Site-Specific Labelling of Native Mammalian Proteins for Single-Molecule FRET Measurements

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking. We address this issue showing the site-specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post-translationally modified proteins on the single-molecule level, among them the hitherto inaccessible human Argonaute2

The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans.

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3'UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo

A specific type of Argonaute phosphorylation regulates binding to microRNAs during C. elegans development

Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs mi-croRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphory-lates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development

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

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

The Tryptophan-binding pockets of ALG-1 are essential to mediate the interaction with AIN-1 and AIN-2 proteins <i>in vitro</i> and <i>in vivo</i>.

<p><b>(A and B) Putative Tryptophan-binding pockets of ALG-1.</b> A homology model (pale cyan) of ALG-1 tryptophan-binding pockets 1 (A) and 2 (B) was generated by Swiss-Model from 4OLB and superposed on the tryptophan (pink)-bound hAgo2 structure (PDB code: 4OLB). hAgo2 is not shown for clarity. <b>(C) Schematic view of ALG-1 structured protein domains.</b> The residues forming the tryptophan-binding pocket 1 and 2 are coloured in green and red respectively and mutated into non-polar amino acid alanine to generate an ALG-1 <u>T</u>ryptophan-binding <u>P</u>ockets mutant (hereafter named TPmut). <b>(D) GST pull-down assays using bacterially expressed GST-Tagged wild-type or tryptophan-binding pocket mutants of ALG-1 protein.</b> An AIN-1 peptide fragment containing the AGO Binding Domain (350-641aa) was purified and co-immunoprecipitated with recombinant GST-tagged ALG-1 or ALG-1(TPmut) protein. The upper part of gel was stained with Coomassie Brilliant Blue and the lower blotted with antibody against AIN-1. <b>(E) Co-Immunoprecipitation of ALG-1 wild-type or mutant and AIN-1 and AIN-2 proteins from whole <i>C</i>. <i>elegans</i> extract.</b> Transgenic animals were generated using MosSCI single copy insertion of mCherry-tagged ALG-1 wild-type or TP mutant (TPmut). Animals were then crossed into <i>alg-1(0)</i> genetic background. ALG-1 proteins from young adults animals was co-immunoprecipitated using ALG-1 specific antibody and AIN-1 and AIN-2 proteins were detected by Western Blotting. Inputs represent the equivalent of 20% and 25% of the total protein extracts used for the IPs for detecting AIN-1 and AIN-2, respectively. Numbers indicate the relative level of proteins in input and Co-IP compared to wt for each strain.</p

The loss of function of ALG-1 tryptophan-binding pockets mutant occurs downstream of the biogenesis of microRNA.

<p><b>(A) Quantification of microRNA expression</b>. The levels of miR-48 and miR-241 were measured by quantitative RT-PCR at mid L3 stage from <i>alg-1TPmut</i> and <i>alg-1(0)</i> whole worm RNA extracts, and were normalized to <i>alg-1(wt)</i>. Small nucleolar RNA sn2841 was used as reference. The error bars represent the 95% confidence interval from three independent experiments and a Student’s two-sided t-test was applied to obtain <i>p</i> values. *<i>p</i><0.05, **<i>p</i><0.005. <b>(B) Western Blot analysis of ALG-1(TPmut) or (wt) associated to lin-4 and let-7 miRISC</b>. Isolation of miRISC was performed using a 2′-O-methyl oligonucleotide (2′-O-Me oligo) column complementary to lin-4 or let-7 mature microRNA. The extract from transgenic animals was first pre-incubated with an unrelated 2′-O-methyl oligonucleotide column (Un) then incubated with the indicated 2′-O-methyl oligonucleotide complementary to lin-4 and let-7 sequences. The purified complexes were run on to an SDS polyacrylamide gel and blotted with an antibody against ALG-1. The dashed line indicates that unrelated lanes have been removed between samples.</p

Impairing the interaction with AIN proteins leads to post-embryonic heterochronic phenotypes.

<p><b>(A) Phenotypic analysis.</b> The <i>alg-1</i> null (<i>alg-1(0)</i>) animals were injected with an array (<i>Ex(array)</i>)containing plasmids expressing a dominant <i>rol-6</i> injection marker (making animals rolling) along with <i>alg-1(wt)</i> or <i>alg-1(TPmut)</i>,expressing vectors. F1 transgenic rollers animals were picked. The number of bursting animals scored (n) 48h later is indicated. The two-tailed <i>p</i> value indicated was measured by Fisher’s exact test. <b>(B) Seam cells lineage of wild type or <i>alg-1</i> mutant animals</b>. The extra-proliferative division pattern at the L3 division stages of seam cells are shown in bold in <i>alg-1</i> mutant. This characteristic of <i>alg-1</i> loss of function pattern result in a retarded heterochronic phenotype observable by specific defect such as alae structure synthesis during the L4-adult transition stages (here represented by three lines). <b>(C) Average and range of seam cells number at adulthood.</b> The indicated strains were crossed with a strain expressing GFP in the seam cells (<i>scm</i>::<i>GFP</i>) and the number of seam cells scored in young adult. The mean value and range covered by the individual counts are indicated. The number of animal scored (n) is indicated. <b>(D) Analysis of animals alae.</b> The structure of alae of young adult <i>alg-1(0)</i> animals expressing either <i>alg-1wt</i> or <i>alg-1(TPmut)</i> single inserted (Si) transgenic allele were assayed by Normarski DIC imaging and compared to <i>alg-1(0)</i>. The number of animal scored (n) is indicated. The two-tailed <i>p</i> values indicated were measured by Fisher’s exact test. <b>(E) Functional analysis using a let-7 microRNA reporter</b>. Animals carrying the <i>GFP</i> reporter transgene under the control of a hypodermis-specific <i>col-10</i> promoter and the <i>lin-41</i> 3’UTR containing the <i>let-7</i> miRNA binding sites (red; diagram) were crossed into either <i>alg-1(0)</i> or <i>alg-1(TPmut)</i> animals. <b>Left:</b> Representative pictures of GFP expression during early larvae stages (L2) and in young adult animals (Adult). Scale bars represent 10μm. <b>Right:</b> Quantification of GFP in adult relative to L2 stage animals. The quantification of the GFP signal was performed by measuring the mean of the GFP detected in five different cells for each animal. The number of animals scored (n) is indicated. The error bars represent the 95% confidence interval from three independent experiments and a Student’s two-sided t-test was applied to obtain <i>p</i> values.</p

The GW182 proteins are not essential for microRNA-dependent silencing pathway during animal embryogenesis.

<p><b>(A) The <i>alg-1</i> tryptophan-binding pocket mutant is not synthetic lethal with <i>alg-2</i></b>. Early L4 staged <i>alg-1(0)</i> animals alone or expressing either <i>alg-1wt</i> or <i>alg-1(TPmut)</i> transgenic allele were picked on <i>alg-2 (+)</i> or <i>control (empty L4440 vector; -)</i> RNAi feeding plate for 24h before being separated onto new RNAi feeding plates. F1 progeny animals were scored for embryonic lethality and post developmental defects. The number of F1 animals scored (n) is indicated. <b>(B) <i>ain-1</i> and <i>ain-2</i> are not essential for <i>C</i>. <i>elegans</i> embryonic development.</b> Early L4 staged <i>ain-1(ku322); ain-2(tm2432)/dpy-5</i> animals were picked on <i>ain-2 (+)</i> or <i>control (empty L4440 vector; -)</i> RNAi feeding plate for 24h before being separated onto new RNAi feeding plates. F1 progeny animals were scored for embryonic lethality and post developmental defects. The number of F1 animals scored (n) is indicated.</p