. c, Structural comparison between the *RuvC* domain of *IS* 110-family Tnp from *IS* 621 (left,
PDB: 8WT7) and the RuvC-like domain from Nop5 (right, PDB: 7XPL). Corresponding secondary structures are coloured similarly for visual comparison and Tnp catalytic residues are shown in purple. d, Structural comparison of IS 110 Tnp (left, PDB: 8WT7), Nop5 (centre, PDB: 7XPL) and Prp31 (right, PDB: 2OZB). Domains are coloured according to the domain annotations in b; the C-terminal disordered region of Prp31 is not resolved. e, Structural comparison of dimeric forms shown as surface representation and coloured as in b, with RNA guides and nucleic acid targets (DNA for IS 110, RNA for Nop5) highlighted in red and blue, respectively. PDB IDs are shown.
<figure><img src="image_16.png" /><figcaption>Fig. 2 | *IS110*-family transposases share a common evolutionary ancestor with *Nop5*-family proteins involved in snoRNP assembly.</figcaption></figure>

a, Phylogenetic tree including IS*110* Tnp, Nop5, Nop56/Nop58 and RuvC-like homologues (RuvC, A22, M3, RuvX/YqgF/Tex) that were included to help with assessing evolutionary relationships. The three protein domains conserved in IS*110* Tnp and Nop5 were separately aligned before concatenating multiple sequence alignments, with rooting on the RuvX/YqgF/Tex family of proteins (Methods). The domain of life origin for each homologue is denoted in the outer ring and UltraFast Bootstrap (UFBoot) values are shown in shades of green. The location of canonical IS*110*-family proteins is also indicated. b, Phylogenetic tree built on the Nop domain of Nop5 (archaeal, green), Nop56/Nop58 (eukaryotic, pink) and Prp31 (eukaryotic, purple) (Methods). The tree is rooted on representative sequences of the IS*110* Tnp (blue) and subsampled to enhance readability; the full version is presented in Extended Data Fig. 5c. UFBoot support values for each branch are indicated in green and are explicitly specified for key branches. c, Parsimonious evolutionary schema illustrating the emergence of Nop5 and Prp31 across the tree of life, from their last common ancestor with the IS*110* Tnp. The Nop5 lineage probably originated in early archaea from an ancestral protein that resembled the IS*110* transposase found in extant bacteria and archaea. Nop5 proteins diverged from the IS*110* Tnp by gaining/losing multiple specific biochemical properties, alongside the transition of bridge RNAs to C/D-box and U4 snRNAs. The Prp31 lineage probably emerged in early eukaryotes via a Nop5 duplication event and further divergence through loss of the N-terminal domain.