AACRNL evolved from virulence factor to epigenetic parasite driving genome expansion in free-living eukaryotes

Domain: neuroscience
Published: 2026-01-29
Editorial outcome: minor_revision

Abstract
Crinkler effectors are iconic toxins deployed by pathogens to suppress host immunity, a function long thought exclusive to pathogenic species. Here, we uncover a striking evolutionary twist: an ADP-ribosyltransferase-associated Crinkler-like protein that has repurposed its toxin-derived activity to function as an epigenetic parasite in free-living eukaryotes, challenging the paradigm that Crinkler effectors are exclusively pathogen-restricted virulence factors. The ADP-ribosyltransferase-associated Crinkler-like protein retains a functional mono-ADP-ribosyltransferase domain homologous to diphtheria toxin, yet exhibits degeneration of N-terminal host-targeting domain, severing ties to canonical pathogenic functions. This activity enables dual subversion of host mechanisms: the ADP-ribosyltransferase-associated Crinkler-like protein mono-ADP-ribosylates the EZH2, reducing H3K27me3-mediated silencing and reactivating its own locus and adjacent transposons to drive replicative amplification; simultaneously, it modifies the immune adaptor TRAF6, suppressing NF-κB and IRF3 pathways to weaken innate immune surveillance and facilitate sustained propagation within the host genome. Notably, this parasitic strategy is countered by host detoxification: TRAF6 ubiquitinates ADP-ribosyltransferase-associated Crinkler-like protein, targeting it for proteasomal degradation. Together, our findings reveal how a toxin-derived effector escaped pathogenic contexts to evolve as a selfish genomic element, leveraging toxin-like activity to subvert epigenetic and immune barriers and redefining the evolutionary potential of Crinkler toxins as drivers of genomic conflict beyond pathogenicity.

Introduction
Pathogenic microbes rely on secreted effector proteins to commandeer host cells and facilitate infection1,2. These virulence factors can disable immune defenses, hijack signaling pathways, and reprogram host physiology to the pathogen’s advantage1,2. In Gram-negative bacteria, type III secretion system effectors are renowned for targeting mitochondria and other organelles to control host cell death and inflammation3. Among oomycetes and fungi, Crinkler (CRN) proteins constitute a prominent effector family that exemplifies the complexity of host manipulation strategies4. First discovered in the potato late blight pathogen Phytophthora infestans, certain CRNs induced a characteristic leaf “crinkling and necrosis” phenotype when expressed in plants, highlighting their virulence potential5.

Structurally, canonical CRN effectors exhibit a modular architecture comprising conserved N-terminal and C-terminal domains that underpin their functional versatility6. The N-terminal domain, ~130 amino acids in length, includes the LXLFLAK motif, ubiquitin-like (UBL) folds, and α-helical structures responsible for host cell translocation4,7. The C-terminal region of CRN encompasses several effector functionalities, including restriction endonuclease (REase), protein kinase, HNH endonuclease, LK nuclease (a RNase), and multiple distinct peptidase domains, predicted to carry their toxicity determinants4,6,8,9. This modularity is thought to enable CRNs to diversify rapidly in response to selective pressures, fueling pathogen adaptation during host-pathogen coevolution.

Unexpectedly, recent comparative genomics surveys have uncovered CRN homologues across multiple free-living eukaryotic lineages, including chlorophyte algae, land plants, amoebozoans, and animals, challenging the long-held notion that these effectors are exclusive to pathogenic taxa7. This unexpected distribution raises fundamental questions about their evolutionary persistence: why do such virulence-associated genes remain embedded in the genomes of non-pathogenic organisms, and what functions, if any, do they perform outside classical pathogenic contexts? Addressing these questions is essential to expanding our understanding of effector evolution and function beyond classical pathogen-host paradigms.

Effector genes often reside within dynamic genomic contexts shaped by transposable elements and mobile genetic elements that facilitate their mobilization and diversification in pathogens10. Such genes are typically subject to strong positive selection because their toxin functions confer clear fitness benefits during infection, facilitating immune suppression and colonization11. By contrast, free-living non-pathogenic eukaryotes are ecologically isolated, limiting horizontal gene transfer opportunities through plasmids, viruses, or symbiotic interactions, which constrains the spread of virulence-associated genes. Furthermore, these sequences are often targets of host epigenetic repression mechanisms—such as H3K27me3-mediated chromatin silencing and DNA methylation—that restrict expression and genomic propagation of potentially invasive elements12,13. These constraints have reinforced the paradigm that effectors are confined to pathogenic contexts and rendered their evolutionary persistence in nonpathogenic genomes largely enigmatic.

Here, we report the discovery and functional characterization of a CRN homologue, ADP-ribosyltransferase-associated CRN-like (AACRNL), whose homologues are broadly distributed across free-living eukaryotic lineages. Unlike canonical CRNs, AACRNL have retained functional toxin-like enzymatic module but exhibit degeneration of N-terminal targeting domain, uncoupling them from classical host translocation. Remarkably, AACRNL co-opts its ART activity to mono-ADP-ribosylate EZH2, eroding H3K27me3-dependent repression and enabling the activation and replication of its own locus and adjacent transposons. Concurrently, AACRNL modifies TRAF6 to suppress NF-κB/IRF3 signaling, creating an immunologically permissive environment for its expansion. This dual strategy reveals how a virulence-derived effector can be repurposed as an epigenetic parasite in free-living eukaryotes, challenging conventional views of CRN biology and illuminating broader principles of genome conflict and adaptation.

Results
Distribution and degeneration of AACRN(L) in free-living eukaryotes
The canonical CRN effector consists of an N-terminal domain responsible for host targeting and a toxin-related C-terminal domain (Fig. 1a). Based on this modular architecture, we identified a distinct type of CRN effectors, termed AACRN, characterized by an N-terminal UBL domain and a C-terminal ADP-ribosyltransferase (ART) domain (Fig. 1b). In addition, we identified a related variant in which the UBL domain has undergone extensive degeneration; we refer to this lineage as AACRN-Like (AACRNL) (Fig. 1b). Although AACRN effectors are typically associated with pathogenic functions, AACRN(L) homologs are also present in several free-living eukaryotes. Specifically, comprehensive search across multiple phyla revealed that the AACRN(L) family were detected in free-living eukaryotes like Porifera, Cnidaria, Echinodermata, Chordata, Rotifera, Nematora, and Arthropoda of animalia (Fig. 1c and Supplementary Fig. 1). Notably, the occurrence of AACRN(L) genes is limited to a minority of taxa and can vary considerably even within the same phylum. For example, within Chordata, the AACRNL family was identified exclusively in Actinopteri, a class of bony fish, which is also the highest living organism distributed by the AACRNL family (Fig. 1c). Given that the N-terminal domain is critical for host targeting, we examined its integrity in these free-living organisms. We observed pronounced degeneration of the N-terminal regions in AACRN effectors. For instance, the AACRNL homologues from Acipenser ruthenus and Porites lobata, the UBL domain has degenerated and lost the essential RGG motif required for ubiquitin chain formation (Fig. 1d, e). Overall, these findings demonstrate that AACRNL homologues persist in the genomes of certain free-living eukaryotes, and their N-terminal domain responsible for host targeting has also degraded, which may reflect a potential functional shift.

AACRNL exerts toxicity in free-living organisms through its ADP-ribosyltransferase activity
To identify the key residues responsible for the ADP-ribosyltransferase activity of AACRN(L), we collected AACRN(L) protein sequences from different phyla, along with two well-characterized bacterial toxins with mono-ADP-ribosyltransferase (mART) activity: diphtheria toxin from Corynebacterium diphtheriae and exotoxin A from Pseudomonas aeruginosa14. Sequence alignment of the mART domain revealed a conserved H-Y-Y motif (Fig. 2a), which is essential for NAD⁺ co-substrate binding15. Next, we extracted the structural domain sequences of Miichthys miiuy AACRNL and C. diphtheriae diphtheria toxin, performing molecular docking with NAD+. The results showed that in both AACRNL and diphtheria toxin, both the “H” and the “Y” residues had interatomic contacts with NAD+ (Fig. 2b). To validate the functional importance of this motif, we generated point mutations in the H-Y-Y motif of M. miiuy AACRNL (Fig. 2c). AACRNL efficiently co-precipitated mono-ADP-ribosylation (mono-ADPr) from cell lysates, but AACRNL-HYY mutants did not (Fig. 2d). Additionally, self-modifying activity tests showed that AACRNL, but not AACRNL-HYY, was modified by mono-ADP-ribose (Fig. 2e). Furthermore, when purified AACRNL or AACRNL-HYY proteins were incubated with biotin-labeled NAD+, only AACRNL’s auto-modification was detected by streptavidin-HRP on western blots (Fig. 2f). Considering the structural homology between AACRNL and diphtheria toxin, as well as the origin of its toxin effector domain, we further investigated whether AACRNL functions as a toxin in fish, the highest-order lineage where AACRNL is distributed. Indeed, we found that AACRNL exerts mART-dependent inhibition of cellular viability (Fig. 2g). In addition, AACRNL suppressed the IRF3 and NF-κB pathways and downregulated the expression of antiviral and pro-inflammatory factors (Fig. 2h–k). Collectively, these findings demonstrate that AACRNL possesses robust mART activity and retains toxin-like functions even in free-living eukaryotes.

