Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function

  1. Adedeji M Aderounmu
  2. P Joseph Aruscavage
  3. Bryan Kolaczkowski
  4. Brenda L Bass

  • Curated by eLife

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    eLife assessment

    This is a valuable paper describing an attempt to reconstruct the evolution of Dicer. Using ancestral reconstruction approaches, the authors carefully examine the biochemical characteristics of reconstructed proteins at various junction points in the animal lineage. They provide solid evidence that the deepest ancestrally reconstructed protein has double-stranded RNA stimulated ATPase activity and that this characteristic was lost along the vertebrate lineage. This paper will be of interest to scientists in the RNA-protein interaction and protein evolution fields.

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Abstract

Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicer’s helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained some ATPase activity; importantly, ATPase activity was undetectable in the vertebrate Dicer-1 ancestor, which had even lower dsRNA affinity. Reverting residues in the ATP hydrolysis pocket was insufficient to rescue hydrolysis, but additional substitutions distant from the pocket rescued vertebrate Dicer-1’s ATPase function. Our work suggests Dicer lost ATPase function in the vertebrate ancestor due to loss of ATP affinity, involving motifs distant from the active site, important for coupling dsRNA binding to the active conformation. By competing with Dicer for viral dsRNA, RIG-I-like receptors important for interferon signaling may have allowed or actively caused loss of ATPase function.

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  1. Version published to 10.7554/elife.85120 on eLife
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    Author Response

    Reviewer #1 (Public Review):

    This paper presents a thorough biochemical characterization of inferred ancestral versions of the Dicer helicase function. Probably the most significant finding is that the deepest ancestral protein reconstructed (AncD1D2) has significant double-stranded RNA-stimulated ATPase activity that was lost later, along the vertebrate lineage. These results strongly suggest that the previously known differences in ATPase activity between extant vertebrates and, for example, extant arthropods is due to loss of the ATPase activity over evolutionary time as opposed to gains in specific lineages. Based on their analysis, the authors also "restore" ATPase function in the vertebrate dicer, but they did so by making many (over 40) mutations in the vertebrate protein, and it is not clear which of these many mutations is required for the restoration of the activity. Thus, it is difficult to discern how the results of this experiment relate to the evolutionary history.

    We completely agree with this reviewer's assessment of our paper. Our Michaelis-Menten analyses raised the intriguing idea that loss of ATPase activity in the helicase domain of the vertebrate ancestor may indicate loss of the ability to couple dsRNA binding to formation of the active conformation. Our rescue experiments support this idea, albeit in future studies we hope to create an active ancestor with fewer amino acid changes. While the rescue experiments validate what these analyses told us, as the reviewer suggests, they do not themselves inform on the evolutionary history.

    A criticism of the paper is the authors' tendency (probably unconscious) to ascribe a purposefulness to evolution. For example, in the introduction, "We speculate that the unique role of the RLR's in the interferon signaling pathway in vertebrates...created an incentive to jettison an active helicase in vertebrates." Although this sentence is clearly labelled as speculation and "incentive" is clearly a metaphor, the implication is that evolution somehow has forethought. (There are other instances of this notion in the paper, for example, in the last line of the abstract). The author's statement also implies that the developing interferon system somehow caused the loss of active helicase, but it seems equally plausible that the helicase function was lost before the interferon system co-opted it.

    We agree with the stated critiques and have rephrased language that suggests that evolution is an active force. In addition to changing the last line of the abstract (page 2, line 35), and removing the quoted sentence from the Introduction, we have included a more nuanced discussion of the order of evolutionary events that may have preceded or followed the loss of helicase function in Dicer (page 18, lines 418-430)

    Reviewer #2 (Public Review):

    The manuscript by Aderounmu presents an interesting attempt to reconstruct evolution of the function of the helicase domain in ancestral Dicers, RNase III enzymes producing siRNAs from long double-stranded RNA and microRNAs from small hairpin precursors. The helicase has a role in long dsRNA recognition and processing and this function could have an antiviral role. Authors show on reconstructed ancestral Dicer variants that the helicase was losing dsRNA binding affinity and ATPase activity during evolution of the lineage leading to vertebrates while an early divergent Dicer-2 variant in Arthropods retained high activity and seemed better adapted for blunt ended long dsRNA, which would be consistent with antiviral function.

