Effect of SAFB1/SAFB2 knockou or ERH knockout on the miRNA transcriptome of murine Baf3 cells

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 two critical factors for this process, SAFB2 (scaffold attachment factor B2) and ERH (enhancer of rudimentary homolog), but it remains unclear to which extent these factors contribute to and are required for cluster assistance. Here, we have used a previously established dual fluorescence reporter system (doi: 10.1016/j.molcel.2020.05.011) to generate loss-of-function clones for SAFB2 and its closely related family member SAFB1 as well as for ERH.Our data indicate that loss of SAFB1/2 and of ERH results in overlapping, but not identical defects in primary miRNA biogenesis. Overall design: Small RNAseq data of Baf3 cell expressing fluorescent reporters for the activity of mutated forms of miR-15a and miR-16 (see doi: 10.1016/j.molcel.2020.05.011 for details) that were transduced with vectors encoding Cas9 and sgRNAs targeting Safb1/2, Erh or murine Rag1 (as a control gene not involved in miRNA biogenesis). The polyclonal population of cells that lost the capability to perform cluster assistance in response to SAFB or ERH gene disruption was identified and sorted out based on the derepression of the dsRed-based miR-15a reporter. 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.