Abstract
RNA-binding proteins (RBPs) are critical regulators of gene expression. An RBP typically binds to multiple mRNAs and modulates their expression. Although loss-of-function experiments on an RBP can infer how it regulates a specific target mRNA, the results are confounded by potential secondary effects due to the attenuation of all other interactions of the target RBP. For example, regarding the interaction between Trim71, an evolutionarily conserved RBP, and Ago2 mRNA, although Trim71 binds to Ago2 mRNA and overexpression of Trim71 represses Ago2 mRNA translation, it is puzzling that AGO2 protein levels are not altered in the Trim71 knockdown/knockout cells. To address this, we adapted the dTAG (degradation tag) system for determining the direct effects of the endogenous Trim71. Specifically, we knocked in the dTAG to the Trim71 locus, enabling inducible rapid Trim71 protein degradation. We observed that following the induction of Trim71 degradation, Ago2 protein levels first increased, confirming the Trim71-mediated repression, and then returned to the original levels after 24 h post-induction, revealing that the secondary effects from the Trim71 knockdown/knockout counteracted its direct effects on Ago2 mRNA. These results highlight a caveat in interpreting the results from loss-of-function studies on RBPs and provide a method to determine the primary effect(s) of RBPs on their target mRNAs.