Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein–protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.

Initially identified as a factor involved in tyrosine kinase receptor signaling, Grb10-interacting GYF protein 2 (GIGYF2) has later been shown to interact with the 5′ cap-binding protein 4EHP as part of a translation repression complex, and to mediate post-transcriptional repression of tethered reporter mRNAs. A current model proposes that GIGYF2 is indirectly recruited to mRNAs by specific RNA-binding proteins (RBPs) leading to translation repression through its association with 4EHP. Accordingly, we recently observed that GIGYF2 also interacts with the miRNA-induced silencing complex and probably modulates its translation repression activity. Here we have further investigated how GIGYF2 represses mRNA function. In a tethering reporter assay, we identify three independent domains of GIGYF2 with repressive activity. In this assay, GIGYF2-mediated repression is independent of 4EHP but largely dependent on the CCR4/NOT complex that GIGYF2 recruits through multiple interfaces. Importantly, we show that GIGYF2 is an RBP and identify for the first time endogenous mRNA targets that recapitulate 4EHP-independent repression. Altogether, we propose that GIGYF2 has two distinct mechanisms of repression: one depends on 4EHP binding and mainly affects translation; the other is 4EHP-independent and involves the CCR4/NOT complex and its deadenylation activity.

Cancer cell survival is highly dependent on its metabolic reprogramming, which supports not only cell growth but also confers to the tumor cells characteristics to initiate migration and colonization. Among the different mechanisms that are involved, translational control plays a significant role in oncogenesis; however, its impact on cancer progression still remains poorly understood. A study by Navickas and colleagues revealed that the RNA-binding protein heterogeneous nuclear ribonucleoprotein C (HNRNPC) functions as a translational regulator, and its downregulation in highly metastatic cells leads to the lengthening of 3′ untranslated regions in HNRNPC-bound mRNAs, resulting in translational repression mediated by the AGO–miRNA RNA-induced silencing complex.

Initially identified as a factor involved in tyrosine kinase receptor signalling, GRB10-interacting GYF protein 2 (GIGYF2) has later been shown to interact with the 5' cap-binding protein m4EHP as part of a translation repression complex, and to mediate post-transcriptional repression of tethered reporter mRNAs. We recently observed that GIGYF2 also interacts with the miRNA-induced silencing complex and modulates its translation repression activity. Here we have further investigated how GIGYF2 represses mRNA function. In RNA tethering reporter assays we show that GIGYF2 exerts its action through a combination of translational repression and stimulated mRNA decay. Using truncation variants we identify two distinct effector domains within GIGYF2. In this assay GIGYF2-mediated repression is independent of m4EHP but dependent on the deadenylation activity of the CCR4/NOT complex. We further show that GIGYF2 interacts with multiple subunits of the CCR4/NOT complex and interestingly depletion of the CNOT1 scaffold subunit does not affect GIGYF2-mediated repression. Finally, we identify endogenous mRNA targets of GIGYF2 that recapitulate m4EHP-independent repression. Altogether, we propose that GIGYF2 has two distinct mechanisms of repression: one depends on m4EHP binding and affects translation, the other is m4EHP-independent and relies on the deadenylation activity of the CCR4/NOT complex.