Proteins are the chemical ‘workhorses’ of the cell: some help maintain a cell’s shape or structure, while others carry out the chemical reactions necessary for life. Organisms therefore need to keep tight control over the production of proteins in their cells, so that the right amount of each protein is made at the right time, in the right place.

Instructions for making new proteins are encoded in a type of molecule called messenger RNA. Each messenger RNA contains the instructions for one protein, which are then ‘read’ and carried out by special cellular machinery called ribosomes. The cell can control how much protein it produces by regulating both the levels of different messenger RNA and the amount of protein ribosomes are allowed to make from those instructions.

The main way to regulate the levels of messenger RNA is through their transcription from the genome. However, this needs fine tuning. Cells can do this in a highly specific way using molecules called microRNAs. A microRNA works by directing a protein called Argonaute to the messenger RNA that it targets. Once Argonaute arrives, it can call in additional ‘helper proteins’ to shut down, or reduce, protein production from that messenger RNA, or alternatively to break down the messenger RNA altogether.

Cells can use an enzyme called CK1α to attach bulky chemical groups onto a specific part of the Argonaute protein, in a reaction termed phosphorylation. The ability to carry out this reaction (and to reverse it) also seems to be important for microRNAs to do their job properly, but why has remained unknown. Bibel et al. wanted to determine what triggers CK1α to phosphorylate Argonaute, and how this affects interactions between microRNAs, Argonaute and their target messenger RNAs.

A series of ‘test tube’ experiments looked at the interaction between purified CK1α and Argonaute under different conditions. These demonstrated that CK1α could only carry out its phosphorylation reaction when Argonaute was already interacting with a microRNA and its corresponding messenger RNA. Further measurements revealed that phosphorylation of Argonaute made it detach from the messenger RNA more quickly. This suggests that phosphorylation might be a way to let Argonaute seek out new messenger RNAs after blocking protein production at its first ‘target’.

These results shed new light on a fundamental mechanism that cells use to control protein production. Bibel et al. propose that this mechanism may be shared across many different species and could one day help guide the development of new medical therapies based on microRNAs.

Most metazoan genes are regulated post-transcriptionally by RNA interference (RNAi) (Friedman et al., 2009). RNAi is a conserved process whereby an Argonaute (Ago) protein binds to a small RNA, such as a microRNA (miRNA) or a small interfering RNA (siRNA), ~22 nt in length, and uses it as a guide to seek out and bind target mRNAs containing regions of partial complementarity (Friedman et al., 2009; Bartel, 2018). Binding of the core RNA-induced silencing complex (RISC), consisting of Ago and a guide (Rivas et al., 2005), to an mRNA can lead to recruitment of cofactors and decreased translation of protein from that mRNA through a number of mechanisms including: preventing translation initiation and elongation; promoting mRNA decay; triggering premature termination and co-translational protein degradation; and sequestering targets from the translational machinery (Jonas and Izaurralde, 2015; Eichhorn et al., 2014). Ago has a high affinity for miRNAs with a very slow off-rate (De et al., 2013; Kingston and Bartel, 2019). Consequently, most Ago/guide complexes are long-lived, with average half-lives on the order of days (Gantier et al., 2011) and some complexes stable for at least 3 weeks (Olejniczak et al., 2013). Thus, once loaded with a guide, Ago can repress countless matching mRNA sites, as long as it is able to efficiently release these targets once silencing is achieved. This turnover is crucial because potential target sites are present in large excess compared to the corresponding miRNAs (Denzler et al., 2014; Denzler et al., 2016). miRNA-binding sites are typically located within the target mRNA’s 3’ UTR and are primarily determined by perfect complementarity in a 6–8 nt ‘seed sequence’ corresponding to nucleotides 2–8 of the guide miRNA (g2–8) (Bartel, 2009), augmented by additional pairing beyond this region. In the core RISC, the subseed region of the miRNA (g2–5) is preorganized for base pairing (Elkayam et al., 2012; Schirle and MacRae, 2012; Klum et al., 2018), which allows Ago to rapidly sample potential target sites (Klum et al., 2018). Binding to a fully complementary seed induces conformational changes in Ago that expose more of the miRNA and widen Ago’s RNA-binding channel, allowing for further pairing (Schirle et al., 2014). Target sites vary in their amount of complementarity to a guide outside of the seed sequence, and additional pairing can influence a site’s repression efficacy (Grimson et al., 2007). This pairing often occurs in the more solvent-exposed 3’ supplementary region (corresponding to g12–17) through a second nucleation event (Bartel, 2018; Sheu-Gruttadauria et al., 2019b). 3’ supplementary pairing, most effectively with 3–4 contiguous base pairs centered around g13–16 (Grimson et al., 2007), can strengthen repression by increasing guide/target affinity through a decrease in K_off (Xiao and MacRae, 2020; Wee et al., 2012; Salomon et al., 2015). It also allows miRNAs of the same family (miRNAs that share the same seed sequence) to preferentially repress different sets of targets (Moore et al., 2015; Broughton et al., 2016). More common for siRNAs, full guide-target complementarity is rare among metazoan miRNA targets, but when present it results in ‘slicing’ by some Ago proteins, a process in which the target mRNA is cleaved between nucleotides across from g10 to g11 (Song et al., 2004; Liu et al., 2004; Meister et al., 2004).

