Target binding triggers hierarchical phosphorylation of human Argonaute-2 to promote target release

  1. Brianna Bibel
  2. Elad Elkayam
  3. Steve Silletti
  4. Elizabeth A Komives
  5. Leemor Joshua-Tor

  • Curated by eLife

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    Evaluation Summary:

    This paper provides well documented and solid biochemical data to show how phosphorylation of AGO2 modulate its binding to target mRNAs, releasing the complex to allow its recycling in the cell via electrostatic repulsion. This result could explain how a small amount of Ago proteins could target a very large number of mRNA molecules . The data support the key claims of the manuscript, and the approaches used are rigorous. This very well-written and elegant study will be of great interest to those working in the miRNA field as it addresses important open questions concerning the dynamic regulation of mIRNA-mediated gene repression.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). Agos bind small RNAs (sRNAs) including small interfering RNAs (siRNAs) and microRNAs (miRNAs) to form the functional core of the RNA-induced silencing complex (RISC). The sRNA is used as a guide to target mRNAs containing either partially or fully complementary sequences, ultimately leading to downregulation of the corresponding proteins. It was previously shown that the kinase CK1α phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Ago2 to target RNA with complementarity to the seed and 3’ supplementary regions of the miRNA primes the EI for hierarchical phosphorylation by CK1α. The added negative charges electrostatically promote target release, freeing Ago to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression.

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  1. Version published to 10.7554/elife.76908 on eLife
  2. eLife

    Evaluation Summary:

    This paper provides well documented and solid biochemical data to show how phosphorylation of AGO2 modulate its binding to target mRNAs, releasing the complex to allow its recycling in the cell via electrostatic repulsion. This result could explain how a small amount of Ago proteins could target a very large number of mRNA molecules . The data support the key claims of the manuscript, and the approaches used are rigorous. This very well-written and elegant study will be of great interest to those working in the miRNA field as it addresses important open questions concerning the dynamic regulation of mIRNA-mediated gene repression.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This work provides solid in vitro evidence supporting the role of the phosphorylation of the EI domain of human AGO2 during the dynamic association/dissociation of the RISC complex to the target mRNAs. The evidence shows that the association of the RISC with a target mRNA, though the seed region, triggers CK1α phosphorylation of the EI region of AGO2, which in turn electrostatically promotes target release and AGO2 recycling. This effect becomes more evident when miRNA/target pairing extends to 3' supplemental regions besides the seed region. Furthermore, the phosphorylation of the EI starts in S828 then acts hierarchically to prime the phosphorylation of the remaining sites in the region. The paper provides strong in vitro evidence that will interest the gene silencing community. Perhaps the weaker aspect of the manuscript is the lack of in vivo evidence to support the conclusion, although this may be hard to achieve.

  4. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Bibel et al. used an in vitro system containing purified Argonaute, CK1, miRNA, and target RNA to investigate the molecular mechanisms underlying the phosphorylation of Argonaute proteins by CK1 and its consequences on the interaction between the RISC and its target RNAs.

    The authors build upon observations by Golden et al, who previously described a phosphorylation/dephosphorylation affecting a cluster of 5 conserved residues (4 serines and 1 threonine) on the EI region of Argonaute.

    Bibel and colleagues show that phosphorylation of these 5 residues by CK1 requires the formation of a Ago-miRNA-targetRNA complex and occurs in a partially hierarchical fashion, with S828 being the first site to be phosphorylated, followed by S831, and then by the three other sites.

    Interestingly, the type of interaction between the Ago-mIR and the target is important. Pairing with the seed and some additional 3' pairing is essential, but perfect or near-perfect complementarity between the miRNA and the target sequence impairs, rather than promoting, phosphorylation by CK1.

    Furthermore, confirming previous cell-based studies by the Mendel group, Bibel and colleagues show that phosphorylation of Ago promotes target dissociation.

    Based on these results the authors propose a model in which binding of the core RISC complex to miRNA target sequence serves as a primer to induce sequential phosphorylation of the 5 residues in the Ago EI region. The consequent increase in negative charge of this region then facilitates the dissociation of the RISC from the negatively charged target mRNA.

    The manuscript is beautifully written, the experiments are clearly described and their results support the model proposed by the authors. The relevant scientific literature is presented and the statistical analysis used is appropriate. These findings are of substantial interest to the scientific community, as they shed light on the emerging importance of the dynamic regulation of the RISC complex.

  5. eLife

    Reviewer #3 (Public Review):

    Using an elegant combination of biochemical, structural and biophysical approaches, the authors nicely demonstrated that binding of miRNA-loaded hAgo2 to its targets triggers phosphorylation of the hAgo2 EI by CK1α.

    They demonstrated an important role for hAgo2 release from its target allowing a turnover of target mRNAs. They also demonstrated a hierarchy in amino acid phosphorylation as well as a role of this phosphorylation in electrostatic repulsion between target mRNA and hAgo2 loaded with a miRNA.

    These results could help to explain how cells could regulate several targets with a small amount of hAgo in comparison to the estimated number of the target.

    They provide a nice view on how Ago proteins that are unable to cleave their target (which is the case with miRNAs and not with piRNAs or siRNAs) could be released from their target via an electrostatic destabilisation.

    Overall the experiments are well done and nicely explained. The manuscript iswas also easy to read. Future experiments should be done in vivo to be fully conclusive about the global role of Ago phosphorylation in the off-rate of association with its target.

  6. Version published to 10.1101/2022.01.06.475261v1 on bioRxiv