Abstract
MicroRNAs (miRNAs) are small evolutionarily conserved regulatory RNAs that modulate mRNA stability and translation in a wide range of cell types. MiRNAs are involved in a broad array of biological processes, including cellular proliferation, differentiation, and apoptosis. To identify previously unidentified regulators of miRNA, we initiated a systematic discovery-type proteomic analysis of the miRNA pathway interactome in human cells. Six of 66 genes identified in our proteomic screen were capable of regulating lethal-7a (let-7a) miRNA reporter activity. Tripartite motif 65 (TRIM65) was identified as a repressor of miRNA activity. Detailed analysis indicates that TRIM65 interacts and colocalizes with trinucleotide repeat containing six (TNRC6) proteins in processing body-like structures. Ubiquitination assays demonstrate that TRIM65 is an ubiquitin E3 ligase for TNRC6 proteins. The combination of overexpression and knockdown studies establishes that TRIM65 relieves miRNA-driven suppression of mRNA expression through ubiquitination and subsequent degradation of TNRC6.