The biochemical basis for the cooperative action of microRNAs

Abstract

In metazoans, microRNAs (miRNAs) act to repress mRNAs through a combination of translational repression and target degradation. miRNAs predominantly pair within the 3' untranslated region (3' UTR) of the mRNA. In cells, closely spaced miRNA target sites within an mRNA can act cooperatively, leading to more repression of the target mRNA than expected by independent action at each site. This dissertation details the use of purified miRNA-AGO2 complexes, synthetic target RNAs, and a purified domain of TNRC6B that is able to simultaneously bind multiple AGO proteins. We examined the target site occupancy and affinities for miRNA-AGO2 binding in the absence and presence of TNRC6B, for target RNAs with a single miRNA site as well as multiple miRNA sites spaced at varying distances. As miRNA-AGOƯbinding to target correlates with target repression, our study assayed target binding. Absent TNRC68, miRNA-AGO2 complexes showed little if any cooperative binding. In the presence of the AGO-binding domain of TNRC6B, we observed strong cooperative binding to dual-site target RNAs. We went on to explore the miRNA site parameters suitable for cooperativity, investigating the spacing between sites as well as different miRNAs working alone or in combination with one another. To interrogate the mechanism by which TNRC6B increases cooperativity, competitive slicing experiments were performed; results indicated that association rates between miRNA-AGO2 complexes and targets were not affected by TNRC68, which implied that the improved affinities were due to reduced dissociation. Thus, the multivalent binding of TNRC6 enables cooperative binding of miRNA-AGO complexes to target RNAs, thereby explaining the basis of cooperative action.