Measuring Proximity-Mediated Function of mRNA Regulatory Proteins by Engineered Tethering.

A powerful approach for studying the functional consequences of site-specific RNA-protein interactions is to artificially tether a protein to a messenger (or noncoding) RNA through a selective, high-affinity interaction. We share a strategy for evaluating the contribution of protein positioning within an mRNA on gene expression. We introduced an RNA hairpin recognition site for the MS2 coat protein into the untranslated regions or coding sequence of mRNAs expressing a luminescent reporter protein, NanoLuc. Effector proteins fused to the MS2 coat protein could thus be targeted to distinct regions across the mRNA. We illustrate this approach using ZFP36L2, which recruits the CCR4-NOT complex for poly(A) tail deadenylation. Tethering ZFP36L2 to the 3'-UTR decreased NanoLuc expression, as expected, given the known interaction of this adapter protein with adenine uridine-rich elements (AREs). Intriguingly, ZFP36L2 also decreased NanoLuc expression when bound within the coding sequence, revealing that ZFP36L2-and potentially many other mRNA regulatory proteins-can function when targeted to diverse locations within an mRNA. This multi-target tethering strategy enables exploration of the interplay between mRNA-protein proximity and gene expression.