Phosphorylation of Argonaute proteins affects mRNA binding and is essential for micro RNA ‐guided gene silencing in vivo

Article23 June 2017free access Source DataTransparent process Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing in vivo Miguel Quévillon Huberdeau Miguel Quévillon Huberdeau orcid.org/0000-0003-4410-906X St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Daniela M Zeitler Daniela M Zeitler Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Judith Hauptmann Judith Hauptmann Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Astrid Bruckmann Astrid Bruckmann Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Lucile Fressigné Lucile Fressigné St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Johannes Danner Johannes Danner Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Sandra Piquet Sandra Piquet St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Nicholas Strieder Nicholas Strieder Department of Statistical Bioinformatics, University of Regensburg, Regensburg, Germany Search for more papers by this author Julia C Engelmann Julia C Engelmann Department of Statistical Bioinformatics, University of Regensburg, Regensburg, Germany Search for more papers by this author Guillaume Jannot Guillaume Jannot St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Rainer Deutzmann Rainer Deutzmann Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Martin J Simard Corresponding Author Martin J Simard [email protected] orcid.org/0000-0002-3189-9309 St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Gunter Meister Corresponding Author Gunter Meister [email protected] orcid.org/0000-0002-2098-9923 Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Miguel Quévillon Huberdeau Miguel Quévillon Huberdeau orcid.org/0000-0003-4410-906X St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Daniela M Zeitler Daniela M Zeitler Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Judith Hauptmann Judith Hauptmann Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Astrid Bruckmann Astrid Bruckmann Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Lucile Fressigné Lucile Fressigné St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Johannes Danner Johannes Danner Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Sandra Piquet Sandra Piquet St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Nicholas Strieder Nicholas Strieder Department of Statistical Bioinformatics, University of Regensburg, Regensburg, Germany Search for more papers by this author Julia C Engelmann Julia C Engelmann Department of Statistical Bioinformatics, University of Regensburg, Regensburg, Germany Search for more papers by this author Guillaume Jannot Guillaume Jannot St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Rainer Deutzmann Rainer Deutzmann Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Martin J Simard Corresponding Author Martin J Simard [email protected] orcid.org/0000-0002-3189-9309 St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada Laval University Cancer Research Centre, Quebec City, Québec, Canada Search for more papers by this author Gunter Meister Corresponding Author Gunter Meister [email protected] orcid.org/0000-0002-2098-9923 Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany Search for more papers by this author Author Information Miguel Quévillon Huberdeau1,2,‡, Daniela M Zeitler3,‡, Judith Hauptmann3, Astrid Bruckmann3, Lucile Fressigné1,2, Johannes Danner3, Sandra Piquet1,2, Nicholas Strieder4, Julia C Engelmann4, Guillaume Jannot1,2, Rainer Deutzmann3, Martin J Simard *,1,2 and Gunter Meister *,3 1St-Patrick Research Group in Basic Oncology, Centre Hospitalier Universitaire de Québec-Université Laval Research Centre (L'Hôtel-Dieu de Québec), Quebec City, Québec, Canada 2Laval University Cancer Research Centre, Quebec City, Québec, Canada 3Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, Regensburg, Germany 4Department of Statistical Bioinformatics, University of Regensburg, Regensburg, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +1 418 525 4444, ext. 15185; E-mail: [email protected] *Corresponding author. Tel: +49 941 943 2847; E-mail: [email protected] The EMBO Journal (2017)36:2088-2106https://doi.org/10.15252/embj.201696386 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans. In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function in vivo. Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA. Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute–mRNA interactions. Synopsis A conserved phosphorylation site in Argonaute 2, a key effector of miRNA-dependent gene regulation, controls mRNA binding in human and worms, revealing that the activity of the RNAi machinery is dynamically regulated. Mapping of post-translational modifications on endogenous Argonaute proteins across species reveal highly conserved phosphorylation events. A conserved phosphorylation cluster on the PIWI domain of Argonaute ALG-1 is essential for microRNA-guided gene regulation in Caenorhabditis elegans. Ago2 mutants mimicking hyper-phosphorylation of the cluster bind miRNAs and are catalytically active. Hyper-phosphorylation of the cluster on human Ago2 alters miRNA target binding. Introduction Argonaute (Ago) proteins form the core of the RNA-induced silencing complex (RISC) and serve as direct binding modules for small RNAs such as short interfering RNAs (siRNAs) or microRNAs (miRNAs) (Hutvagner & Simard, 2008; Meister, 2013). Ago proteins (or the Ago clade) represent a specific and highly conserved branch of the larger Argonaute protein family, which also contains the so-called PIWI proteins that associate with piRNAs and silence repetitive sequences in the germline. Ago proteins contain four domains (Jinek & Doudna, 2009; Ipsaro & Joshua-Tor, 2015). At the N-terminus, the N domain has been implicated in loading of small RNAs onto Ago proteins (Kwak & Tomari, 2012). The following PAZ domain anchors the 3′ end within a defined binding pocket and the MID domain binds the 5′ end of the small RNA (Lingel et al, 2003; Song et al, 2003; Yan et al, 2003; Ma et al, 2004, 2005; Parker et al, 2005). These two domains serve as a molecular “ruler” only accepting small RNAs within a very defined length range, typically between 19 and 25 nucleotides (nt). The C-terminal PIWI domain is structurally very similar to RNase H, which cleaves RNA molecules in DNA–RNA hybrids (Song et al, 2004; Yuan et al, 2005). Some Ago proteins indeed possess cleavage activity and cleave target RNAs complementary to the bound small RNA. In human, four different Ago proteins exist (Ago1–4), but only Ago2 is catalytically active and thus often referred to as “slicer” (Liu et al, 2004; Meister et al, 2004). Minor structural changes inactivate Ago1, Ago3, and Ago4 but they nevertheless bind small RNAs with equal efficiency and fulfill cleavage-independent miRNA functions (Faehnle et al, 2013; Hauptmann et al, 2013, 2014; Nakanishi et al, 2013; Schurmann et al, 2013). Ago proteins receive their small RNA partner from double-stranded (ds) precursor RNAs, which are processed by the RNase III enzyme Dicer. Dicer produces a short dsRNA but only one strand of the duplex, referred to as the guide strand, finds its way onto the Ago protein. During a specific loading step, Ago is kept in an open conformation and the small dsRNA Dicer product binds to the Ago protein (Dueck & Meister, 2014; Kobayashi & Tomari, 2016). One strand is now displaced (the passenger strand) and the guide strand stably associates with the Ago protein. In many organisms, a so-called RISC loading complex containing Dicer and its co-factor TRBP, an Ago protein, heat shock protein 90 (HSP90) and a number of co-chaperones have been suggested (Iwasaki et al, 2010; Kawamata & Tomari, 2010; Martinez et al, 2013). Ago2 cleaves the passenger strand and can displace it faster than other Ago proteins. Loading is completed by shifting the Ago protein to a closed conformation that is now ready for target binding. Likewise, generation of the closed confirmation requires ATP hydrolysis most likely by HSP90 (Dueck et al, 2014). The bound small RNA strand guides Ago to complementary target sites on mRNAs. MiRNA target sites are typically located on the 3′ untranslated region (UTR) of target mRNAs and are only partially complementary to the miRNA (Bartel, 2009). On the mRNA, Ago proteins recruit a member