Divergent C. elegans toxin alleles are suppressed by distinct mechanisms
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eLife Assessment
This study provides valuable insights into a new toxin-antidote element in C. elegans, the first naturally occurring unlinked toxin-antidote system where endogenous small RNA pathways post-transcriptionally suppress the toxin. The strength of evidence is solid, using a combination of genomic and experimental methods. Enthusiasm, however, is tempered by its reliance on meta-analysis of existing data sets and limited experimental evaluation.
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Abstract
Toxin-antidote elements (TAs) are selfish DNA sequences that bias their transmission to the next generation. TAs typically consist of two linked genes: a toxin and an antidote. The toxin kills progeny that do not inherit the TA, while the antidote counteracts the toxin in progeny that inherit the TA. We previously discovered two TAs in C. elegans that follow the canonical TA model of two linked genes: peel-1/zeel-1 and sup-35/pha-1 . Here, we report a new TA that exists in three distinct states across the C. elegans population. The canonical TA, which is found in isolates from the Hawaiian islands, consists of two genes that encode a maternally deposited toxin (MLL-1) and a zygotically expressed antidote (SMLL-1). The toxin induces larval lethality in embryos that do not inherit the antidote gene. A second version of the TA has lost the toxin gene but retains a partially functional antidote. Most C. elegans isolates, including the standard laboratory strain N2, carry a highly divergent allele of the toxin that has retained its activity, but have lost the antidote through pseudogenization. We show that the N2 toxin allele has acquired mutations that enable piRNA binding to initiate MUT-16-dependent 22G small RNA amplification that targets the transcript for degradation. The N2 haplotype represents the first naturally occurring unlinked toxin-antidote system where the toxin is post-transcriptionally suppressed by endogenous small RNA pathways.
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eLife Assessment
This study provides valuable insights into a new toxin-antidote element in C. elegans, the first naturally occurring unlinked toxin-antidote system where endogenous small RNA pathways post-transcriptionally suppress the toxin. The strength of evidence is solid, using a combination of genomic and experimental methods. Enthusiasm, however, is tempered by its reliance on meta-analysis of existing data sets and limited experimental evaluation.
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Reviewer #1 (Public review):
Summary:
The article by Zdraljevic et al. reports the discovery of a third toxin-antidote (TA) element in C. elegans, composed of the genes mll-1 (toxin) and smll-1 (antidote). Unlike previously characterized TA systems in C. elegans, this element induces larval arrest rather than embryonic lethality. The study identifies three distinct haplotypes at the TA locus, including a hyper-divergent version in the standard laboratory strain N2, which retains a functional toxin but lacks a functional antidote. The authors propose that small RNA-mediated silencing mechanisms, dependent on MUT-16 and PRG-1, suppress the toxicity of the divergent toxin allele. This work provides insights into the evolutionary dynamics of TA elements and their regulation through RNA interference (RNAi).
Overall, there are many things to …
Reviewer #1 (Public review):
Summary:
The article by Zdraljevic et al. reports the discovery of a third toxin-antidote (TA) element in C. elegans, composed of the genes mll-1 (toxin) and smll-1 (antidote). Unlike previously characterized TA systems in C. elegans, this element induces larval arrest rather than embryonic lethality. The study identifies three distinct haplotypes at the TA locus, including a hyper-divergent version in the standard laboratory strain N2, which retains a functional toxin but lacks a functional antidote. The authors propose that small RNA-mediated silencing mechanisms, dependent on MUT-16 and PRG-1, suppress the toxicity of the divergent toxin allele. This work provides insights into the evolutionary dynamics of TA elements and their regulation through RNA interference (RNAi).
Overall, there are many things to like about this paper and only a few small quibbles, which will not require more than a little rewriting or relatively minor analyses.
Strengths:
(1) The discovery of a maternally deposited TA element with delayed toxicity due to delayed mRNA translation of the maternally deposited toxin mRNA is a significant addition to the literature on selfish genetic elements in metazoans.
(2) Identifying three haplotypes at the TA locus provides a snapshot of potential evolutionary trajectories for these elements, which are often inferred but rarely demonstrated in naturally occurring strains. The genomic analysis of 550 wild isolates contextualizes the findings within natural populations, revealing geographic clustering and evolutionary pressures acting on the TA locus.
(3) The study employs various techniques, including CRISPR/Cas9 knockouts, FISH, long-read RNA sequencing, and population genomics. The use of inducible systems to confirm toxicity and antidote functionality is particularly robust. This multifaceted approach strengthens the validity of the findings.
(4) The authors provide compelling evidence that small RNA pathways suppress toxin activity in strains lacking a functional antidote. This highlights an alternative mechanism for neutralizing selfish genetic elements.
Weaknesses:
(1) The introduction focuses strongly (for good reason) on bacterial TA systems and then jumps to TA systems in C. elegans. It's unclear why TA systems in other eukaryotes are not discussed.
(2) Similarly, there is a missed opportunity to discuss an analogy between the suppressor mechanism discovered here and the hairpin RNA suppressors of meiotic drive identified by Eric Lai and colleagues. Discussing these will provide a fuller context of the present study's findings and will not affect their novelty.