    The work is consistent with apparent adaptation of vertebrate Dicers for miRNA biogenesis and two known modes of substrate loading: "bottom up" dsRNA threading through the helicase domain where the helicase domain recognizes the end of dsRNA and feeds it into the enzyme and "top-down" where the substrate is first anchored in the PAZ domain before it locks into the enzyme. Some extant Dicer variants are known to be adapted for just one of these two modes while Dicer in C. elegans exemplifies an "ambidextrous" variant. The reconstruction of the helicase domain complex enabled authors to test how well would be ancestral helicases supporting the "bottom up" feeding of long dsRNA and whether the helicase would be distinguishing blunt-end dsRNA and 3' 2 nucleotide overhang. Although the reconstruction of an ancestral protein from highly divergent extant sequences yields just a hypothetical ancestor, which cannot be validated, the work provides remarkable data for interpreting evolutionary history of the helicase domain and RNA silencing in more general. While it is not surprising that the ancestral helicase was a functional ATPase stimulated by dsRNA, particularly new and interesting are data that the decline of the helicase function started already at the level of the common deuterostome ancestor and the helicase was essentially dead in the vertebrate ancestor. It has been reported two decades ago that human Dicer carries a helicase, which has highly conserved critical residues in the ATPase domain but it is non-functional (10.1093/emboj/cdf582). Recently published mouse mutants showed that these highly conserved residues are not important in vivo (10.1016/j.molcel.2022.10.010). Aderounmu et al. now suggest that Dicer carried this dead ATPase with conserved residues for over 500 million years of vertebrate evolution.

    I do not have any major comments to the biochemical analyses and while I think that the ancestral protein reconstruction could yield hypothetical sequences, which did not exist, I think they represent reasonable reconstructions, which yielded data worth of interpretations. My major criticism of the work concerns clarity for the readership and interpretations of some results where I wish authors would clarify/revise the text. The following three examples are particularly significant:

    1. It should be explained to which common ancestor during metazoan evolution belongs the ancestral helicase AncD1D2 or at least what that sequence might represent in terms of common ancestry during metazoan evolution.

    We thank the reviewer for bringing this issue to our attention, and we have now included a brief discussion of the complexity in identifying AncD1D2’s exact position in metazoan evolution (page 6, lines 124-134). Our maximum likelihood phylogeny is constructed from Dicer’s helicase and DUF283 subdomains which evidently do not contain enough phylogenetic signal to resolve the finer details of early metazoan evolutionary events surrounding the divergence of non-bilaterians: Porifera, Ctenophora, Cnidaria and Placozoa. In our tree, Cnidaria even diverges later than the Nematode bilaterian branch reflecting the fact that our reported phylogeny does not match consensus species relationships, especially in the invertebrate clades. This means we cannot pinpoint AncD1D2’s exact position with certainty. While we do not intend to overinterpret the evolutionary trends from these hypothetical ancestral constructs, we believe the functional differences in biochemical activity are meaningful and correspond to big-picture changes over evolutionary time. AncD1D2 thus corresponds to some early metazoan ancestor that existed before the divergence of bilaterians from non-bilaterians. In support of this interpretation, when the phylogeny is constrained such that the bilaterian branches match the consensus species tree (Figure 1-figure supplement 2A) we observe that AncD1D2 is ancestral to the bilaterian ancestor, AncD1BILAT (now labeled on the figure), but retains 95% identity to the version of AncD1D2 constructed from the maximum likelihood phylogeny (Figure 1-figure supplement 3B).

    1. This is linked to the first point - authors work with phylogenetic trees reconstructed from a single protein sequence, which are not well aligned with predicted early metazoan divergence (https://doi.org/10.1098/rstb.2015.0036). While their sequence-based trees show early branching of Dicer-2 as if the two Dicers existed in the common ancestor of almost all animals (except of Placozoa), I do not think there is sufficient support for such a statement, especially since antiviral RNAi-dedicated Dicers evolve faster and Dicer-2 is restricted to a few distant taxonomic group, which might be better explained by independent duplications of ambidextrous ancestral Dicers. I would appreciate if authors would discuss this issue in more detail and make readers more aware of the complexity of the problem.