of the GW protein family, which are characterized by multiple tryptophanes (W) often flanked by glycines (G) (Jakymiw et al, 2005; Liu et al, 2005; Meister et al, 2005; Rehwinkel et al, 2005). Two specific tryptophan residues that are located in the N-terminal half of GW proteins directly bind into specific binding pockets on the surface of the PIWI domain of the Ago protein to form a stable complex (Schirle & MacRae, 2012; Pfaff et al, 2013; Jannot et al, 2016; Kuzuoglu-Ozturk et al, 2016). In mammals, the GW proteins are referred to as TNRC6A, B, and C, in Drosophila GW182 and in Caenorhabditis elegans AIN-1 and AIN-2. GW proteins are largely unstructured and serve as scaffolds to accommodate downstream enzymes and other protein-binding partners. For example, GW proteins contact the poly(A) binding protein on the poly(A) tail of the mRNA. Furthermore, they recruit deadenylation enzymes such as PAN2/3 or the CCR4/NOT complex that remove the poly(A) tail of the mRNA. After deadenylation, decapping enzymes are recruited and the mRNA is degraded in 5′ to 3′ direction (Jonas & Izaurralde, 2015). Ago proteins are at the heart of small RNA-guided gene silencing and therefore subject to intensive regulation. Ago proteins can be post-translationally modified at various positions with different consequences on their functions (Jee & Lai, 2014). For example, in the absence of miRNAs (e.g., in cells or tissues with impaired miRNA biogenesis), Ago proteins are ubiquitinated and degraded (Smibert et al, 2013). Interestingly, in mice miRNAs appear to be globally downregulated upon T-cell stimulation, which is also mediated by Ago ubiquitination and proteasomal degradation (Bronevetsky et al, 2013). Another modification affecting Ago stability has been found almost a decade ago. Hydroxylation of proline 700 leads to increased Ago stability and enhanced siRNA-mediated target RNA cleavage (Qi et al, 2008). Poly-ADP-ribosylation (PARylation) is a common regulator of protein function in various organisms. It has been found that under stress conditions, specific enzymes such as poly(ADP) polymerases are localized to stress granules, where Ago proteins are concentrated as well. Ago proteins are PARylated, and this modification correlates with inhibition of Ago-mediated repression in stress granules (Leung et al, 2011). The most widespread protein modification, however, is phosphorylation. Phosphorylations have been characterized at different residues of Ago proteins. It has been demonstrated that Ago2 S387 is phosphorylated by the MAPK signaling pathway, and this modification affects cellular localization of endogenous Ago proteins into processing bodies (P bodies) (Zeng et al, 2008). Very recently, this modification has been associated with sorting of Ago2 into exosomes (McKenzie et al, 2016). Modification at the same position by Akt3 appears to shift Ago2 activity from cleavage to translational repression (Horman et al, 2013). Under hypoxic stress conditions, epidermal growth factor receptor, a known oncogene in many human cancers, phosphorylates Ago2 at S393 leading to a reduction of a number of tumor-suppressive miRNAs (Shen et al, 2013). In addition, it has been reported that protein tyrosine phosphatase 1B (PTP1B) de-phosphorylates Ago2 S393 under specific conditions (Yang et al, 2014). Finally, a distinct tyrosine within the 5′ phosphate-binding pocket of the MID domain has been found as potential phosphorylation site (Rudel et al, 2011). The negative charge within this pocket inhibits access of the 5′ phosphate of the miRNA and thus blocks miRNA binding. In macrophages, this modification contributes to a relief of miRNA-guided repression of pro-inflammatory cytokine mRNAs (Mazumder et al, 2013). Although various modifications mainly on human Ago2 have been reported, a global and quantitative picture of phosphorylation on animal Ago proteins is still missing. Here, we report a systematic and unbiased mass spectrometry analysis of endogenous Ago phosphorylation sites. We find that various phosphorylation sites are highly conserved between species. The phosphorylation of a specific serine/threonine cluster located on a loop on the surface of the PIWI