Effect of the deletion of the reported ERH binding site embedded in the DGCR8 N-terminus and/or global ERH knockdown on the expression of mature miRNAs

MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of most mammalian genes. They are generated in a multi-step process initiated by the Microprocessor, a protein complex composed of DROSHA and DGCR8. Recent studies have described the phenomenon of “cluster assistance”, in which a prototypic primary miRNA hairpin can license the Microprocessor-mediated processing of a clustered suboptimal hairpin in cis. Mechanistic analyses led to the identification of ERH (enhancer of rudimentary homolog) as a critical factor in this process. Notably, ERH has been demonstrated to associate with the N-terminus of DGCR8, but it has not been demonstrated that this complex is required for cluster assistance. Here, we have used prime editing to generate alleles that encode a DGCR8 ?103-126Q mutant that cannot bind ERH anymore. In contrast to global ERH knockdown, disruption of the ERH binding motif in DGCR8 affects processing of only a subset of cluster assistance-unrelated pri-miRNAs. Thus, ERH-mediated cluster assistance is independent of its described association with DGCR8, indicating that ERH confers two distinct roles in primary miRNA biogenesis: One driven by its direct binding to the Microprocessor via DGCR8 and the other as a DGCR8/ERH complex-independent factor in cluster assistance. Overall design: Wild-type 293T cell clones and clones in which the DGCR8 locus had been modified by prime editing to delete the reported ERH binding site were transiently transfected with vectors encoding non-targeting shRNA 1 or 2 as well as with shRNAs targeting ERH. Transfected cells were sorted on day 5 based on expression of a fluorescent marker and directly subjected to total RNA isolation. Total RNA of the respective cell populations was prepared with Trizol, mixed with defined amounts of spike-in control RNAs and subjected to small RNA library preparation.