Separable roles for Microprocessor and its cofactors, ERH and SAFB1/2, during microRNA cluster assistance.

While most conserved microRNA (miRNA) transcripts harbor a suite of features that mediate their efficient biogenesis into small RNAs, some loci bear suboptimal attributes that enable additional layers of processing regulation. A notable example is cluster assistance, whereby a miRNA hairpin with suboptimal nuclear biogenesis can be enhanced by an optimal neighbor. This process involves local transfer of the Microprocessor complex, composed of the RNase III enzyme Drosha and its partner, DGCR8, in concert with cofactors such as ERH and SAFB1/2. However, the mechanisms that underlie miRNA cluster assistance remain largely unclear. Here, we gained insights into this process by integrating mutant cells of Microprocessor and its cofactors with analysis of miRNA structure-function variants, biochemical tests, and genome-wide profiling. We defined features of suboptimal miRNAs that render them dependent on cluster assistance and distinguished among a network of proposed interactions among Microprocessor and its cofactors to reveal a subset that is critical for cluster assistance. Most importantly, we used epistatic tests to separate and order the functional requirements for ERH and SAFB1/2 into a pathway. Our data indicate that ERH may engage in the process of Microprocessor transfer between hairpins, while SAFB factors (especially SAFB2) mediate recognition and stable binding of a suboptimal miRNA hairpin after Microprocessor transfer. Finally, we show how cluster assistance integrates into a feedback regulatory loop on Microprocessor via Drosha-mediated cleavage of a suboptimal miRNA hairpin in the DGCR8 transcript. Altogether, our findings reveal complex regulatory transactions during biogenesis of clustered miRNAs.