AACRNL replicates in teleost via adjacent transposable elements
We further assessed the distribution and abundance of AACRNL genes in teleost, the highest-level vertebrate group in which AACRNL homologs have been identified so far. AACRNL exhibits lineage-specific expansion and contraction in bony fish, with Cyprinus carpio displaying the highest number of AACRNL copies, although their transcriptional activity is yet to be elucidated (Fig. 3a). In contrast, Takifugu rubripes is the only bony fish lacking AACRNL (Fig. 3a). Through a comprehensive analysis of chromosome-level whole genome data from seven fish species (Scleropages Formosus, M. miiuy, Pleuronectes platessa, Oryzias latipes, Oreochromis niloticus, Alosa alosa, and Danio rerio), we found that phylogenetic analysis of AACRNLs from these fish species revealed significant clustering into three lineages, which we refer to as AACRNLα, AACRNLβ, and AACRNLγ (Fig. 3b). The linear relationship around the AACRNLα gene is relatively conservative and single copy, indicating that it has lost its replication ability (Fig. 3b, c). However, AACRNLβ and AACRNLγ exhibit a very limited linear relationship and multiple copies, indicating that they have undergone replication (Fig. 3b, c).

To investigate the replication mechanisms of AACRNLβ and AACRNLγ, we examined their surrounding genomic sequences. Among the three copies identified in M. miiuy, AACRNLα was unable to transpose, while the other two included AACRNLβ and another copy that had become inactive, which we designated pAACRNLβ (Fig. 3c). Based on their linear arrangement, the right flank of pAACRNLβ was identical to that of AACRNLβ, both containing the hce1 and lce1 genes, indicating that pAACRNLβ originated from the duplication of AACRNLβ (Fig. 3d). Notably, we observed terminal inverted repeats (TIRs) of a non-autonomous hAT_Charlie transposable element flanking both sides of the AACRNLβ and lce1 loci, suggesting that the replication and transposition of AACRNLβ may have been mediated by the hAT_Charlie (Fig. 3d). We also found that autonomous mariner transposon elements are present on both sides of the AACRNLβ1 and AACRNLβ3 genes in P. platessa. Upstream of AACRNLβ1, there is the gtse1 gene, and within the last intron, the 5′ TIR and transposase of an autonomous mariner transposon are present. At the 3′ flanking region of AACRNLβ3, the 3′ TIR of an autonomous mariner transposon is detected. Analysis of the flanking regions of the AACRNLβ4, AACRNLβ5, and AACRNLβ6 genes in P. platessa also revealed autonomous mariner transposon elements together with the last exon of gtse1, confirming that these genes indeed originated through transpositional duplication from AACRNLβ1, AACRNLβ3, and related loci (Fig. 3e). Similarly, TIR sequences of non-autonomous hAT transposons were also detected flanking the AACRNLγ1 locus in zebrafish, further supporting our hypothesis (Fig. 3f). In summary, our research found that AACRNL can rely on neighboring transposable elements for selfish propagation in teleost genomes.

AACRNLβ abrogates H3K27me3-dependent repression of its locus and flanking hAT elements
Given that transposable elements in the host genome are generally maintained in a silenced state, yet replicative transposition of AACRNL is remarkably prevalent, this suggests that AACRNL may possess the capacity to activate these elements. Sequence alignment of the conserved “HGT” motif, which is essential for mART activity15, revealed that AACRNLα has lost this enzymatic function, whereas AACRNLβ and AACRNLγ retain intact catalytic domains (Fig. 4a). Consistent with their mobility, AACRNLα was unable to undergo replication, whereas AACRNLβ and AACRNLγ exhibited active transposition (Fig. 3b–f). These observations suggested that the intrinsic mART activity of AACRNL might be required for its selfish propagation (Fig. 4a). H3K27me3 and H3K9me3 are key chromatin modifications that silence transposable elements and repress gene transcription16. We found that M. miiuy AACRNLα, which lacks mART activity, was unable to suppress H3K27me3 levels, whereas AACRNLβ effectively reduced this repressive mark (Fig. 4b). Consistently, the AACRNLβ-HYY mutant, which has lost mART activity, also failed to inhibit H3K27me3 deposition (Fig. 4c). Given that H3K27me3 is deposited by the Polycomb Repressive Complex 2 (PRC2), we examined whether AACRNLβ interacts with PRC2 components. Co-immunoprecipitation experiments revealed that AACRNLβ specifically associated with EZH2, the catalytic subunit of PRC2, but not with SUZ12 (Fig. 4d). Moreover, immunoblotting with mono-ADP-ribose–specific antibodies confirmed in vivo mono-ADP-ribosylation of EZH2 (Fig. 4e). In vitro assays further demonstrated that AACRNLβ directly mediates mono-ADP-ribosylation of EZH2 (Fig. 4f, g). Beyond modifying EZH2, AACRNLβ also promotes the degradation of EZH2 protein in an enzymatic activity–dependent manner, without affecting ezh2 mRNA levels (Fig. 4h–j). Therefore, AACRNLβ can suppress H3K27me3 modification levels by inhibiting EZH2.

To determine whether AACRNLβ-driven reduction of H3K27me3 could relieve silencing at its own locus and neighboring hAT elements, we designed four pairs of ChIP-qPCR primers spanning the region from the AACRNLβ promoter to the 5′ TIR (Fig. 4k). Chromatin immunoprecipitation revealed that both AACRNLβ and the adjacent hAT sequences were enriched in H3K27me3 marks (Fig. 4l). Overexpression of EZH2 further increased H3K27me3 deposition, whereas co-transfection of wild-type AACRNLβ, but not the catalytically inactive mutant, effectively abrogated EZH2-mediated enrichment at these loci (Fig. 4m). Functionally, EZH2 suppressed AACRNLβ mRNA expression and decreased the recruitment of POLR2A, a core subunit of RNA polymerase II, to the AACRNLβ promoter (Fig. 4n, o). In contrast, co-transfection of AACRNLβ reversed this repression, suggesting that AACRNLβ is capable of self-activation (Fig. 4o). Consistently, siRNA knockdown of AACRN reduced POLR2A binding at its promoter (Fig. 4p). Collectively, these results demonstrate that AACRNLβ relieves H3K27me3-mediated silencing at its locus and neighboring hAT elements by targeting and modifying EZH2. This establishes a permissive chromatin environment that facilitates both self-activation and replicative transposition, thereby linking AACRNL’s mART enzymatic activity to its capacity for selfish genomic spread.

Viral infection triggers AACRNL activation and subsequent genomic instability
To investigate the activation dynamics and functional consequences of AACRNL, we analyzed its expression during zebrafish embryogenesis to capture baseline developmental activity, and upon viral infection to assess stress-induced activation. Single-cell analysis across early (4–6 h), mid (8–12 h), and late (24 h) embryonic stages showed that all five AACRNLγ members were transcriptionally silent, with AACRNLγ2, γ3, and γ5 loci enriched for H3K27me3, indicative of developmental repression (Fig. 5a). Notably, this silencing was reversible: SVCV infection strongly induced AACRNLγ2, γ3, and γ5, whereas γ1 and γ4 remained undetectable, suggesting potential loss of transcriptional competence in these loci (Fig. 5b). Similarly, M. miiuy AACRNLβ was markedly upregulated upon SCRV infection, as shown by RNA-seq and validated by qPCR in spleen, kidney, and MKC cells (Fig. 5c–f). Functional assays demonstrated that siRNA-mediated knockdown of AACRNLβ restored H3K27me3 levels suppressed by viral infection and prevented DNA damage, as indicated by tp53, p21, and gadd45a induction, revealing that infection-induced AACRNLβ promotes genomic instability (Fig. 5g, h). This function required AACRNLβ’s mART activity (Fig. 5i). Finally, wild-type AACRNLβ, but not the catalytically inactive AACRNLβ-HYY mutant, significantly increased the expression of transposon RNAs known to respond to pathogen challenge, including LINEs and ERVs (Fig. 5j). These findings indicate that pathogen-induced AACRNL activation promotes DNA damage and creates a permissive chromatin environment for transposon expression.