Ago proteins are part of a larger Ago superfamily, which includes Piwi- and Wago-clade proteins. Members of the superfamily show high structural conservation throughout evolution and have four main structural domains: N, PAZ, MID, and PIWI (Song et al., 2004). The MID and PIWI domains form an almost continuous lobe which, together with the N domain, forms a crescent-shaped base above which the PAZ domain is able to move more freely (Song et al., 2004; Ming et al., 2007). The 5’ end of the small RNA is bound by a pocket created by the MID and PIWI domains (Frank et al., 2010) and the 3’ end is held by the PAZ domain (Song et al., 2003; Ma et al., 2004), with the length of the small RNA cradled in a groove traversing the crescent (Elkayam et al., 2012; Schirle and MacRae, 2012; Wang et al., 2008). The PAZ domain is the most flexible and, sometimes in concert with the N domain, undergoes rigid body movements along linker regions (L1 and L2) in order to accommodate RNA (Schirle et al., 2014; Sheu-Gruttadauria et al., 2019b; Ming et al., 2007; Faehnle et al., 2013). Humans have four Ago orthologs (hAgo1–4) in addition to four Piwi-clade Agos, with hAgo2 being the most highly expressed in most tissues (Valdmanis et al., 2012; Hauptmann et al., 2015). Although particular functions have been attributed to specific hAgo proteins, hAgo1–4 are largely considered functionally redundant (Su et al., 2009). The lone exception is hAgo2; knock-out of hAgo2 is lethal in early mouse development (Liu et al., 2004; Morita et al., 2007), whereas knock-out of the other hAgos, and even knock-out of all three, is not (Hajarnis et al., 2018). This essentiality is attributed largely to the fact that, under most circumstances, only hAgo2 has slicer activity (Song et al., 2004; Liu et al., 2004; Meister et al., 2004). Although slicing is not required for miRNA-mediated regulation, it is necessary for the maturation of rare but important Dicer-independent miRNAs including miR-451 (Cheloufi et al., 2010) and miR-486 (Jee et al., 2018), which play essential roles in erythrocyte development.

Although the core architecture is conserved among all Ago proteins, they are subject to multiple types of post-translational modification, including ubiquitylation, PARylation, and phosphorylation (Jee and Lai, 2014; Gebert and MacRae, 2019 ). For example, S837 undergoes stress-induced p38/MAPK-mediated S837 phosphorylation, which, among other effects, is thought to contribute to hAgo2 localization in processing bodies (P-bodies) (Zeng et al., 2008). There is a span of residues on the surface of the PIWI domain in eukaryotic miRNA-specialized Ago proteins that is absent in prokaryotic Ago proteins (Figure 1a). This region, corresponding to residues ~820–837 in hAgo2 numbering, is termed the eukaryotic insertion (EI), and it contains four to five highly conserved potential phosphorylation sites: four serine residues (in hAgo2 numbering, S824, S828, S831, and S834) as well as a threonine (T830) which is only conserved in Ago2 homologs. This cluster was recently found to be heterogeneously phosphorylated in multiple Ago homologs in a variety of cell types and organisms (Golden et al., 2017; Quévillon Huberdeau et al., 2017) and its phosphorylation and dephosphorylation were attributed to the kinase CK1α and the phosphatase PP6/ANKRD52, respectively (Golden et al., 2017).

