Dual function of ERH in primary miRNA biogenesis
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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 . Genetic screening and mechanistic analyses led to the identification of two critical factors for this process, SAFB2 (scaffold attachment factor B2) and ERH (enhancer of rudimentary homolog), which have been shown to associate with the N-termini of DROSHA and DGCR8, respectively, but also form a complex with each other. However, it remains unclear how SAFB2 and ERH can alter the Microprocessor substrate specificity, and whether the described protein-protein interactions are required for cluster assistance.
In this study, we determined the crystal structure of the SAFB2-ERH complex, revealing the interaction interface and show that mutation of critical residues weakens complex formation but does not affect cluster assistance. Accordingly, loss of SAFB1 and 2 or of ERH results in overlapping, but not identical defects in primary miRNA biogenesis, suggesting that both factors confer also unique roles in this process. Moreover, our data indicate that SAFB1/2 and ERH are required for efficient Microprocessor feedback regulation, uncovering a clear physiological function of cluster assistance impacting global miRNA abundance. Most importantly, we demonstrate that ERH-mediated cluster assistance does not require its described association with DGCR8, and show that its disruption affects only a small subset of cluster assistance-unrelated pri-miRNAs. Thus, this study reveals dual roles of ERH in primary miRNA biogenesis, one driven by its direct binding to DGCR8, and the other in cluster assistance that does not require DGCR8 binding.
Highlights
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SAFB2 and ERH interact through a defined motif, but formation of this complex is largely dispensable for miRNA cluster assistance.
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Loss of SAFB1/2 and ERH, respectively, induces overlapping, but not identical defects in primary miRNA biogenesis.
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Both SAFB2 and ERH are involved in Microprocessor feedback regulation.
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Processing of suboptimal Microprocessor substrates requires SAFB2 and ERH, suggesting their direct involvement.
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ERH-mediated cluster assistance does not require its binding to the DGCR8 N-terminus.