AACRNLβ mono-ADP-ribosylates TRAF6
Given that transposon activity and exogenous genetic elements often trigger host immune surveillance, we investigate how AACRNLβ creates an immune permissive environment to promote selfish reproduction. Co-immunoprecipitation results showed that AACRNLβ specifically binds to TRAF6, not to TRAF3, TBK1, TAK1, or NEMO proteins (Fig. 6a). Immunofluorescence revealed that AACRNLβ is in both the cytoplasm and nucleus, while TRAF6 is in the cytoplasm (Fig. 6b). Endogenous co-IP demonstrated that TRAF6 specifically interacts with AACRNLβ, and AACRNLβ and its mutants interact with TRAF6 with the same binding affinity (Fig. 6c, d). Mono-ADPr antibodies confirmed that TRAF6 undergoes mono-ribosylation in vivo (Fig. 6e), and AACRNLβ can mediate TRAF6 mono-ribosylation in vitro (Fig. 6f, g). To identify deribosylases that reverse this modification, we examined TRAF6-interacting proteins in our mass spectrometry data and discovered ARH3, a poly-ADP ribose glycohydrolase17,18. In addition, we also constructed a Macro-3 domain plasmid from the ARTD8 protein of M. miiuy responsible for specifically removing mono-ribosylation modifications18. His-pulldown results indicated that both ARH3 and Macro3 can interact with TRAF6 (Fig. 6h). Overexpression of AACRNLβ significantly promoted the binding of Macro3, but not ARH3, to TRAF6, while catalytically inactive AACRNLβ-HYY did not affect their interaction (Fig. 6i, j). These findings strongly support that AACRNLβ induces TRAF6 mono-ADP-ribosylation19.

AACRNLβ inhibits TRAF6-mediated ubiquitin signaling
To define how AACRNLβ-mediated mono-ADP-ribosylation affects TRAF6 function, we first examined its impact on TRAF6 stability. Knockdown or overexpression of AACRNLβ increased or decreased TRAF6 protein levels without affecting TRAF6 mRNA levels (Fig. 7a, b). During SCRV or V. anguillarum infection, AACRNLβ consistently suppressed TRAF6 protein expression (Fig. 7c). The AACRNLβ-HYY mutant also significantly degraded TRAF6, though less effectively (Fig. 7d). Cycloheximide (CHX) assays confirmed that AACRNLβ shortened the half-life of endogenous TRAF6 protein (Fig. 7e). To determine if AACRNLβ-mediated TRAF6 degradation was dependent on ubiquitin-proteasome, autophagosome, or lysosomal pathways, MKC cells were treated with MG132, 3-Methyladenine (3-MA), and NH4Cl. Results indicated AACRNLβ‘s interference with TRAF6 expression is ubiquitin-dependent (Fig. 7f). Next, we investigated the effect of AACRNLβ on the ubiquitination modification of TRAF6. AACRNLβ knockdown inhibited, while overexpression promoted, TRAF6 ubiquitination, which depended on AACRNLβ’s ADP-ribosylation activity (Fig. 7g, h). AACRNLβ overexpression significantly inhibited K27-, K33-, K48-, and K63-linked polyubiquitination of TRAF6 compared to other types of linkage (Fig. 7i, Supplementary Fig. 2a), and this regulation required AACRNLβ‘s ADP-ribosylation activity (Fig. 7j, k, Supplementary Fig. 2b, c). Given that TRAF6-mediated K27-linked ubiquitination of TAK1, and K63-linked ubiquitination of TBK1 or NEMO are crucial for responding to pathogenic infections20–22, AACRNLβ inhibits this ubiquitination activity through its ADP-ribosylation activity, thereby suppressing the immune signaling (Fig. 7l–n). These results suggest that AACRNLβ induces mono-ADP-ribosylation of TRAF6, facilitating its ubiquitin–proteasome–dependent degradation and impairing its function as a central E3 ligase in immune signaling, thereby contributing to toxicity and selfish reproduction.

TRAF6 ubiquitinates and degrades AACRNLβ as an antidote
As AACRNLβ remains toxic in the host, the host may implement counteracting detoxification functions to sustain immune equilibrium. Upon exposure to MG132, 3-MA, NH4Cl, and CQ, only MG132—a 26S proteasome inhibitor—significantly elevated AACRNLβ protein expression, indicating that the ubiquitin-proteasome system likely serves as a detoxifying pathway for AACRNLβ (Fig. 8a). Our research suggests that AACRNLβ, derived from AACRN effector, can inhibit the E3 activity of TRAF6. We then explored whether TRAF6 could use its ubiquitination activity to degrade AACRNLβ and act as an antidote. Results showed that knockdown or overexpression of TRAF6 increased or decreased AACRNLβ protein levels, supporting our hypothesis (Fig. 8b, c). Transfection of TRAF6 or its truncated mutants in MKC cells revealed that the TRAF6ΔRing and TRAF6ΔZF mutants, which lack E3 activity, could not degrade AACRNLβ (Fig. 8d). This degradation was independent of binding interactions since both TRAF6 and its mutants still interacted with AACRNLβ (Fig. 8e). Additionally, knockdown or overexpression of TRAF6 significantly reduced or enhanced the K48 ubiquitin modification of AACRNLβ (Fig. 8f, g). These findings suggest that TRAF6 can degrade AACRNLβ to counteract its inhibition, and serve as an antidote in the host’s body.

Methods
Identification of AACRN(L) proteins in organisms
BLASTP and TBLASTN searches were conducted on the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and target sequences with e-values above 10−10 and proteins with unrelated domain were discarded. These target sequences were used to perform iterative-BLAST to find more AACRN(L) proteins in eumycota and animalia.

Alignment, phylogenetic, and synteny analysis
Protein and DNA sequences were aligned by MUSCLE software (v3.8.31) for its high accuracy and speed, and Jalview software (v2.10) is used for visualizing comparison results. Maximum likelihood (ML) phylogenetic trees were generated using MEGA7, with WAG+G model and 1000 bootstrap replicates. Neighbor-Joining (NJ) trees were generated using MEGA7, with JTT model and 1000 bootstrap replicates. ITOL website (https://itol.embl.de/) were used to beautify the above phylogenetic tree. For gene synteny analysis, AACRNL genes were used as focal reference gene to TBLASTN the genome of seven indicated fish (S. Formosus, M. miiuy, P. platessa, O. latipes, O. niloticus, A. alosa, and D. rerio).

Domain, tertiary structure, and molecular docking analysis
The protein domain structure was predicted by the SMART website (http://smart.embl-heidelberg.de/). The tertiary structure of AACRNL proteins were predicted by AlphaFold Server (https://golgi.sandbox.google.com/). The molecular docking between mART domains and NAD+ were performed by AlphaFold Server website (AlphaFold Server (google.com)) and PyMOL software.

Identification of transposable elements flanking the AACRNL gene
To investigate the replicative transposition mechanism of AACRNL, ~5 kb of genomic DNA flanking the AACRNL gene was extracted. Then we used the BLAST tool at FishTEDB (https://www.fishtedb.com/) and Dfam website (https://dfam.org/search/sequence) to analyze potential transposon components.

Fish and challenge
M. miiuy (~50 g) was obtained from Zhoushan Fisheries Research Institute, Zhejiang Province, China. Fish was acclimated in aerated seawater tanks at 25°C for 6 weeks before experiments. In the SCRV injection group, fish was challenged with 200 μl SCRV at a multiplicity of infection (MOI) of 5 through intraperitoneal. As a comparison, 200 μl of physiological saline was used to challenge the individuals. Afterwards, the fish were sacrificed at different time points, and kidney and spleen tissues were collected for RNA extraction. SCRV virus was isolated as described previously39.

Cell culture
M. miiuy kidney cells (MKCs) were cultured in L-15 medium (HyClone) supplemented with 15% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin at 26°C. MKC cells were prepared from the kidney of M. miiuy as previously described40. EPC cells (ATCC, RRID: CVCL_4361) were maintained in medium 199 (HyClone) supplemented with 10% FBS (Gibco), 2 mM of L-glutamine, 100 U/mL of penicillin, and 100 mg/mL of streptomycin under humidified conditions at 26°C with 5% CO2. HEK293T cells (ATCC, RRID: CVCL_0063) were cultured in DMEM (HyClone) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% CO2. All cells were tested negative for mycoplasma contamination and morphologically confirmed. No cell line was used for more than 20 passages after thawing from frozen stocks.

Cells transfection
Cells were transiently transfected with siRNA in 24-well plates using Lipofectamine RNAiMAX (Invitrogen) and with DNA plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. For functional analyses, the overexpression plasmid (500 ng per well) and siRNA (100 nM) were transfected into cells in a culture medium and then harvested for further detection.

RNA interference
The AACRNLβ-specific small interfering RNA (siRNA; si-AACRNLβ-1 and si-AACRNLβ-2) sequences were 5′-GGAUGUUUCAACAAGAUCATT-3′ and 5′-GGACUUCUGUUGAAAGAAATT-3′, respectively. The TRAF6-specific small interfering RNA (si-TRAF6) is as follows: 5′-ACACCAUGCAAGGAGCUUUTT-3′. The scrambled control RNA sequences were 5′-UUCUCCGAACGUGUCACGUTT-3′.