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This cycle of phosphorylation of Ago’s EI was implicated in regulatory control of Ago itself (Golden et al., 2017; Quévillon Huberdeau et al., 2017). Golden et al. found that decreased EI phosphorylation led to an increase in target RNA association with the Ago-guide complex in cell culture, as measured by qt-RT-PCR of immunoprecipitated hAgo2, whereas increased EI phosphorylation or its mimicking had the opposite effect (Golden et al., 2017). Additionally, disruption of the hAgo EI phosphorylation cycle led to dysregulation of mRNA levels, as determined by RNA-seq. In the absence of EI phosphorylation, hAgo2 associated with a more diverse pool of mRNA targets, but with decreased coverage of individual targets as determined by eCLIP (Golden et al., 2017). Furthermore, reporter assays showed that increased phosphorylation or its mimicking led to reduced repression of reporter targets in cell culture (Cheloufi et al., 2010 and in Caenorhabditis elegans Jee et al., 2018). These silencing defects could be rescued by tethering Ago to the target, indicating that phosphorylation impaired a step upstream of repression, preventing productive Ago-guide/target interactions (Golden et al., 2017; Quévillon Huberdeau et al., 2017). Together with their findings that miRNA production and guide association appeared unaffected, these results point to a role of EI phosphorylation in mediating Ago-guide/target interactions, but key questions remained about the molecular events associated with this process.

Using a combination of biochemical and biophysical assays, we determined that binding of miRNA-bound hAgo2 to targets containing 3’ supplementary pairing triggers hierarchical phosphorylation of the EI by CK1α. The added negative charge electrostatically repels the negatively charged target RNA, leading to target release. Such regulation could help explain how cells maintain tight and consistent miRNA-mediated regulation despite there being a large excess of miRNA target sites compared to Ago and miRNA molecules (Denzler et al., 2014; Denzler et al., 2016).

miRNA-mediated gene repression is a widespread mode of regulation in metazoans. Most human genes contain at least one evolutionarily conserved miRNA target site, and many genes are under regulation by multiple families of miRNAs. Each miRNA family, in turn, typically has hundreds of targets; the 90 most broadly conserved miRNA families have over 400 conserved targets each (Friedman et al., 2009). At the same time, the number of miRNA target sites is much greater than the number of miRNA-Ago complexes (Denzler et al., 2014) and the half-lives of mRNAs are, on average, significantly shorter than those of RISC complexes (Kingston and Bartel, 2019). Therefore, it appears that each core RISC targets multiple mRNAs throughout its lifetime. On the other hand, repression efficacy is largely related to binding stability, with stronger binding correlating with enhanced repression (McGeary et al., 2019). Thus, ensuring robust mRNA repression at the global level is a balancing act between enabling strong binding at individual sites and binding to a large number of sites. 3’ supplementary pairing increases binding affinity and thus can lead to enhanced repression at cognate sites (Becker et al., 2019), but high affinity may come with the risk of effectively sequestering RISC. CK1α-mediated EI phosphorylation provides a mechanism to counterbalance this added affinity, freeing RISC to repress additional targets. Indeed, Golden et al. found that hAgo2 which could not be phosphorylated in the EI associated with a more diverse pool of mRNA targets, but with decreased coverage of individual targets, accompanied by global dysregulation of miRNA-mediated repression (Golden et al., 2017).

This may help explain discrepancies between the in vitro and in vivo effects of 3’ supplementary pairing. In vitro, 3’ supplementary pairing can significantly enhance the stability of Ago/target interactions, with 3–4 contiguous canonical base pairs in the supplementary region increasing affinity over 20-fold for some miRNA/target pairs (Sheu-Gruttadauria et al., 2019b; Xiao and MacRae, 2020; Salomon et al., 2015). This effect is more pronounced for miRNAs with weak, low GC-content, seeds (Xiao and MacRae, 2020; Salomon et al., 2015). However, several reports concluded that the contributions of supplementary pairing in vivo and in cell culture are less substantial (Grimson et al., 2007; Wee et al., 2012; Salomon et al., 2015). Some of this discrepancy has been attributed to differences in cell types and measurement conditions, as well as the large effects of a small number of miRNA/targets being diluted out in large datasets (Xiao and MacRae, 2020). Additionally, the precise position and composition of optimal pairing in the 3’ region has been found to differ for different miRNAs (McGeary et al., 2022), complicating classifications and predictions.

However, as we have shown in this study, supplementary pairing triggers phosphorylation of the EI, which could offset added binding affinity. Therefore, in vivo, the overall effects of supplementary pairing on target repression would appear to be less pronounced. This would be consistent with supplementary pairing functioning primarily to distinguish between target sites for the same miRNAs. Phosphorylation of targets with supplementary pairing after repression is established, freeing RISC to repress additional targets, would allow for the most efficient use of supplementary pairing for such discriminatory purposes.