    We agree with the reviewer that in our initial submission we did not properly address the incongruence between our maximum likelihood phylogeny and the consensus species tree of life. We have now addressed this by revisions that discuss the difficulty in using a single gene or protein to accurately date ancient evolutionary events, especially in the case of Dicer, a protein whose evolutionary history is littered with multiple duplication events (page 6, lines 124-147, beginning with “Importantly, we observed multiple instances…”; page 16, lines 365-371, sentence beginning with “Uncertainty in the single gene or protein phylogeny…”). Our assumption that an early gene duplication produced the arthropod Dicer-2 clade is consistent with previous Dicer phylogenies that have been constructed with maximum likelihood algorithms with different parameters (https://doi.org/10.1371/journal.pone.0095350, https://doi.org/10.1093/molbev/msx187, https://doi.org/10.1093/molbev/mss263) using full length Dicer sequences with different taxon sampling depths and tree construction parameters. Removing other fast evolving taxa with long branch lengths from the sequence alignment still resulted in arthropod Dicer-2 branching out early in metazoan phylogeny (https://doi.org/10.1093/molbev/mss263).

    In analyses not included in our manuscript, we also independently constructed trees using full-length metazoan Dicers, helicase and DUF-283 subdomains using both RAXML-NG and MrBayes. We tried different taxon sampling depths and tried rooting the tree using either a non-bilaterian outgroup or a fungal outgroup and also tried breaking up potential long-branch attraction with deep taxon sampling. In every iteration, the arthropod Dicer-2 clade diverged early in animal evolution at some point before or during non-bilaterian evolution. We recognize that all these efforts are still prone to long-branch attraction that may cause the rapidly evolving Dicer-2 clade to artificially cluster with distant outgroups, but so far, the only evidence to support an arthropod-specific duplication event is parsimony. This parsimony model is plausible and one might expect a recently duplicated arthropod Dicer-2 to cluster closely with nematode Dicer-1, another antiviral Dicer that would have descended from a common ecdysozoan ancestor but this is not the case. The nematode HEL-DUF clade does get attracted to non-bilaterian Cnidaria clade in our ML tree, but unlike the arthropod Dicer-2 clade, this position varied depending on the parameters of phylogenetic analysis, and so we cannot conclude that arthropod Dicer-2’s position is due to long branch attraction. More sophisticated phylogenetic and statistical tools are needed to answer this question definitively, so we decided to proceed with the highest scoring maximum-likelihood phylogeny generated by our analysis.

    While we have now included a short discussion on the nature of this uncertainty in the revised manuscript (page 6, line 124., page 16, lines 365-371), we have excluded these additional details (paragraph above) from the main text in an attempt to prioritize readability for the generalist reader, and we hope that more specialized readers will find this discussion in the public comments helpful.

    1. Authors should take more into the account existing literature and data when hypothesizing about sequences of events. Some decline of the helicase activity is apparent in AncD1DEUT suggesting that it initiated between AncD1D2 and AncD1DEUT. This implies that a) antiviral role of Dicer was becoming redundant with other cellular protein sensors by then and b) Dicer was already becoming adapted for miRNA biogenesis, which further progressed in the lineage leading to vertebrates to the unique top-down loading with the distinct pre-dicing state where the helicase forms a rigid arm. Authors even cite Qiao et al. (https://doi.org/10.1016/j.dci.2021.103997) who report primitive interferon-like system in molluscs - this places the ancestry of the interferon response upstream of AncD1DEUT and suggests that this ancestral protein-based system was taking over antiviral role of Dicer much earlier. In fact, a bit weaker performance of AncD1LOPH/DEUT combined with the aforementioned interferon-like system and massive miRNA expansion in extant molluscs (10.1126/sciadv.add9938) suggests that molluscs possibly followed a convergent path like mammals. While I am missing this kind of discussion in the manuscript, I think that the model where "interferon appears ..." in AncD1VERT (Fig. 6) is incorrect and misleading.

    This comment is similar to others, including point 3 of Essential revisions, and we have revised our model in Figure 6 accordingly. We agree with the reviewer that we did not sufficiently explore the significance of the decline in Dicer helicase function between AncD1D2 and AncD1DEUT. In addition to the changes noted in point 3 of Essential revisions, we have corrected this by adding or modifying sentences in the Results (page 9, sentence beginning on line 197 “This reduction in ATP hydrolysis efficiency prior to deuterostome divergence may have coincided with…”, and page 11, sentence beginning on line 247 “One possibility is that between AncD1D2 and the deuterostome ancestor…”).