Plasmids construction
The full-length coding sequence (CDS) of expression proteins of miiuy croaker (M. miiuy) were generated by PCR and cloned into pcDNA3.1 (Invitrogen) with specific tags. HA-tagged wild-type ubiquitin (UB-HA) and HA-tagged ubiquitin mutant expression plasmids were constructed with the template of pRK5-HA-ubiquitin-WT (Addgene, Cat# 17608) using ClonExpress II One Step Cloning Kit (Vazyme). Site or deletion mutants of AACRNLβ and TRAF6 were amplified by PCR with specific primers based on the recombinant plasmid. For protein purification, expression plasmids with 8× His tag was constructed based on pcDNA3.1. The correct construction of the recombinant plasmids was verified through Sanger sequencing and extracted using an Endotoxin-Free Plasmid DNA Miniprep Kit (Tiangen, China). The primers are listed in Supplementary Data 1.

Cell viability
Cell viability was assessed 48 h post-transfection in MKCs using the Cell-Titer-Glo luminescent assay (Promega), following the manufacturer’s protocol.

Protein purification and analysis
(His8)-tagged expression plasmids of M. miiuy were transiently overexpressed in 293T cells and lysed in a binding buffer (500 mM NaCl, 20 mM Tris/HCl [pH 7.4], 20 mM Imidazole, and 1% Triton X-100) including protease inhibitor cocktail (Roche). The lysate was incubated overnight at 4°C with anti-His beads (Solarbio). Anti-His beads were washed subsequently with washing buffer (500 mM NaCl, 20 mM Tris/HCl [pH 7.4], 20 mM Imidazole, and 1% Triton X-100). Proteins were eluted by addition of elution buffer (500 mM NaCl, 20 mM Tris/HCl [pH 7.4], 500 mM Imidazole, and 1% Triton X-100) to the beads. The purity of recombinant proteins was determined by SDS-PAGE separation or ponceau S. The purified AACRNLβ and AACRNLβ-HYY proteins were used to perform in vitro ADP-ribosylation assays.

RNA extraction and quantitative real-time PCR
The TRIzol Reagent (Invitrogen) was utilized for total RNA extraction, and subsequent cDNA synthesis was performed using the FastQuant RT Kit (Tiangen). The expression profiles of each gene were conducted by using the SYBR Premix Ex Taq™ (Takara). Quantitative real-time PCR was carried out using an Applied Biosystems® QuantStudio 3 (Thermo Fisher Scientific). β-actin served as the internal control for mRNA. The primer sequences are detailed in Supplementary Data 1.

Western blotting
Cellular lysates were prepared using 1× SDS-PAGE loading buffer. Protein extraction from cells was conducted, and quantification was performed using the BCA Protein Assay kit (Vazyme). The proteins were separated by SDS-PAGE (10%) and transferred onto PVDF membranes using a semidry blotting technique (Bio-Rad Trans Blot Turbo System). The membranes were then blocked with 5% BSA and probed with various antibodies. The antibody against AACRNLβ was diluted at 1:1000 (Customized in Genscript); The antibody against TRAF6 was diluted at 1: 500 (Boster, A00185); The antibody against IκBα was diluted at 1:500 (absin, abs106057); The antibody against TNF-α was diluted at 1:500 (ABclonal); The antibody against H3K27me3 (Beyotime, AF5710), H3K9me3 (Beyotime, AF5707), H3 (Beyotime, AF7101), and EZH2 (Beyotime, AF6828) were diluted at 1:500 (Beyotime); anti-Flag (Beyotime, AF519), anti-Myc (Beyotime, AF2864), anti-HA (Beyotime, AH158) and anti-Tubulin (Beyotime, AT819) monoclonal antibodies were diluted at 1: 1000; and HRP-conjugated anti-rabbit IgG (Abbkine, A25022) or anti-mouse IgG (Abbkine, A25012) at 1:5000. The immunoreactive proteins were visualized using Western Bright TM ECL (Advansta), and digital imaging was conducted with a cold CCD camera.

Co-immunoprecipitation
The cells were lysed in the cold IP buffer. The supernatant from cell lysates was incubated with indicated antibodies. Then, the protein A/G beads were incubated with the lysates overnight at 4°C. The beads were washed three times with cold lysis buffer and resuspended in the protein loading buffer. The immunoprecipitates and whole-cell lysates were followed by western blotting analysis.

Ni2+-NTA pull-down assay
Ni2+-NTA pull-down assay was done as described previously41. Cells cultured in 10-cm plates were transfected with the indicated plasmids. Cells from each plate were washed and collected with ice-cold PBS and divided into two aliquots. One sample was lysed with NP-40 lysis buffer and analyzed by immunoblotting directly to examine the expression of indicated proteins. Another sample was lysed in buffer A (8 M urea, 50 mM NaHPO4.H2O, 300 mM NaCl, 10 mM Tris-HCl, pH 8.0, 10 mM imidazole) and subjected to sonication for 20 s. Cell lysates were incubated with 50-µL pre-equilibrated Ni2+-NTA beads (denaturation type) for 8 h at 4°C. Then, beads were washed four times sequentially with buffer A and B (same as A except with 20 mM imidazole). Beads bound with His-tagged proteins were boiled with 2× SDS loading buffer containing 200 mM imidazole and were subjected to immunoblot.

Immunofluorescence
Cells cultured on glass coverslips in 24-well plates were fixed with 4% paraformaldehyde for 15 min following a triple wash with PBS. The cells were permeabilized with 0.5% Triton X-100 in PBS (PBST) for 15 min. After a 60-min block with 5% BSA, the cells were subjected to a 10-h incubation with the primary antibody at 4°C. Following three washes with PBS, the cells were exposed to a fluorescent-dye conjugated secondary antibody for 60 min at room temperature. Post a 10-min DAPI counterstain, the fluorescence signals of the cells were captured using a Leica fluorescence microscope.

In vitro ADP-ribosylation assay
After obtaining the purified proteins, ADP-ribosylation assays were carried out in 30-ml reaction buffer (50 mM Tris, pH 8.0, 0.2 mM DTT, 4 mM MgCl2) with 100 mM NAD+ (Macklin, N814818) or 100 mM Bio-NAD+ (Mreda, M256875) for 45 min at room temperature. The reactions were stopped by addition of SDS sample buffer, and the products were probed with mono-ADP-Ribose antibody (BIO-RAD, AbD33204) or streptavidin–HRP (Beyotime, A0303).

In vivo ADP-ribosylation analysis
MKC cells were transfected with AACRNLβ-Myc or AACRNLβ-HYY-Myc plasmids, then cell lysate was immunoprecipitated by Myc antibody and the interacted proteins with mono-ADPr modification were immunoblotted with mono-ADPr antibody to analyse the ADP-ribosylation effects of AACRNLβ in vivo. To detect whether TRAF6 and EZH2 have mono-ADPr modification in vivo, MKC cell lysate was immunoprecipitated by IgG antibody or mono-ADP-Ribose antibody, then TRAF6 and EZH2 antibody were used to detect mono-ADP-ribosylation.

Chromatin immunoprecipitation (ChIP) assay
MKC cells were seeded in 10 cm² dishes and transfected with plasmids once the cell density reached 5.0 × 10⁶. For chromatin immunoprecipitation (ChIP), cells were cross-linked with 1% formaldehyde at 37°C for 10 min, then quenched with glycine for 5 min at room temperature. After two washes with PBS, cells were lysed on ice, collected, and sonicated to shear chromatin. Lysates were clarified by centrifugation at 12,000 rpm (~13,800 × g) for 10 min at 4°C. The resulting supernatant was incubated with the appropriate antibody overnight at 4°C with rotation. Protein A/G agarose beads were added and incubated for 2 h at 4°C. After centrifugation at 6000 rpm (~3500 × g) for 5 min, the supernatant was discarded. Beads were sequentially washed with low-salt wash buffer (twice), LiCl wash buffer, and TE buffer. Immunocomplexes were eluted with elution buffer and diluted to 500 ml using diffusion buffer. Cross-links were reversed by adding 5 M NaCl and incubating at 65°C for 12 h, followed by treatment with 0.5 M EDTA, 1 M Tris-HCl, and 20 mg/ml Proteinase K at 45°C for 2 h.

Statistical analysis
Statistical analyses were performed using Prism v8 (GraphPad Software). Data are expressed as the mean ± SE from at least three independent experiments, each performed with three technical replicates. Comparisons between two groups were performed using a two-tailed unpaired Student’s t-test. For comparisons involving more than two groups, one-way ANOVA followed by Duncan’s post-hoc multiple comparison test was conducted. The relative gene expression data was acquired using the 2−ΔΔCT method42. Effect sizes (Cohen’s d for t-tests and η² for ANOVA) were calculated within Prism v8. A value of p < 0.05 was considered significant.