CK1α phosphorylation likely occurs in a distributive manner (Gebel et al., 2020). Because phosphorylation of multiple residues would require CK1α to actively interact with Ago multiple times, supplementary pairing might be expected to increase phosphorylation by way of added affinity. However, we found that target retention time is not the primary driver of CK1α-mediated EI phosphorylation, as demonstrated by the minimal levels of phosphorylation observed upon binding to the fully complementary or TDMD-like target. Our findings instead suggest that the conformation of the RISC complex is of primary importance with regard to triggering phosphorylation. Compared to 3’ supplementary targets, fully complementary targets require different conformational changes due to base pairing of the central region (Sheu-Gruttadauria et al., 2019b), and these changes might make Ago’s EI a poorer substrate for CK1α.

Recently, a study linked a variety of germline single amino acid mutations in hAgo2 to RNAi deficits and neurological symptoms (Lessel et al., 2020). These mutations clustered in helix 7, L1, and a loop in the PIWI domain, and, when expressed in cell culture, some of the mutated Agos had decreased EI phosphorylation and a slight increase in quantity of select target mRNAs. This, combined with molecular dynamics simulations, led the authors to propose that reduced phosphorylation prevents efficient target release. Based on our findings that target binding promotes phosphorylation, prolonged target binding in the usual conformation would be expected to increase rather than decrease phosphorylation. Therefore, it appears possible that the identified mutations may cause hAgo2 to adopt a suboptimal target-bound conformation which disfavors, or at least fails to promote, phosphorylation.

There are multiple examples in the literature where substrate tertiary structure has been shown to be critical for promoting CK1-mediated phosphorylation. For example, T-antigen is phosphorylated by CK1 at non-canonical sites at its N-terminus in a manner that is dependent on proper folding and an intact C-terminus (Cegielska et al., 1994). Similarly, conformational changes induced by target binding may cause Ago to adopt a conformation which is structurally preferable for CK1α. However, this preference cannot solely be attributed to the changes making the EI more accessible. Despite the high degree of flexibility and solvent accessibility of the EI and the surrounding area in the RNA-free hAgo2, as seen from our HDX-MS results (Figure 3 and Figure 4—figure supplement 1) and through limited proteolysis (Elkayam et al., 2012), we were unable to phosphorylate it in vitro (Figure 1). This suggests there must be an additional factor required. Since the EI shows no significant changes in deuterium exchange neither upon guide- nor upon target-binding, the additional information needed to enable phosphorylation stems from outside of the EI. Such information could come from a different part of the hAgo2 protein that is now in a more suitable conformation to interact with the kinase. Alternatively and/or additionally, factors outside of hAgo2 may assume this role, potentially the target RNA itself.

As we show in this study, target binding triggers the CK1α-mediated phosphorylation of S828 (Figure 1). However, this residue is a non-canonical target for CK1α, deviating from its preferred consensus sequence, as determined largely through peptide-based studies. Numerous examples have now come to light in which CK1α phosphorylates non-canonical sites, and additional consensus sequences have been identified (Marin et al., 2003). Nevertheless, CK1 kinases are thought to generally prefer primed substrates. Binding of the phosphorylated, priming residue, or a cluster of acidic residues to a basic patch on the kinase, named Anion Binding Site 1, is thought to stabilize the kinase in an active conformation and position the downstream phosphorylation site in the active site (Gebel et al., 2020; Philpott et al., 2020; Longenecker et al., 1996). However, phosphorylation was not impacted when we neutralized the acidic residues previously proposed to serve as a priming cluster (Golden et al., 2017; Figure 5—figure supplement 1). We would like to suggest that it is the target RNA’s backbone, being negatively charged, that may be able to bind to this site and prime for S828 phosphorylation. The need for seed + supplementary pairing as opposed to other complementarity combinations in order to trigger robust phosphorylation supports a role for conformational change. These changes might position the RNA to interact with the kinase properly, rather than making the EI more accessible.

The need for target binding could explain findings from previously reported in vitro phosphorylation assays testing CK1α’s ability to phosphorylate peptide substrates spanning hAgo2’s EI region (Golden et al., 2017). Golden et al. reported that an unphosphorylated EI peptide could not be phosphorylated by recombinant CK1α, leading them to propose that contextual features in the full-length hAgo2 were required in order to make S828 a suitable CK1α substrate. Our work shows that it is not only full-length hAgo2 that is required, but rather hAgo2 bound to a guide and target RNA with seed and supplementary region pairing.

