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The microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here, we identify Enhancer of Rudimentary Homolog (ERH) as a new component of Microprocessor. Through a crystal structure and biochemical experiments, we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8, in a 2:2 stoichiometry. Knock-down of ERH or deletion of the DGCR8 N-terminus results in a reduced processing of suboptimal pri-miRNAs in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate ‘cluster assistance’ in which Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA biogenesis pathway.

Summary Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.

The Microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here we identify Enhancer of Rudimentary Homolog (ERH) as a new component of the Microprocessor. ERH binds to a conserved region in the N-terminus of DGCR8. Knockdown of ERH or deletion of the DGCR8 N-terminus results in a decrease of processing of primary miRNAs with suboptimal hairpin structures that reside in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate “cluster assistance” in which the Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA pathway.

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons, and may explain preferential evolutionary emergence of miRNA operons.

Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.