Ethics statement
All animal experimental procedures were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Research Ethics Committee of Shanghai Ocean University (No. SHOU-DW-2025-033).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the discovery of a ‘selfish’ gene product that is endemic to a variety of organisms within multiple phyla is reported. The authors describe AACRN as a Crinkler family protein product encoded in host genomes that resembles pathogenic gene products. In this manuscript the authors describe the distribution and frequency of AACRN genes in a number of kingdoms and phyla. The authors go on to characterize the genetic structure of AACRN families and conclude that beta and gamma members in fish species are preferentially replicating themselves within their hosts genome. They elucidate that AACRN is often within a non-autonomously replicating transposon and that AACRN is able to interact with EZH2 to inhibit histone methylation of the transposon and thereby increase their expression and subsequent ability to propagate with the transposon. Given that these types of pathogenic gene products and transposition mechanisms activate host immune mechanisms the authors investigated how AACRN manipulates host defense mechanisms. To this end they describe how AACRN can ADP ribosylate TRAF6 and target it for ubiquitylation and degradation.\nOverall, this is a well written and intriguing body of work describing a novel form of selfish gene or pathogenic gene product that has persisted in host genomes. The data are of high quality and the experiments, their interpretation, and conclusions are well reasoned and appropriate. I have a few suggests of places to improve the manuscript before publication. They are ranked roughly in order of importance for improving the manuscript.\n1) The authors conclude that AACRN is strictly a remnant of pathogenesis and its perpetuation is an example of a gene product that is functionless to the host but who’s mechanism of action and gene structure ensures propagation. It struck me that many examples of repetitive elements in mammals are described as junk or remnants of ancient infections until they are discovered to have an endogenous function. The authors discuss examples of this (eg RAG proteins) but conclude AACRN is just a pathogen. I think this conclusion is premature. There is no investigation of how this gene could have an endogenous function that is beneficial to their host fish species. This issue is potentially addressable. Morpholino knock downs are routine in zebrafish embryos and the authors indicate that Danio rario has an AACRN gamma family of genes. If knock down compromised development then the host has exapted AACRN function. Without an experiment such as this to address the hosts relationship with AACRN I think it is premature to discuss it as purely a pathogen and the discussion should instead indicate that this is an open question.\n2) My biggest criticism of the data as presented is the lack of investigation of endogenous AACRN. Much of the transfection based approaches to investigate ubiquitination and ribosylation make sense to me, but the data that implicates endogenous AACRN in these types of activities is lacking. Does endogenous AACRN bind to TRAF6? The siRNA knock down of AACRN in Fig. 5G is not the most convincing with regards to TRAF6 levels being dependent on AACRN expression. Similarly, showing endogenous stabilization of AACRN by MG132 instead of transfection in Fig. 6A is key to validating the more artificial and transfection based experiments that ensue. There are key endogenous level observations of AACRN that are needed to cement how it works as relying entirely on transfection is risky.\n3) I thought it was odd that the endogenous TRAF6 blot that demonstrates MG132 increases its expression and implicates regulation by ubiquitylation was in supplemental figure 2. It is more important the downstream details of attachment points for ubiquitin in Fig. 5K. Furthermore, the conclusion that K48 and K63 are the most common point of attachment seems inappropriate based on this figure. The blot shown is too exposed to rule out a similar effect of AACRN on K6 and K29, while K33 and K27 seem to look just like K63. This experiment doesn’t rationalize the use of K63 Ub in later experiments, it is just one attachment point among many.\n4) The inclusion of TACRN in Fig.1 and its occasional mention in the results and discussion is a distraction. It isn’t studied sufficiently to add to the work and is a distracting tangent in what is otherwise almost a very complete look at AACRN.\n5) My reading of the manuscript indicates that most experiments take place in fish cells of the appropriate species and that cDNAs were generated from it for most transfection experiments. However the methods and figure legends allude to the occasional use of 293T cells and the difficulty of finding those experiments more clearly is troubling. It would be better if these specific experiments (ie. panels within figures) were specifically indicated in the results narrative so they can be readily seen as heterologous.\n6) Throughout reading the paper is struck me that a final published version of this work would be very unlikely to include figures in the form that they appear in this submission. Many figures are a full page in landscape mode. Figure 1 exemplifies this as in addition to occupying this much space it’s panels are extensively text dependent and already too small to read. I’m not sure what to suggest but it this needs extensive reorganization to be readable in a pdf.", "reviewer_3": "Crinkler (CRN) effector proteins represent a family of pathogen-secreted molecules that manipulate host cell immune defenses during infection. This manuscript demonstrates that ADP-ribosyltransferase-associated CRN (AACRN), a CRN homolog, has evolved beyond its pathogenic origins to function as an epigenetic parasite in free-living eukaryotes. The authors present molecular evidence showing that AACRN mono-ADP-ribosylates both EZH2 and TRAF6 via its mART domain, thereby disrupting H3K27me3-mediated chromatin silencing and suppressing NF-κB/IRF3 signaling to establish an immunologically permissive environment for expansion. The data presented supports the authors' conclusions; however, several critical issues require attention.\nMajor Comments:\n1. The fundamental question remains: why do these degenerated effector proteins maintain intact mART activity and continue expanding in free-living eukaryotes? This evolutionary paradox deserves deeper and mechanistic explanation.\n2. Mechanism of EZH2 Protein Reduction- The observed decrease in EZH2 protein expression following AACRN β-Myc expression (Figure 4H) requires mechanistic clarification. Specifically, does this reduction result from decreased EZH2 mRNA transcription or enhanced protein degradation, similar to the TRAF6 degradation demonstrated in this study?\n3. What is the impact of AACRNβ mutation in host chromatin regulation and gene expression? Understanding these broader genomic impacts would strengthen the conclusions regarding AACRN's role as an epigenetic modifier. This comment is mostly related to my point no. 1.\nMinor comments:\n1. Line 55, translocation word is written twice.\n2. In fig. 1C, classes without AARCNs are highlighted with black color whereas in line 146, authors state that ‘AACRN exhibits lineage-specific expansion and contraction in bony fish, with Cyprinus carpio displaying the highest number of AACRN’. I am not clear, whether the bony fish has AACRNs or not.\n3. Line 149 is sentence incomplete, please complete it.\n4. Line 237, (Fig. 6H) should be (Fig. 5H).\n5. Provide references to this statement in line 250, ‘TRAF6-mediated K63-250 linked ubiquitination of TBK1 or NEMO is crucial for responding to pathogenic infections’.\n6. Line 263, the statement is incorrect. It should be- ‘Results showed that knockdown or overexpression of TRAF6 increased or decreased AACRNβ protein levels, supporting our hypothesis (Fig. 6B and 6C)’.", "reviewer_5": "This manuscript describes the discovery of proteins which have similarity to functional domains found in some CRN effector proteins from pathogenic eukaryotes. The authors focus most of their efforts on characterising one of these, AACRN, and show that it has ADP-ribosylation activity. Intriguingly, it appears to use this toxin-like activity to promote its own expression and replication, by suppressing the epigenetic mechanisms that cells would normally use to control mobile elements. The host genome has evolved an antidote to AACRN to keep its activity in check. This was a very interesting manuscript to read.\nCRN effectors were first described from oomycete plant pathogens. The unifying feature of these candidate effectors is a highly conserved N terminal peptide sequence, while the C terminal section of these proteins can be highly diverse. Since the proteins studied in this manuscript lack the classical N terminal leader (header) characteristic of CRN proteins, it seems misleading to call them as such. I would strongly suggest that the AACRN proteins be renamed throughout the manuscript. This will also impact on the title for the manuscript, which will need to be changed accordingly.\nGenerally, the work has been done well and I have only relatively minor issues for the authors to address. Several of the western blot figures show uneven band intensity, suggesting technical issues with protein transfer to membranes, and making it difficult to interpret the results. Some of these figures would also benefit from signal quantification to more accurately determine the significance of the results. Inclusion of replicate western blot results in the supplementary information would also help the reader.\nLine 240: write out the name of 3-MA in full here. Correct the formatting for NH4Cl.\nLine 262: this should be ‘ubiquitination activity’.\nLine 298: the paper cited here (ref 28) is about oomycetes, not fungi.\nLine 349-354: Which versions of the software packages were used?