All the in vitro phosphorylation experiments that have been reported point to a central importance of S828. Our finding that S828 phosphorylation is required for subsequent hierarchical phosphorylation by CK1α in full-length hAgo2 (Figure 5) is consistent with previous experiments using phosphorylated peptide substrates (Golden et al., 2017). Golden et al. showed that peptides phosphorylated at S828 were able to be phosphorylated at S831 and those phosphorylated at S831 were able to be phosphorylated at S834. They suggested that pS828 primes for hierarchical phosphorylation of the EI at S831 and S834, consistent with our experiments with full-length hAgo2. This was further supported by their finding that S828A mutation, but not a single A mutation at any of the other phosphorylatable residues in the EI, eliminated detectable phosphorylation of hAgo2 in HCT116 cells lacking expression of ANKRD52, a regulatory subunit of PP6 (Golden et al., 2017). The central importance of S828 is also reflected in functional data from in vivo experiments. Huberdeau et al. found that mutating the corresponding serine in the C. elegans hAgo2 homolog ALG-1 (S992) to alanine led to severe developmental defects similar to those seen when mutating all EI phosphorylation sites to alanine (Quévillon Huberdeau et al., 2017). Furthermore, Golden et al. reported that expression of hAgo2(S828A), but not wt hAgo2, was able to rescue repression of a miR-19 EGFP reporter in ANKRD52-deficient cells. Phosphorylation of S828 thus appears to serve as a key gatekeeper regulating further phosphorylation and its consequent functional effects, implying its regulation would be critical. We now show that this gatekeeper is directly regulated by target binding.

Because miRNA-mediated repression does not involve slicing of the targets, additional cofactors are required to carry out silencing of miRNA targets. After miRNA-bound Ago binds a target mRNA, Ago binds a scaffolding protein of the GW182 family (TNRC6A/B/C in humans), which in turn recruits deadenylation complexes (PAN2-PAN3 and/or CCR4-NOT) and decapping complexes to promote target degradation (Jonas and Izaurralde, 2015). Previous studies have found that EI phosphorylation does not impact hAgo2/GW182 interaction (Golden et al., 2017; Quévillon Huberdeau et al., 2017). It is possible that one of the recruited proteins recruits CK1α. However, since we are able to observe Ago phosphorylation in the absence of additional proteins, they are not required for CKα-mediated phosphorylation. Importantly, because we did not observe phosphorylation until after target binding, and even then, can only detect initial phosphorylation of a single site, the in vitro system appears to recapitulate a regulated system having high specificity.

Because Ago must be dephosphorylated by PP6 before it can optimally repress additional targets, phosphorylation also has the potential to serve as a global regulatory measure. Knocking out PP6 in human cell lines led to a global decrease in RISC binding to mRNA (Golden et al., 2017). Under normal conditions, however, CK1α-mediated EI phosphorylation is rapidly countered by dephosphorylation (Golden et al., 2017). Despite this, neither 5XA nor 5XE hAgo2 could rescue miRNA-mediated repression in Ago2^-/- cells, indicating that the phosphorylation cycling process itself is important, not merely the presence or absence of phosphorylation.

Our findings that only S828 can be phosphorylated in our in vitro system without priming phosphorylation, and that this phosphorylation only occurs after binding to targets meeting certain criteria reveal novel structural and sequence determinants of highly specific CK1α-mediated phosphorylation. We therefore propose that target binding serves as a means for regulating CK1α-mediated phosphorylation of Ago, which, in turn, regulates RISC turnover and thus miRNA-mediated mRNA repression. The presence of multiple phosphorylation sites with decreasing target affinity may merely reflect the biophysical requirements for establishing sufficient negative charge to repel the target RNA. However, it might also provide a way for a cell to titrate the Ago/target affinity and/or to serve as a ‘checkpoint’ in order to ensure that targets aren’t spuriously released. Our work doesn’t address the temporal dynamics of phosphorylation, dephosphorylation, and repression. However, given the previous in vivo findings showing widespread regulatory defects with both hyper- and hypophosphorylation (Golden et al., 2017; Quévillon Huberdeau et al., 2017), we speculate that phosphorylation, or at least phosphorylation of enough sites to efficiently promote target release, happens after target repression is established. The requirement for multiple phosphorylations could help ensure that there is enough time for the initiation of repression to occur before the target is released. Direct tethering of GW182 family proteins to mRNA can bypass the need for RISC (Behm-Ansmant et al., 2006; Zipprich et al., 2009), suggesting that if enough Ago-independent interactions between repressive machinery and the mRNA are formed, Ago may even be able to be released while repression is ongoing.