    We did not intend to suggest that this loss of Dicer helicase function was unique to vertebrates, but we focused on the deuterostome-to-vertebrate transition for the following reasons:

    a) The mollusk clade in our analysis is incongruent with its expected species position as a protostome. In our tree it clusters with deuterostomes instead. On one hand, this is probably an artefact of incomplete lineage sorting or long branch attraction. On the other hand, it is possible that this clade’s position is an underlying signal of the convergent evolution proposed by the reviewer. In support of the latter, some extant mollusk Dicer helicases (ACCESSION: XP_014781474, ACCESSION: XP_022331683) show a loss of amino acid conservation in Dicer’s ATPase motifs implying that extant mollusks have also lost Dicer helicase function like vertebrates. However, this is in contrast to vertebrate Dicer helicase where loss of function exists, but ATPase motifs remain conserved. We do not discuss this in the paper because the evidence remains inconclusive until extant mollusk Dicers can be functionally characterized, similar to Human Dicer and Drosophila Dicer-1, to determine that they are truly specialized for miRNA processing to the detriment of helicase function.

    b) Caenorhabditis elegans Dicer is an example of an ambidextrous Dicer, that processes both miRNAs, with the top-down mechanism, and viral dsRNAs, with the bottom-up mechanism. Recently, work has been published that suggests that C. elegans also possesses a protein-based innate immune defense mechanism, but instead of competing with the RNA interference mechanism, both mechanisms seem to work in concert and even share a protein in both pathways: DRH-1, a RIG-I-Like receptor homolog (https://doi.org/10.1128/JVI.01173-19). Furthermore, a protein-based pathway has also been reported in Drosophila and in this scenario Drosophila Dicer-2 is the dsRNA sensor that is common to both pathways (https://doi.org/10.1371/journal.pntd.0002823). This collaboration observed in ecdysozoan invertebrates is different from the competition that has been well established in vertebrates. More data is needed to understand whether a model of competition or collaboration exists in lophotrochozoan invertebrates like mollusks.

  3. eLife

    eLife assessment

    This is a valuable paper describing an attempt to reconstruct the evolution of Dicer. Using ancestral reconstruction approaches, the authors carefully examine the biochemical characteristics of reconstructed proteins at various junction points in the animal lineage. They provide solid evidence that the deepest ancestrally reconstructed protein has double-stranded RNA stimulated ATPase activity and that this characteristic was lost along the vertebrate lineage. This paper will be of interest to scientists in the RNA-protein interaction and protein evolution fields.

  4. eLife

    Reviewer #1 (Public Review):

    This paper presents a thorough biochemical characterization of inferred ancestral versions of the Dicer helicase function. Probably the most significant finding is that the deepest ancestral protein reconstructed (AncD1D2) has significant double-stranded RNA-stimulated ATPase activity that was lost later, along the vertebrate lineage. These results strongly suggest that the previously known differences in ATPase activity between extant vertebrates and, for example, extant arthropods is due to loss of the ATPase activity over evolutionary time as opposed to gains in specific lineages. Based on their analysis, the authors also "restore" ATPase function in the vertebrate dicer, but they did so by making many (over 40) mutations in the vertebrate protein, and it is not clear which of these many mutations is required for the restoration of the activity. Thus, it is difficult to discern how the results of this experiment relate to the evolutionary history.

    A criticism of the paper is the authors' tendency (probably unconscious) to ascribe a purposefulness to evolution. For example, in the introduction, "We speculate that the unique role of the RLR's in the interferon signaling pathway in vertebrates...created an incentive to jettison an active helicase in vertebrates." Although this sentence is clearly labelled as speculation and "incentive" is clearly a metaphor, the implication is that evolution somehow has forethought. (There are other instances of this notion in the paper, for example, in the last line of the abstract). The author's statement also implies that the developing interferon system somehow caused the loss of active helicase, but it seems equally plausible that the helicase function was lost before the interferon system co-opted it.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript by Aderounmu presents an interesting attempt to reconstruct evolution of the function of the helicase domain in ancestral Dicers, RNase III enzymes producing siRNAs from long double-stranded RNA and microRNAs from small hairpin precursors. The helicase has a role in long dsRNA recognition and processing and this function could have an antiviral role. Authors show on reconstructed ancestral Dicer variants that the helicase was losing dsRNA binding affinity and ATPase activity during evolution of the lineage leading to vertebrates while an early divergent Dicer-2 variant in Arthropods retained high activity and seemed better adapted for blunt ended long dsRNA, which would be consistent with antiviral function.

