MicroRNA (
miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an
miRNA-containing precursor
miRNA hairpin from a large
primary miRNA transcript (
pri-miRNA) by the Microprocessor, which consists of the nuclear
RNase III Drosha and the double-stranded RNA-binding domain
protein DGCR8 (
DiGeorge syndrome critical region
protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the
pri-miRNA stem-loop followed by Drosha recruitment and
pri-miRNA cleavage. Because countless
RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between
RNA binding properties of DGCR8 and specific
pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds
RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known
RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.