This mechanism appears specialized for repression mediated by miRNA, as opposed to siRNA, as binding to fully complementary sites only triggers minimal phosphorylation (Figure 2b) and phosphomimicking does not impair slicing (Figure 2—figure supplement 2). This is consistent with the evolutionary conservation of the phosphosites among miRNA-specialized Ago proteins (e.g. D. melanogaster Ago1 [dmAgo1]) but not among siRNA-specialized Agos (e.g. dmAgo2), and our work provides a mechanistic explanation. With siRNA, slicing of the fully complementary target promotes its release by destabilizing the guide/target duplex (Salomon et al., 2015), but partially-complementary miRNAs with high affinity, such as those with 3’ supplementary pairing, could get stuck on a target and phosphorylation could serve as a mechanism to promote their release in the absence of slicing. However, we found that extending 3’ pairing in the absence of central pairing also decreased EI phosphorylation (Figure 2b). Such targets can induce TDMD in vivo, which resolves the interaction through degradation of the RISC complex (Shi et al., 2020; Han et al., 2020), and therefore these targets would not need phosphorylation-promoted release to resolve RISC/target interactions.

Though we showed that hAgo1 and hAgo3 are also subject to target-binding-triggered CK1α-mediated phosphorylation in vitro similar to hAgo2, we did not investigate the functional impact of this phosphorylation; however, based on the high homology between the human Ago’s and their apparent redundancy in most instances (Su et al., 2009), we would expect the effects of hAgo1 and hAgo3 phosphorylation to be similar to those we observed with hAgo2. Therefore, this regulatory mechanism is likely shared by these miRNA-handling Ago orthologs, suggesting a potentially broad impact on post-transcriptional regulation.

In conclusion, our findings show that Ago EI phosphorylation is a post-target-binding regulatory mechanism that promotes target release, freeing guide-bound Ago, namely RISC, from the mRNA target and dissuading it from binding more targets until it gets dephosphorylated by PPP6C/ANKRD52 (Golden et al., 2017). This dephosphorylation was previously shown to be rapid (Golden et al., 2017), therefore the released Ago-guide complex is likely to quickly be able to seek out and repress additional targets. Although we did not investigate in vivo temporal dynamics, we suggest that phosphorylation occurs once repression has already been established and Ago’s presence is no longer required. As long as the cycle of phosphorylation/dephosphorylation is not disrupted, this would allow cells to strike a balance between the increased efficacy and specificity provided by stronger binding sites and the quest for efficiency, allowing a single miRISC to repress multiple mRNAs (Figure 7). This mechanism of promoting miRNA target release appears specialized for miRNA-mediated repression and the broad conservation of the phosphorylation sites suggests that its use is widespread among miRNA-handling Ago proteins.

Figure 7

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All raw files and final state data for the HDX-MS experiments have been deposited in the MASSive public repository at massive.ucsd.edu with the dataset number MSV000088648. Individual uptake plots can be viewed by opening the state data files with DECA which is available at https://github.com/komiveslab/DECA, (copy archived at swh:1:rev:ec7a72e54aa1d025342fcdcb3f61840e08cd456f).

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Author details

1. Brianna Bibel

   1. Cold Spring Harbor Laboratory School of Biological Sciences, Cold Spring Harbor, United States
   2. Howard Hughes Medical Institute, W. M. Keck Structural Biology Laboratory, Cold Spring Harbor, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8543-151X

2. Elad Elkayam

   Howard Hughes Medical Institute, W. M. Keck Structural Biology Laboratory, Cold Spring Harbor, United States

   Present address

   Ventus Therapeutics, Waltham, Massachusetts, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Writing – review and editing

   Competing interests

   is affiliated with Ventus Therapeutics. The author has no financial interests to declare

   "This ORCID iD identifies the author of this article:" 0000-0001-9266-7002

3. Steve Silletti

   Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States

   Contribution

   Data curation, Formal analysis, Resources, Software

   Competing interests

   No competing interests declared

4. Elizabeth A Komives

   Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States

   Contribution

   Formal analysis, Methodology, Resources, Software, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

5. Leemor Joshua-Tor

   1. Cold Spring Harbor Laboratory School of Biological Sciences, Cold Spring Harbor, United States
   2. Howard Hughes Medical Institute, W. M. Keck Structural Biology Laboratory, Cold Spring Harbor, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   leemor@cshl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8185-8049

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