    The work is consistent with apparent adaptation of vertebrate Dicers for miRNA biogenesis and two known modes of substrate loading: "bottom up" dsRNA threading through the helicase domain where the helicase domain recognizes the end of dsRNA and feeds it into the enzyme and "top-down" where the substrate is first anchored in the PAZ domain before it locks into the enzyme. Some extant Dicer variants are known to be adapted for just one of these two modes while Dicer in C. elegans exemplifies an "ambidextrous" variant. The reconstruction of the helicase domain complex enabled authors to test how well would be ancestral helicases supporting the "bottom up" feeding of long dsRNA and whether the helicase would be distinguishing blunt-end dsRNA and 3' 2 nucleotide overhang. Although the reconstruction of an ancestral protein from highly divergent extant sequences yields just a hypothetical ancestor, which cannot be validated, the work provides remarkable data for interpreting evolutionary history of the helicase domain and RNA silencing in more general. While it is not surprising that the ancestral helicase was a functional ATPase stimulated by dsRNA, particularly new and interesting are data that the decline of the helicase function started already at the level of the common deuterostome ancestor and the helicase was essentially dead in the vertebrate ancestor. It has been reported two decades ago that human Dicer carries a helicase, which has highly conserved critical residues in the ATPase domain but it is non-functional (10.1093/emboj/cdf582). Recently published mouse mutants showed that these highly conserved residues are not important in vivo (10.1016/j.molcel.2022.10.010). Aderounmu et al. now suggest that Dicer carried this dead ATPase with conserved residues for over 500 million years of vertebrate evolution.

    I do not have any major comments to the biochemical analyses and while I think that the ancestral protein reconstruction could yield hypothetical sequences, which did not exist, I think they represent reasonable reconstructions, which yielded data worth of interpretations. My major criticism of the work concerns clarity for the readership and interpretations of some results where I wish authors would clarify/revise the text. The following three examples are particularly significant:

    1. It should be explained to which common ancestor during metazoan evolution belongs the ancestral helicase AncD1D2 or at least what that sequence might represent in terms of common ancestry during metazoan evolution.

    2. This is linked to the first point - authors work with phylogenetic trees reconstructed from a single protein sequence, which are not well aligned with predicted early metazoan divergence (https://doi.org/10.1098/rstb.2015.0036). While their sequence-based trees show early branching of Dicer-2 as if the two Dicers existed in the common ancestor of almost all animals (except of Placozoa), I do not think there is sufficient support for such a statement, especially since antiviral RNAi-dedicated Dicers evolve faster and Dicer-2 is restricted to a few distant taxonomic group, which might be better explained by independent duplications of ambidextrous ancestral Dicers. I would appreciate if authors would discuss this issue in more detail and make readers more aware of the complexity of the problem.

    3. Authors should take more into the account existing literature and data when hypothesizing about sequences of events. Some decline of the helicase activity is apparent in AncD1DEUT suggesting that it initiated between AncD1D2 and AncD1DEUT. This implies that a) antiviral role of Dicer was becoming redundant with other cellular protein sensors by then and b) Dicer was already becoming adapted for miRNA biogenesis, which further progressed in the lineage leading to vertebrates to the unique top-down loading with the distinct pre-dicing state where the helicase forms a rigid arm. Authors even cite Qiao et al. (https://doi.org/10.1016/j.dci.2021.103997) who report primitive interferon-like system in molluscs - this places the ancestry of the interferon response upstream of AncD1DEUT and suggests that this ancestral protein-based system was taking over antiviral role of Dicer much earlier. In fact, a bit weaker performance of AncD1LOPH/DEUT combined with the aforementioned interferon-like system and massive miRNA expansion in extant molluscs (10.1126/sciadv.add9938) suggests that molluscs possibly followed a convergent path like mammals. While I am missing this kind of discussion in the manuscript, I think that the model where "interferon appears ..." in AncD1VERT (Fig. 6) is incorrect and misleading.

  6. Version published to 10.1101/2022.12.30.522297v1 on bioRxiv