Engineering double-stranded RNA binding activity into the Drosha double-stranded RNA binding domain results in a loss of microRNA processing function

• Published in: PloS one 2017-08, Vol.12 (8), p.e0182445-e0182445
• Main Authors: Kranick, Joshua C, Chadalavada, Durga M, Sahu, Debashish, Showalter, Scott A
• Format: Article
• Language: English
• Subjects: Acids; Amino acid composition; Amino Acid Sequence; Amino acids; Binding; Biochemistry; Biology and life sciences; Cell cycle; Computer and Information Sciences; Conserved Sequence; Double-stranded RNA; Double-Stranded RNA Binding Motif - genetics; Double-Stranded RNA Binding Motif - physiology; Electrophoretic Mobility Shift Assay; Engineering; Enzymes; Escherichia coli; Genetic aspects; Genetic Engineering; HEK293 Cells; Humans; Kinases; Microprocessors; MicroRNA; MicroRNAs; MicroRNAs - metabolism; miRNA; Models, Molecular; Molecular biology; Mutation; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Nuclei; Nucleotide sequence; Physiological aspects; Protein Conformation, alpha-Helical - genetics; Protein Conformation, alpha-Helical - physiology; Proteins; Research and Analysis Methods; Ribonuclease; Ribonuclease III; Ribonuclease III - genetics; Ribonuclease III - metabolism; Ribonucleic acid; RNA; RNA processing; RNA, Double-Stranded - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0182445

Canonical processing of miRNA begins in the nucleus with the Microprocessor complex, which is minimally composed of the RNase III enzyme Drosha and two copies of its cofactor protein DGCR8. In structural analogy to most RNase III enzymes, Drosha possesses a modular domain with the double-stranded RNA binding domain (dsRBD) fold. Unlike the dsRBDs found in most members of the RNase III family, the Drosha-dsRBD does not display double-stranded RNA binding activity; perhaps related to this, the Drosha-dsRBD amino acid sequence does not conform well to the canonical patterns expected for a dsRBD. In this article, we investigate the impact on miRNA processing of engineering double-stranded RNA binding activity into Drosha's non-canonical dsRBD. Our findings corroborate previous studies that have demonstrated the Drosha-dsRBD is necessary for miRNA processing and suggest that the amino acid composition in the second α-helix of the domain is critical to support its evolved function.