domain is essential for miRNA function in animals. In the hyper-phosphorylated state, Ago proteins cannot associate with mRNAs but are nevertheless fully functional when artificially tethered to mRNAs. Interestingly, mutants that do not bind miRNAs and thus cannot get in touch with mRNAs or mutants that do not bind to TNRC6 proteins are hypo-phosphorylated at this cluster. In agreement with these findings in human cells, the phosphorylation of this serine/threonine cluster is essential for miRNA-mediated gene regulation in C. elegans. Therefore, we propose a model in which we suggest that phosphorylation states of that particular region on Ago proteins modulate mRNA binding. Results Mapping of conserved Ago phosphorylation sites To generate a comprehensive overview of the phosphorylation sites on Ago proteins and to elucidate their potential functional roles, we isolated endogenous Ago proteins from tissue and cell lysates (Fig 1). To purify all four human Ago proteins at the same time, we used antibodies against the associated TNRC6 proteins (Schraivogel et al, 2015). The corresponding Ago band was excised from the SDS gel, and Ago phosphorylation was measured using mass spectrometry (Dataset EV1). To eliminate technical artifacts, we performed our measurements in triplicates and only overlapping, and therefore reliable, phospho-sites were considered (Fig 1A, left). We find several phosphorylation hotspots, which are conserved in all four human Ago proteins (Fig 1A, right). First, we observe phosphorylated peptides at the very N-terminus although at somewhat different residues. Second, a tyrosine (Y) in the PAZ domain is found phosphorylated in all four Argonaute proteins. Third, a larger cluster, in which individual residues are repeatedly phosphorylated, is found between threonine (T) 530 and 556. Forth, a cluster, from which multiple phosphorylated peptides (up to four phospho-sites on one peptide) are identified, is found at the very C-terminus of all four human Ago proteins ranging from serine (S) 824 to 834. Phospho-sites that are specific to individual Ago proteins are only found in Ago2, among them the well-characterized S387 (Zeng et al, 2008). Of note, Ago4 is very low abundant in these cells and phospho-sites could have escaped detection due to low amounts. Figure 1. Identification and conservation of phosphorylation sites in Ago proteins Mass spectrometric measurements of human Ago proteins. Left: Venn diagrams depict overlapping phosphorylation sites of biological replicates. Striped area indicates overlaps between three biological replicates. Right: Schematic representation of phosphorylation sites in Ago proteins. Scheme depicts the human Ago domain structure and phosphorylation sites that were detected by mass spectrometry after anti-TNRC6 co-IP/FLAG-Ago-APP. Green bars represent phosphorylation sites of Ago1–4 measured in at least three biological replicates, and phosphorylated sites that are measured in only two biological replicates are indicated in orange. Ago protein purification from different species. To analyze conservation of Ago phosphorylation sites, endogenous proteins were purified by Ago-APP, FLAG-APP, and Ago/ALG-1-IP from mouse tissue, rat H32 cells, zebrafish embryos, and a mixed population of worms. Samples were eluted with Laemmli buffer, separated by electrophoresis, and stained with Coomassie Blue. Conservation of Ago2 phosphorylation sites in different species. Overview of conserved phosphorylation sites of Ago proteins from various species. Green bars represent phosphorylation sites that are measured in at least three biological replicates, and phospho-sites that are measured in two biological replicates are indicated in orange. Hs: Homo sapiens, Mm: Mus musculus, Rn: Rattus norvegicus, Dr: Danio rerio, Ce: Caenorhabditis elegans. Position of MID domain phospho-sites on the human Ago2 structure. Location of the residues 555:61 are shown in red, based on the model 4W5O (Schirle & MacRae, 2012). Position of the C-terminal cluster phosphorylation. The structure is based on the model 4W5O (Schirle & MacRae, 2012). Residues 824:34 are located on a flexible loop (not shown) from E821 to G836. The miRNA