Abstract
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.
Highlights
Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology
In the miRNA-induced silencing complex, Ago is involved in post-transcriptional repression of mRNA function, while in the RISC-loading complex (RLC), Ago gets loaded with miRNAs and interacts with factors stimulating this process
Focusing on protein–protein interactions (PPI) involved in the miRNA-mediated silencing pathway, we show that it allows very-high-resolution proteomics of functional complexes
Summary
Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. The AP-MS approaches, suffered from two main limitations: (1) important functional factors such as the CCR4/ NOT complex, which is directly recruited by TNRC6 and is required for efficient miRNA-mediated silencing[5,6,7] were notably absent from the AP-MS data sets, and (2) it is not possible to assign novel identified proteins to a specific step of the pathway as Ago is part of both RLC and miRISC. To address the former, we decided to use the recently described BioID technique[8]. Another study recently described an alternative splitBioID assay[12], suggesting that BirA* can be split at multiple sites
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