The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein

Braden M Roth,Daniella Ishimaru,Mirko Hennig

doi:10.1074/jbc.m112.446880

Braden M Roth, Daniella Ishimaru + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m112.446880

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Abstract

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.

Highlights

Double-stranded RNA-binding domain-containing proteins play key roles in microRNA biogenesis
Our findings indicate that DGCR8core exchanges between and among pri-mir-16lower-binding sites on the intermediate chemical shift time scale leading to differential line broadening
This observation confirms earlier reports by Han et al [10] and suggests that association of Drosha and DGCR8 does not depend on the availability of RNA substrate molecules

Summary

Background

Double-stranded RNA-binding domain-containing proteins play key roles in microRNA biogenesis. Most Caenorhabditis elegans pri-miRNAs apparently lack determinants required for processing in human cells, yet one-fifth of all human pri-miRNAs lack primary sequence determinants such as downstream SRp20 binding, basal UG, and the apical GUG motifs, recently described by Bartel and co-workers [19] This leaves 70-nt hairpins with unpaired flanking regions as the only feature and common denominator across Microprocessor substrates. Most dsRBDs have been reported to bind duplex RNA as the name implies, unpaired ssRNA in loops, bulges, and mismatched pairs can be recognized (20 –23) This flexibility in substrate recognition presents a challenge in identifying both protein and RNA features required for specific proteinRNA interaction in the absence of structural data. Our results point to an intricate relationship between DGCR8 and Drosha in which both proteins are required for binding and processing, rather than a hierarchical DGCR8-mediated primiRNA recognition and subsequent Drosha recruitment model

EXPERIMENTAL PROCEDURES

RESULTS

Summary of filter binding experiments

DISCUSSION