\nLine 356: please give the names of the seven indicated fish.\nLine 374: most of this line seems to have been duplicated from the previous line.\nLine 382-383: the syntax in this sentence seems strange and should be corrected.\nLine 395: scientific name for miiuy croaker?\nLine 398: Addgene plasmid number?\nLines 487 and 490: centrifugation conditions should be expressed as x g.\nFigure 4B+C which shows a western blot analysis of AACRN protein activities would benefit from quantification of the signal on the blots.\nIn Figure 5G, the bands in the uppermost panel show uneven signal within each band, so it is impossible to determine whether the siRNA treatments differ from the controls. Again, quantification of the signal from the western blots would help to interpret the figure.\nIn Figure 5K, it is difficult to see obvious differences in polyubiquitination for K48 and K63. Again, the samples seem to be unevenly transferred, leading to uneven signals. (it would also have been helpful if the authors had submitted their other replicates of western blots)"}, "author_response": "Point-by-point response\nWe thank the reviewers for their time and for their insightful and constructive comments. We\nare encouraged that Reviewer #1 considered this “a well written and intriguing body of work, …the\ndata are of high quality and the experiments, their interpretation, and conclusions are well reasoned\nand appropriate”, that Reviewer #3 found that “the data presented supports the authors’ conclusions”,\nand that Reviewer #5 commented that “this was a very interesting manuscript to read, …the work\nhas been done well”.\nThree reviewers provided professional suggestions to improve the quality of the manuscript,\nwe have carefully addressed all points raised by the reviewers through additional experiments and\nhave substantially revised the manuscript, including the addition of 23 new figure panels and\nimprovements to the clarity of the text. Point-by-point responses to all comments are provided below,\nand all revisions in the manuscript are marked using track changes.\nReviewer #1:\nIn this manuscript, the discovery of a ‘selfish’ gene product that is endemic to a variety of\norganisms within multiple phyla is reported. The authors describe AACRN as a Crinkler family\nprotein product encoded in host genomes that resembles pathogenic gene products. In this\nmanuscript the authors describe the distribution and frequency of AACRN genes in a number of\nkingdoms and phyla. The authors go on to characterize the genetic structure of AACRN families\nand conclude that beta and gamma members in fish species are preferentially replicating themselves\nwithin their hosts genome. They elucidate that AACRN is often within a non-autonomously\nreplicating transposon and that AACRN is able to interact with EZH2 to inhibit histone methylation\nof the transposon and thereby increase their expression and subsequent ability to propagate with the\ntransposon. Given that these types of pathogenic gene products and transposition mechanisms\nactivate host immune mechanisms the authors investigated how AACRN manipulates host defense\nmechanisms. To this end they describe how AACRN can ADP ribosylate TRAF6 and target it for\nubiquitylation and degradation.\ngene or pathogenic gene product that has persisted in host genomes. The data are of high quality\nand the experiments, their interpretation, and conclusions are well reasoned and appropriate. I have\na few suggests of places to improve the manuscript before publication. They are ranked roughly in\norder of importance for improving the manuscript.\nResponse: We sincerely thank Reviewer #1 for the thoughtful, detailed, and highly professional\nevaluation of our manuscript. We are very pleased that the reviewer considered our study to be “well\nwritten,” “intriguing,” and supported by high-quality data and well-reasoned conclusions. We truly\nappreciate this positive recognition.\nWe are equally grateful for the reviewer’s insightful and constructive suggestions. These\ncomments were extremely valuable for refining our analyses and improving the overall clarity and\nrigor of the manuscript. In response, we have carefully addressed all points through additional\nexperiments, strengthened data interpretation, and substantive revisions to the text. Additionally,\nfollowing Reviewer #5’s suggestion regarding nomenclature, we have updated “AACRN” to\n“AACRN-like (AACRNL)” throughout both the revised manuscript and our responses to reviewers\nto ensure consistency and avoid any confusion. All corresponding updates have been incorporated\ninto the revised manuscript, and detailed point-by-point responses are provided below.\nQ1: The authors conclude that AACRN is strictly a remnant of pathogenesis and its perpetuation is\nan example of a gene product that is functionless to the host but who’s mechanism of action and\ngene structure ensures propagation. It struck me that many examples of repetitive elements in\nmammals are described as junk or remnants of ancient infections until they are discovered to have\nan endogenous function. The authors discuss examples of this (eg RAG proteins) but conclude\nAACRN is just a pathogen. I think this conclusion is premature. There is no investigation of how\nthis gene could have an endogenous function that is beneficial to their host fish species. This issue\nis potentially addressable. Morpholino knock downs are routine in zebrafish embryos and the\nauthors indicate that Danio rario has an AACRN gamma family of genes. If knock down\ncompromised development then the host has exapted AACRN function. Without an experiment such\nas this to address the hosts relationship with AACRN, I think it is premature to discuss it as purely\n2\na pathogen and the discussion should instead indicate that this is an open question.\nResponse: We thank the reviewer for raising this insightful point. To further assess the possibility\nthat AACRNL may have endogenous, host-beneficial functions, we performed additional analyses\nusing publicly available zebrafish datasets. Single-cell RNA-seq profiles indicate that members of\nthe Danio rario AACRNLγ family show no detectable transcription during embryogenesis (early\n4–6 h, mid 8–12 h, and late 24 h stages), likely due to H3K27me3-mediated repression (Fig. 5a),\nsuggesting that these loci are unlikely to play a developmental role. However, their inactivity during\ndevelopment does not exclude context-dependent functions. Indeed, Danio rario AACRNLγ2, γ3,\nand γ5 are rapidly induced following SVCV infection (Fig. 5b), and M. miiuy AACRNLβ is\nsimilarly activated upon SCRV infection (Fig. 5c–5f). Upon activation, AACRNLβ exhibits ADP-\nribosyltransferase activity that promotes DNA damage and derepresses multiple transposon RNAs\n(LINEs and ERVs) (Fig. 5g–5j), further supporting its selfish, pathogen-like behavior.\nWhile our current data reinforce the interpretation that AACRNL functions primarily as a self-\npropagating, toxin-derived element, we fully agree with the reviewer that potential host-beneficial\nroles cannot be ruled out. We have revised the Discussion to clearly state that the possibility of\nbeneficial or adaptive host roles remains open and warrants future investigation (Lines 374-379).\nQ2: My biggest criticism of the data as presented is the lack of investigation of endogenous AACRN.\nMuch of the transfection-based approaches to investigate ubiquitination and ribosylation make sense\nto me, but the data that implicates endogenous AACRN in these types of activities is lacking. Does\nendogenous AACRN bind to TRAF6? The siRNA knock down of AACRN in Fig. 5G is not the\nmost convincing with regards to TRAF6 levels being dependent on AACRN expression. Similarly,\nshowing endogenous stabilization of AACRN by MG132 instead of transfection in Fig. 6A is key\nto validating the more artificial and transfection-based experiments that ensue. There are key\nendogenous level observations of AACRN that are needed to cement how it works as relying entirely\non transfection is risky.\nResponse: We sincerely thank the reviewer for this insightful and important comment. To directly\naddress the concern regarding endogenous AACRNL, we generated a custom antibody against M.\nmiiuy AACRNLβ through GenScript Co.. The specificity of this antibody was confirmed by siRNA-\n3\nmediated knockdown, which markedly reduced the endogenous AACRNLβ signal (Fig. 7a).\nUsing this endogenous AACRNLβ antibody, we supplemented and repeated several key\nexperiments, including those shown in Figs. 6c, 7a, and 8a–8d. Our results demonstrate that\nendogenous AACRNLβ can specifically bind TRAF6 (Fig. 6c), and that si- AACRNLβ knockdown\nsignificantly increases TRAF6 protein levels (Fig. 7a), confirming that TRAF6 expression is\ndependent on AACRNLβ. Furthermore, treatment with MG132, as well as TRAF6 knockdown,\noverexpression of wild-type plasmid, and mutant plasmids, all affected endogenous AACRNLβ\nprotein expression as expected (Fig. 8a-8d), providing further validation of the observations\npreviously made using transfection-based approaches. Together, these data now provide strong\nevidence that endogenous AACRNLβ is functionally active in regulating TRAF6 and support the\nconclusions drawn from the transfection experiments.\nQ3: I thought it was odd that the endogenous TRAF6 blot that demonstrates MG132 increases its\nexpression and implicates regulation by ubiquitylation was in supplemental figure 2. It is more\nimportant the downstream details of attachment points for ubiquitin in Fig. 5K. Furthermore, the\nconclusion that K48 and K63 are the most common point of attachment seems inappropriate based\non this figure. The blot shown is too exposed to rule out a similar effect of AACRN on K6 and K29,\nwhile K33 and K27 seem to look just like K63. This experiment doesn’t rationalize the use of K63\nUb in later experiments, it is just one attachment point among many.