is indicated in orange, and parts of the mRNA are shown in green. Download figure Download PowerPoint Next, we analyzed which phosphorylation events are conserved between species (Fig 1B). We isolated Ago proteins from mouse tissues (e.g., liver tissue), the rat cell line H32, from zebrafish embryos as well as C. elegans lysates and analyzed phosphorylation sites by mass spectrometry. We find that all phosphorylated areas of Ago2 mentioned above are conserved between these species except for Y322, which is only found in human (Fig 1C). Two conserved phosphorylation clusters caught our attention. First, single phosphorylations between T555 and S561 are located around the binding pocket of the target mRNA that is opposite to the first miRNA nucleotide which is tightly bound into the 5′ binding pocket of the MID domain and is not engaged in RNA–RNA interactions (Fig 1D and E; Elkayam et al, 2012; Schirle et al, 2014). Second, the hyper-phosphorylated cluster at the very C-terminus of Ago proteins is located on an exposed loop, which is not resolved in the current Ago2 crystal structures (Schirle & MacRae, 2012). However, the loop is resolved in the Neurospora crassa Argonaute protein QDE-2 crystal structure (Boland et al, 2011) and modeling this structure into Ago2 reveals that it has the potential to come close to the site where the mRNA is hybridized to the bound miRNA (Appendix Fig S1A). Overall, we have mapped phosphorylation sites on endogenous Ago proteins from different species, which reveals two phosphorylated areas located close to the mRNA in the Ago2 structure that could be involved in the regulation of mRNA binding. The Argonaute 555:61 and 824:34 phosphorylation cluster We further examined the most prominent phosphorylations located between residues 555:61 and 824:34. In the 555:61 area, we only found peptides with single phosphorylations (Fig 2A, indicated by individual boxes). Compared to all other phosphorylation sites that we measured, we always found peptides containing S824:834 with multiple phosphate groups (Fig 2A, one box indicates hyper-phosphorylation). To rule out technical issues, we had a closer look into our mass spectrometry data (Fig 2B and C). Indeed, on the mass spectra, single phosphorylated peptide peaks were observed for the 555:61 cluster (Fig 2B) and peaks corresponding to multiply phosphorylated peptides can be clearly identified for the 824:34 cluster (Fig 2C). Figure 2. Spectra of specific phosphorylation sites S556 and 824:34 cluster and tethering assay Schematic representation of the Ago2 domain organization. The positions of identified phosphorylation sites are shown in red bars. The single phosphorylation sites of 555:61 and the cluster phosphorylations of 824:34 are highlighted in blue boxes. Representative CID fragment spectra of phosphorylated human Ago2 peptides from a LC-UHR-QTOF run. Monophosphorylated Ago2 peptide TpTPQTLSNLCLK. Monoisotopic mass: 1397.65 Da, Mascot ion score: 45, expectation value: 3.2e-005, Δ mass: 0.01 Da. Quadruplyphosphorylated Ago2 peptide YHLVDKEHDpSAEGpSHTpSGQpSNGR. Phospho-site localization at positions S824, S828, S831, S834 according to Mascot. Monoisotopic mass: 2829.98 Da, Mascot ion score: 31, expectation value: 0.00085, Δ mass: 0.0017 Da. Tethering assays with F/H-tagged wt Ago2 as well as several mutants. Renilla luciferase (RNL) activity was detected in extracts of HeLa cells. Cells were co-transfected with constructs expressing the RNL-5BoxB reporter, firefly luciferase (FF) and λNAgo2 and phospho-mutants. λNTNRC6 served as positive control. The expression levels of Renilla luciferase were normalized to co-transfected firefly luciferase signals (n ≥ 3, SEM). Download figure Download PowerPoint To get a first glimpse on potential functions of the identified Ago phosphorylation sites, we focused our work on the 555:61 region and the 824:34 cluster. We generated phospho-mimicking (E) and phospho-lacking (A) mutants and physically tethered them to a luciferase reporter mRNA using the widely used LambdaN(λN)/Box-B system (Gehring et al, 2003; Fig 2D). Using this approach, we find that all single mutants (both A and E of residues located in both regions) are