Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory

To assess whether AGO-associated stem-loop containing mRNAs are regulated by the microprocessor complex, we performed siRNA-mediated knockdown of DROSHA and DGCR8 in HEK293T cells and assessed changes in gene expression (Figs. 5H-J) . We observed robust knockdown of both DROSHA and DGCR8 protein levels (Fig. 5H) . Consistent with the role of DROSHA in regulating DGCR8 expression, we found that knockdown of DROSHA increased DGCR8 protein and RNA expression (Fig. 5H-I) . To determine whether DGCR8 and/or DROSHA participate in the regulation of AGO-associated stem-loop containing mRNAs, we performed mRNA-seq and RT-qPCR on knockdown RNA samples. To our surprise, the majority of mRNAs hosting AGO-associated stemloops were unaffected by knockdown of components of the microprocessor. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/294488 doi: bioRxiv preprint (Table S7 and Fig. 5I ). In fact, the only gene tested by RT-qPCR that was significantly affected was GLUL, but knockdown of either DGCR8 or DROSHA decreased the level of GLUL expression, in contrast to the expected role of the microprocessor. These data suggest that cleavage of AGO-associated stemloops does not have a significant impact on the expression of the host mRNA because cleavage events are rare, or because this process is independent of the microprocessor, with neither model being mutually exclusive.