\nResponse: We thank the reviewer for this insightful comment. To better emphasize the regulation\nof endogenous TRAF6 by AACRNLβ, we have moved the data previously shown in Fig. S2 to the\nmain figures (now Figs. 7b, 7c, 7e, and 7f), where the key observations are presented more clearly.\nIn response to the reviewer’s concern regarding the interpretation of ubiquitin linkage\nspecificity, we optimized the experimental conditions and re-examined the effects of AACRNLβ on\nmultiple ubiquitin linkage types (Fig. 7i). Following Reviewer #5’s suggestion, we have also\nincluded independent experimental repeats in Fig. S2a. These data show that AACRNLβ\noverexpression markedly increases K27, K33, K48, and K63 ubiquitination of TRAF6, whereas K6,\nK11, and K29 ubiquitination remain unchanged. We interpret this global increase as consistent with\nour proposed model in which AACRNLβ inhibits TRAF6-mediated chain transfer, causing ubiquitin\n4\nchains to accumulate on TRAF6 itself.\nTo address the reviewer’s question about the rationale for focusing on K63 ubiquitination, we\nhighlight that K63-linked chains represent the most functionally relevant modification catalyzed by\nTRAF6 during innate immune signaling (DOI: 10.1128/mcb.00270-07; DOI:\n10.3389/fimmu.2024.1339510). Accordingly, we examined whether AACRNLβ affects TRAF6-\nmediated K63 ubiquitination of downstream substrates and found that AACRNLβ significantly\ninhibits the K63-linked modification of TBK1 and NEMO (Figs. 7m and 7n), thereby blocking\ndownstream signaling. To further support the chain-retention mechanism, we performed additional\nfunctional assays using K27-linked ubiquitin, given that TRAF6 has been reported to mediate\nnoncanonical K27 ubiquitination of TAK1 (DOI: 10.4049/jimmunol.1900083). Consistent with our\nmodel, AACRNLβ suppresses TRAF6-mediated K27 ubiquitination of TAK1 (Fig. 7l).\nTogether, these revised data provide a more comprehensive and mechanistically coherent basis\nfor how AACRNLβ modulates TRAF6 ubiquitination and justify our focus on TRAF6 K63- and\nK27-linked activities in downstream signaling.\nQ4: The inclusion of TACRN in Fig.1 and its occasional mention in the results and discussion is a\ndistraction. It isn’t studied sufficiently to add to the work and is a distracting tangent in what is\notherwise almost a very complete look at AACRN.\nResponse: We thank the reviewer for this valuable comment. In response, we have removed all\nreferences to TACRN in Fig. 1, as well as in the Results and Discussion sections, to maintain a\nclearer focus on AACRN and avoid any distraction from the main story.\nQ5: My reading of the manuscript indicates that most experiments take place in fish cells of the\nappropriate species and that cDNAs were generated from it for most transfection experiments.\nHowever, the methods and figure legends allude to the occasional use of 293T cells and the difficulty\nof finding those experiments more clearly is troubling. It would be better if these specific\nexperiments (ie. panels within figures) were specifically indicated in the results narrative so they\ncan be readily seen as heterologous.\nResponse: Thank you for your insightful comment. After carefully reviewing the manuscript, I\n5\nfound that 293T cells were only used during the protein purification step. Specifically, His-tagged\nplasmids were transfected into 293T cells to purify AACRN and AACRN-HYY proteins, which\nwere then used in the subsequent in vitro ADP-ribosylation assays. Therefore, the use of 293T cells\nis limited to this part of the experiment, and it may not be entirely appropriate to label the figures\nrelated to the in vitro ribosylation assays as involving 293T cells.\nTo improve the clarity of the manuscript, we have added a more detailed description of the\npurpose of protein purification (Lines 470-471). Additionally, we have updated the figure legends\nof Figs. 2e, 2f, 4f, 4g, 6f, and 6g to explicitly indicate the use of 293T cells for protein production.\nWe appreciate the reviewer’s suggestion, which has helped make the manuscript clearer and more\nprecise.\nQ6: Throughout reading the paper is struck me that a final published version of this work would be\nvery unlikely to include figures in the form that they appear in this submission. Many figures are a\nfull page in landscape mode. Figure 1 exemplifies this as in addition to occupying this much space\nit’s panels are extensively text dependent and already too small to read. I’m not sure what to suggest\nbut it this needs extensive reorganization to be readable in a pdf.\nResponse: Thank you very much for your valuable suggestion. We have made significant revisions\nto the figures to ensure better clarity for the final published version. Specifically, Figures 1, 2, 3, 4,\nand 8 have been reorganized into a more square layout to reduce excessive horizontal space. In\nresponse to your comment regarding Fig. 1, we have also removed the TACRN-related content, as\nper your recommendation, to reduce crowding and improve readability. Additionally, due to the\ninclusion of new experiments and the transfer of some data from Figure S2 to the main figures (as\nsuggested), we found that Fig. 5, with its large number of panels, required further splitting. As a\nresult, we have separated it into Fig. 6 and Fig. 7 to make the presentation clearer and more\nmanageable. We believe these changes substantially enhance figure clarity and overall readability,\nensuring the manuscript is well-prepared for publication.\nReviewer #3:\nmanipulate host cell immune defenses during infection. This manuscript demonstrates that ADP-\nribosyltransferase-associated CRN (AACRN), a CRN homolog, has evolved beyond its pathogenic\norigins to function as an epigenetic parasite in free-living eukaryotes. The authors present molecular\nevidence showing that AACRN mono-ADP-ribosylates both EZH2 and TRAF6 via its mART\ndomain, thereby disrupting H3K27me3-mediated chromatin silencing and suppressing NF-κB/IRF3\nsignaling to establish an immunologically permissive environment for expansion. The data\npresented supports the authors' conclusions; however, several critical issues require attention.\nResponse: We thank the reviewer for the insightful summary and for recognizing the significance\nof our findings on AACRN’s dual roles in chromatin regulation and immune signaling. We\nappreciate the constructive comments and have carefully addressed all critical issues raised. In\nresponse, we performed additional experiments, revised the figures, and refined the manuscript for\nimproved clarity and rigor. Additionally, following Reviewer #5’s suggestion regarding\nnomenclature, we have updated “AACRN” to “AACRN-like (AACRNL)” throughout both the\nrevised manuscript and our responses to reviewers to ensure consistency and avoid any confusion.\nDetailed responses are provided below.\nMajor Comments:\nQ1: The fundamental question remains: why do these degenerated effector proteins maintain intact\nmART activity and continue expanding in free-living eukaryotes? This evolutionary paradox\ndeserves deeper and mechanistic explanation.\nResponse: We appreciate the reviewer’s insightful question regarding why these degenerated\neffector proteins retain intact mART activity and continue to expand in free-living eukaryotes. To\naddress this evolutionary paradox, we incorporated new analyses and expanded our discussion.\nFirst, by examining publicly available transcriptomic datasets, we found that all five zebrafish\nAACRNLγ members remain largely silent during embryogenesis, whereas three members\n(AACRNLγ2, 3, and 5) are strongly induced following SVCV infection (Figs. 5a and 5b). Similarly,\nin M. miiuy, AACRNLβ expression is markedly upregulated upon SCRV infection and coincides\nwith increased DNA damage and heightened transposon activity (Figs. 5c-5j). These results indicate\n7\nthat pathogen infection is a key trigger for AACRNL activation, while under basal conditions\nAACRNL remains repressed to minimize host fitness costs.\nSecond, our findings support a model in which AACRNL family expansion reflects a balance\nbetween host-driven degeneration and the element’s intrinsic capacity for self-propagation.\nConsistent with host-imposed silencing, several AACRNL homologs show evidence of functional\ndecay—for example, zebrafish AACRNLγ1 and AACRNLγ4 retain intact coding sequences but\nappear transcriptionally inactive (Figs. 5a and 5b), and multiple fish species (including M. miiuy, A.\nalosa, and P. platessa) harbor clearly degenerated AACRNL copies (Fig. 3c). Nevertheless, viral\ninfection provides episodic opportunities for reactivation. Once induced, AACRNL not only\npromotes genomic instability but also suppresses immune signaling, generating a transient\n“replication window” that facilitates transposon mobilization and ensures propagation of active\nAACRNL elements.\nThus, we propose that AACRNL preserves mART catalytic activity because this activity\nremains essential for its occasional but crucial bursts of replication under infection-induced\npermissive conditions. We have now incorporated these data and mechanistic considerations into\nthe revised Discussion (Lines 317-334).\nQ2: Mechanism of EZH2 Protein Reduction- The observed decrease in EZH2 protein expression\nfollowing AACRN β-Myc expression (Figure 4H) requires mechanistic clarification. Specifically,\ndoes this reduction result from decreased EZH2 mRNA transcription or enhanced protein\ndegradation, similar to the TRAF6 degradation demonstrated in this study?\nResponse: We appreciate the reviewer’s insightful comment regarding the mechanism underlying\nthe reduction of EZH2 protein levels upon AACRNβ-Myc expression. To clarify this point, we\nperformed additional experiments. Quantitative RT–PCR analysis showed that overexpression of\nAACRNβ-Myc does not alter EZH2 mRNA levels, indicating that transcriptional regulation is not\ninvolved (Fig. 4i). Furthermore, cycloheximide (CHX) chase assays demonstrated that AACRNβ-\nMyc accelerates the degradation of EZH2 protein (Fig. 4j). These results support that AACRNβ\nprimarily reduces EZH2 abundance by promoting its protein degradation rather than affecting its\ntranscription, consistent with our observations on AACRNβ-mediated regulation of TRAF6 stability.