indistinguishable from wild-type (wt) Ago2. Furthermore, mutating all potentially phosphorylated residues of the 824:34 cluster at the same time did not abolish silencing activity when tethered to the reporter mRNA (824:34A and E). Importantly, this suggests that all mutants used are most likely not mis-folded due to the introduced mutations and are therefore functional. In addition, our data also point toward a potential role of the detected phospho-sites upstream of deadenylation and translational repression (i.e., miRNA loading or mRNA binding) since upon tethering, downstream silencing is not affected. Of note, when we mutate all phospho residues within the 555:61 region to E, silencing activity is strongly impaired (Fig 2D, 555:61E). Consistently, the 555:61E mutant does not localize to P bodies, and it does not bind to TNRC6 proteins and also not to mRNAs (Appendix Fig S1B). Its catalytic cleavage activity is also impaired, but miRNA binding is unaffected (Appendix Fig S1C–E). The 555:61A mutant is indistinguishable from wt Ago2 (Fig 2D and Appendix Fig S1B–E). Since we never found a multiple phosphorylated peptide originating from this region in our mass spectrometry data and we did not see a phenotype with single mutants, we did not study the phosphorylation at the 555:61 region further. Phosphorylation of the 824:34 cluster does not affect miRNA loading and Argonaute localization Phosphorylations can be rare events, and in most studies, in which phospho-specific antibodies are used, it often remains unknown how much of the investigated protein is actually phosphorylated. Therefore, we performed absolute quantification by mass spectrometry. We spiked in non-phosphorylated peptides, a peptide carrying a phosphate at position S824 or a peptide containing four phosphates at the conserved serines shown in Fig 2C and quantified the amount of non-phosphorylated and phosphorylated Ago proteins in different mouse tissues (Fig 3A). Interestingly, the well-known S387 (correspond to S388 in mouse Ago2) phosphorylation is rather low and often below 1% of total Ago2. However, phosphorylation at position S824 (S825 in mAgo2) is between 5 and 10% and reaches almost 25% in testis. Ago proteins carrying four phosphates are between 5 and 10% as well, except in spleen where the levels are noticeably lower. Of note, multiple phosphorylations might be lost more easily during extract preparation or mass spectrometry and could therefore be even higher. These data suggest that a significant portion of Ago2 is phosphorylated at the 824:34 (825:35 in mouse) cluster in lysates from mouse tissues. Figure 3. Functional analysis of the phosphorylation cluster 824:34 Quantification of Ago2 phosphorylation levels in primary mouse tissues. The two most prominent phosphorylation sites, pS388 and the cluster pS825:35, were analyzed by quantitative mass spectrometric measurements (SRMs). Therefore, Ago1–4 were purified by FLAG-APPs. Isotope-labeled phosphorylated peptides were spiked into the tryptic digest. Bars represent the ratio phosphorylated:non-phosphorylated peptide (p/n.p.). S388 is phosphorylated at very low levels (1%, white bars), whereas S825 is phosphorylated at around 10–25% (light gray bars). The cluster peptide with phosphorylations at S825, S829, S832, and S835 can be detected in high levels (dark gray). Immunofluorescence of overexpressed Ago2 variants. HeLa cells were transfected with F/H-Ago2 wt and 824:34 mutants. Ago2 and mutants were detected in immunofluorescence with anti-HA antibody staining (shown in green). For co-localization studies, LSm4, a P body marker, was stained with a specific antibody (shown in red). DAPI staining (blue) indicates the nucleus. Co-immunoprecipitation of TNRC6 proteins and miR-19b. F/H-Ago2 wt and 824:34 mutants were overexpressed in HEK 293T cells, immunopurified by anti-FLAG-IP, separated on a SDS–PAGE, and analyzed by Western blotting. Co-immunoprecipitated TNRC6 proteins were detected by pan-TNRC6-antibody, clone 7A9, and F/H-Ago2 were detected by anti-HA antibody. Co-immunoprecipitated miR-19b, miR-21, and let-7a were detected by Northern blotting. In vitro cleavage assay of Ago2 cluster mutants. Cleavage ac