(3) While the evidence for RNAi-mediated suppression is strong, the claim that positive selection drove diversification at piRNA binding sites requires further discussion and clarification. The elevated dN and dS are unusual (how unusual relative to other genes in vicinity? What is hyper-divergent statistically speaking?), but there is no a priori reason that there would be selection on piRNA binding sites within the mll-1 transcript to facilitate its recognition by endogenous RNAi machinery; what is the selective pressure for mll-1 to do so? Most TA systems would like to avoid being suppressed by the host. One cannot make the argument that this was motivated by the loss of the antidote because the loss of the antidote would be instantly suicidal, so the cadence of events described requiring hypermutation of the mll-1 transcript does not work.
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Reviewer #2 (Public review):
Summary:
In the manuscript by Walter-McNeill, Kruglyak, and team, the authors provide solid evidence of another toxin-antidote (TA) system in C. elegans. Generally, TA systems involve selfish and linked genetic elements, one encoding a toxin that kills progeny inheriting it, unless an antidote (the second element) is also present. Currently, only two TA systems have been characterized in this species, pointing to the importance of identifying new instances of such systems to understand their transmission dynamics, prevalence, and functions in shaping worm populations.
Strengths:
This novel TA system (mll-1/smll-1) was identified on LGV in wild C. elegans isolates from the Hawaiian islands, by crossing divergent strains and observing allele frequency distortions by high-throughput genome sequencing after 10 …
Reviewer #2 (Public review):
Summary:
In the manuscript by Walter-McNeill, Kruglyak, and team, the authors provide solid evidence of another toxin-antidote (TA) system in C. elegans. Generally, TA systems involve selfish and linked genetic elements, one encoding a toxin that kills progeny inheriting it, unless an antidote (the second element) is also present. Currently, only two TA systems have been characterized in this species, pointing to the importance of identifying new instances of such systems to understand their transmission dynamics, prevalence, and functions in shaping worm populations.
Strengths:
This novel TA system (mll-1/smll-1) was identified on LGV in wild C. elegans isolates from the Hawaiian islands, by crossing divergent strains and observing allele frequency distortions by high-throughput genome sequencing after 10 generations. These allele frequency distortions were subsequently confirmed in another set of crosses with a separate divergent strain, and crosses of heterozygous males or hermaphrodites resulted in a pattern of L1 lethality in progeny (with a rod arrest phenotype) that suggested the maternal transmission of this TA system from the XZ1516 genetic background. By elegantly combining the use of near-isogenic lines, CRISPR editing to generate knock-outs, and a transgene rescue of the antidote gene, the authors identified the genes encoding the toxin and the antidote, which they refer to as mll-1 and smll-1. Moreover, the specific mll-1 isoform responsible for the production of the toxin was identified and mll-1 transcripts were observed by FISH in early and late embryos, as well as in larvae. Inducible expression of the toxin in various strains resulted in larval arrest and rod phenotypes. The authors then characterized the genetic variation of 550 wild isolates at the toxin/antidote region on LGV and distinguished three clades: (1) one with the conserved TA system, (2) one having lost the toxin and retaining a mostly functional antidote, and (3) one having lost the antidote and retaining a divergent yet coding toxin (this includes the reference strain Bristol N2, in which the homologous toxin gene has acquired mutations and is known as B0250.8). Further, the authors show that this region is under positive selection. These data are compelling and provide very strong evidence of a new TA system in this species.
Weaknesses:
The question remained as to how one clade, including N2, could retain the toxin gene but not possess a functional antidote. In the second part of the manuscript, the authors hypothesized that small RNA targeting (RNAi) of the toxin transcript could provide the necessary repression to allow worms to survive without the antidote. Through a meta-analysis of multiple small RNA datasets from the literature, the authors found evidence to support this idea, in which the toxin transcript is targeted by 22G siRNAs whose biogenesis is dependent on the Mutator foci protein, MUT-16. They note that from previous studies, mut-16 null mutants displayed a varied penetrance of larval arrest. In their own hands, mut-16 mutants displayed 15% varied larval arrest and 2% rod phenotypes. In an attempt to link B0250.8 to mut-16/siRNAs, they made a double mutant and examined body length as a proxy for developmental stage. Here, they observed a partial rescue of the mut-16 size defect by B0250.8 mutation. Finally, the authors also highlight data from further meta-analysis, which predicts the recognition of B0250.8 by several piRNAs. Also based on existing data from the literature, the authors link loss of Piwi (PRG-1), which binds piRNAs, to a depletion of 22G-RNAs targeting B0250.8 and an upregulation of B0250.8 expression in gonads, suggesting that piRNAs are the primary small RNAs that target B0250.8 for downregulation. The data in this portion of the manuscript are intriguing, but somewhat preliminary and incomplete, as they are based on little primary experimentation and a collection of different datasets (which have been acquired by slightly different methods in most cases). This portion of the study would require subsequent experimentation to firmly establish this mechanistic link. For example, to be able to claim that "the N2 toxin allele has acquired mutations that enable piRNA binding to initiate MUT-16-dependent 22G small RNA amplification that targets the transcript for degradation" the identified piRNA sites should be mutated and protein and transcript levels analysed in wild-type and in the strain with mutated piRNA sites. At a minimum, the protein levels in wild-type and mut-16, prg-1, and/or wago-1 mutants should be measured by western blot and/or by live imaging (introducing a GFP or some other tag to the endogenous protein via CRISPR editing) to show that the toxin is not accumulated as a protein in wt, but increases in levels in these mutants. mRNA levels in Figure S5A suggest there is still some expression of the B0250.8 transcript in a wild-type situation.
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