\nUnderstanding these broader genomic impacts would strengthen the conclusions regarding\nAACRN's role as an epigenetic modifier. This comment is mostly related to my point no. 1.\nResponse: We thank the reviewer for raising this important point. To address the broader chromatin\nand transcriptional impacts of AACRNLβ, we performed additional analyses focusing on key\nrepressive epigenetic marks and transposon activity.\nFirst, we examined whether AACRNLβ affects other major heterochromatic modifications\nbeyond H3K27me3. Our results show that AACRNLβ does not alter the levels of H3K9me3,\nindicating that its impact on host chromatin is selective rather than globally disruptive (Fig. 4b).\nIn addition, as noted in our response to Comment 1, AACRNL is transcriptionally activated during\nviral infection. We further demonstrate that AACRNL activation during virus infection correlates\nwith increased DNA damage and reactivation of multiple transposable elements, supporting its role\nas a stress-responsive epigenetic disruptor (Fig. 5). Importantly, knockdown of AACRNLβ rescues\nthe SCRV-induced loss of H3K27me3, confirming that AACRNLβ is important for virus-mediated\nrelease of Polycomb repression. Mechanistically, we show that AACRNLβ enhances the expression\nof canonical DNA-damage–responsive genes (tp53, p21, gadd45a) in a mono-ADP-\nribosyltransferase–dependent manner, and similarly promotes the transcription of LINE and ERV\nretroelements through its enzymatic activity. These results indicate that AACRNLβ exerts broad\neffects on chromatin stability and genome regulation, primarily through its catalytic function and its\nability to antagonize H3K27me3-mediated silencing during viral infection.\nTogether, these findings demonstrate that AACRNLβ functions as a potent epigenetic disruptor\nthat promotes DNA damage, removes H3K27me3-dependent repression, and activates transposon\nexpression in an mART-dependent manner, particularly under viral infection.\nMinor comments:\nQ1: Line 55, translocation word is written twice.\nResponse: Thank you for taking the trouble to point out our mistakes. The duplicate word\n“translocation” has been removed (Line 57).\nauthors state that ‘AACRN exhibits lineage-specific expansion and contraction in bony fish, with\nCyprinus carpio displaying the highest number of AACRN’. I am not clear, whether the bony fish\nhas AACRNs or not.\nResponse: Thank you for pointing out our mistake caused by carelessness, we appreciate the\nreviewer’s comment. The inconsistency arose because the panel was mistakenly labeled as Fig. 1c\ninstead of Fig. 3a, which resulted in the apparent mismatch. We have corrected the figure label and\nupdated the corresponding text (Line 145).\nQ3: Line 149 is sentence incomplete, please complete it.\nResponse: Thank you for taking the trouble to point out our mistakes. The incomplete sentence has\nbeen completed and revised for clarity (Lines 146-151).\nQ4: Line 237, (Fig. 6H) should be (Fig. 5H).\nResponse: Thank you for pointing this out. Because the figures have been substantially reorganized\nduring revision, the previous Fig. 5h has now been updated to Fig. 7d.\nQ5: Provide references to this statement in line 250, ‘TRAF6-mediated K63-250 linked\nubiquitination of TBK1 or NEMO is crucial for responding to pathogenic infections’.\nResponse: Thank you for your advice. Supporting references for the statement on TRAF6-mediated\nK63-linked ubiquitination of TBK1/NEMO have been added (Line 271).\nQ6: Line 263, the statement is incorrect. It should be- ‘Results showed that knockdown or\noverexpression of TRAF6 increased or decreased AACRNβ protein levels, supporting our\nhypothesis (Fig. 6B and 6C)’.\nResponse: Thank you for taking the trouble to point out our mistakes. The sentence has been\ncorrected to: “Results showed that knockdown or overexpression of TRAF6 increased or decreased\nAACRNLβ protein levels, supporting our hypothesis (Fig. 8b and 8c).”\nThis manuscript describes the discovery of proteins which have similarity to functional\ndomains found in some CRN effector proteins from pathogenic eukaryotes. The authors focus most\nof their efforts on characterising one of these, AACRN, and show that it has ADP-ribosylation\nactivity. Intriguingly, it appears to use this toxin-like activity to promote its own expression and\nreplication, by suppressing the epigenetic mechanisms that cells would normally use to control\nmobile elements. The host genome has evolved an antidote to AACRN to keep its activity in check.\nThis was a very interesting manuscript to read.\nCRN effectors were first described from oomycete plant pathogens. The unifying feature of\nthese candidate effectors is a highly conserved N terminal peptide sequence, while the C terminal\nsection of these proteins can be highly diverse. Since the proteins studied in this manuscript lack\nthe classical N terminal leader (header) characteristic of CRN proteins, it seems misleading to call\nthem as such. I would strongly suggest that the AACRN proteins be renamed throughout the\nmanuscript. This will also impact on the title for the manuscript, which will need to be changed\naccordingly.\nGenerally, the work has been done well and I have only relatively minor issues for the authors\nto address. Several of the western blot figures show uneven band intensity, suggesting technical\nissues with protein transfer to membranes, and making it difficult to interpret the results. Some of\nthese figures would also benefit from signal quantification to more accurately determine the\nsignificance of the results. Inclusion of replicate western blot results in the supplementary\ninformation would also help the reader.\nResponse: We sincerely thank the reviewer for the insightful and constructive comments. We fully\nagree that referring to these proteins as CRN homologs was potentially misleading given the absence\nof the canonical N-terminal leader. In response, we have renamed AACRN as AACRN-like\n(AACRNL) throughout the manuscript (Fig. 1b), including the title, to clearly distinguish them from\nclassical CRN effectors.\npanels. We have optimized the experimental and transfer conditions, repeated the relevant\nexperiments, and updated the figures accordingly. In addition, representative replicate blots have\nbeen included in the supplementary information to improve clarity and reproducibility.\nWe are grateful for the reviewer’s positive evaluation of our work and hope that the revised\nmanuscript satisfactorily addresses all concerns.\nQ1: Line 240: write out the name of 3-MA in full here. Correct the formatting for NH Cl.\nfor NH₄Cl has been corrected (Line 262).\nQ2: Line 262: this should be ‘ubiquitination activity’.\nResponse: Revised to “ubiquitination activity.” (Line 283)\nQ3: Line 298: the paper cited here (ref 28) is about oomycetes, not fungi.\nResponse: The reference has been checked; we now clarify that ref. 28 pertains to oomycetes (Line\n339).\nQ4: Line 349-354: Which versions of the software packages were used?\nResponse: The specific software versions have been added (Lines 391-393).\nQ5: Line 356: please give the names of the seven indicated fish.\nResponse: The seven indicated fish species are now listed by name (Lines 397-398).\nQ6: Line 374: most of this line seems to have been duplicated from the previous line.\nResponse: The duplicated portion has been removed for clarity (Line 418).\nQ7: Line 382-383: the syntax in this sentence seems strange and should be corrected.\nResponse: The sentence syntax has been corrected (Lines 433-434).\nResponse: The scientific name of miiuy croaker (M. miiuy) has been added (Line 447).\nQ9: Line 398: Addgene plasmid number?\nResponse: The Addgene plasmid number has been included (Line 450).\nQ10: Lines 487 and 490: centrifugation conditions should be expressed as ×g.\nResponse: Centrifugation conditions are now expressed in ×g (Lines 545 and 548).\nQ11: Figure 4B+C which shows a western blot analysis of AACRN protein activities would benefit\nfrom quantification of the signal on the blots.\nResponse: We thank the reviewer for the suggestion. We have improved the experimental\nconditions, including increasing the antibody concentration, and have repeated the western blot\nexperiments for Figs 4b and 4c. The panels have been replaced with higher-quality images.\nQ12: In Figure 5G, the bands in the uppermost panel show uneven signal within each band, so it is\nimpossible to determine whether the siRNA treatments differ from the controls. Again, quantification\nof the signal from the western blots would help to interpret the figure.\nResponse: We thank the reviewer for this helpful comment. We have repeated the western blot\nexperiment in Figure 5G under optimized conditions to ensure clearer and more uniform signal\npresentation across all bands. The updated panel now more clearly reflects the differences between\ncontrol and siRNA-treated samples (Fig. 7a). As the experiment has been fully repeated with\nimproved clarity, we have not added additional quantification.\nQ13: In Figure 5K, it is difficult to see obvious differences in polyubiquitination for K48 and K63.\nAgain, the samples seem to be unevenly transferred, leading to uneven signals. (it would also have\nbeen helpful if the authors had submitted their other replicates of western blots)\nResponse: We thank the reviewer for this valuable comment. We have repeated the experiment\n13\nshown in Figure 5k and replaced the panel with a higher-quality blot to ensure even signal and\nclearer visualization of the ubiquitination patterns (Fig. 7i). In addition, an independent replicate of\nthis experiment has been provided in the supplementary figure (Fig. S2a) as requested. The results\nconsistently demonstrate that AACRNLβ promotes TRAF6 K27, K33, K48, and K63\npolyubiquitination.\n14"}, {"round": 2, "reviewer_comments": {"reviewer_1": "My requested revisions have been adequately addressed."}, "author_response": ""}]

Decision letter