Human DROSHA Protein in Complex with the C-Terminal Tail of DGCR8

Alexandra Hall '19 and Hannah Wendlandt '19

Contents:

I. Introduction

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RNA interference (RNAi) is the process by which small double-stranded noncoding RNAs influence gene expression through either mRNA degradation, translational inhibition, or chromatin remodeling. DROSHA is a 159 kDa protein that interacts with two subunits of DGCR8, also known as PASHA, in the heterotrimeric Microprocessor complex. The Microprocessor complex functions to prepare endogenous RNAs (microRNAs) for use in RNAi pathways. In the classical model of miRNA processing, DROSHA cuts the RNA to yield a small hairpin structure, which is then cleaved again by DICER.

DROSHA function is critical for successful completion of RNAi pathways , which are an important mode of gene regulation. DROSHA's wide-ranging effects in vivo are not yet fully understood, however it is becoming more apparent that its function influences many cellular- and organismal-level systems. For instance, mutation in the RNase III catalytic center has recently been correlated with increased risk of colorectal cancer.

II. General Structure

DROSHA and DGCR8 together form the , a heterotrimeric complex of one DROSHA and two DGCR8 (C-terminal tails shown) . Human DROSHA contains a ribonuclease III (RIIID) dimer of and , , , , and .

The RIIID dimer (RIIIDa and RIIIDb ) forms one processing center, containing the catalytic sites for double-stranded RNA cleavage. The PAZ-like domain is similar to the PAZ domain found in Dicer, which recognizes the 3' terminus of the substrate double-stranded RNA, positioning the active sites 22 nucleotides away. Part of the PAZ-like domain structure was unable to be resolved due to structural flexibility. Its exact function is not yet known since double-stranded RNA is prevented from reaching the PAZ-like domain in DROSHA. The dsRBD's exact function is not known, but it may interact with the GUG motif in the double-stranded RNA. The connector helix links the RIIID dimer and the platform. The platform and connector helix together are bent towards the dsRNA, helping to block it and giving DROSHA its stooped conformation.

III. Complex Stability

Two DGCR8 subunits directly bind to DROSHA through interaction between a helix and an RIIID subunit, respectively. The two DGCR8 binding sites are asymmetric on the the RIIID subunits; DGCR8 binds RIIIDa on helices 3,4, and 9, while it binds RIIIDb on helices 1,5, and 8 (not shown). The RIIIDb-CTT (of DGCR8) interaction is more important than that with RIIIDa for microprocessor complex stability and formation. Two surprising may play an important role in stability by bridging different structural modules. contains His1026, Cys536, Cys536, and His549. contains His609, His680, Cys676, and Cys561.

IV. Pre-miRNA Processing

Unlike the well-described DICER system, it is not entirely clear how DROSHA functions to process miRNAs. However, some predictions may be made based on a model of DROSHA-RNA binding. A prior model of two subunits of with a manufactured RNA substrate shows orientation within the complex. Within the DROSHA complex, the apical loop of the dsRNA segment orients towards the RNase III domains, with the basal segments of the RNA positioned towards the center domain (CED) of the protein. The ( RIIIDa and RIIIDb ) of the RNase domains indicate that RIIIDa cleaves the 3’ strand of the RNA while RIIIDb cleaves the 5’ strand. The of the RNase domain is positioned next to the last base pair of the dsRNA region. , specifically, is highly conserved and its mutation inhibits cleavage activity. It is possible that the bump helix allows DROSHA to function as a “ruler,” measuring out 11 base pairs from the last base pair before cleavage.


V. References

Davidson, Beverly L. and Paul B. McCray. 2011. Current Prospects for RNA Interference -Based Therapies. Nature Reviews Genetics. 12:329-340.

Gan, Jianhua, Joseph E. Tropea, Brian P. Austin, Donald L. Court, David S. Waugh, and Xinhua Ji. 2006. Structural Insight into the Mechanism of Double-Stranded RNA Processing by Ribonuclease III. Cell. 124:355-366.

Kwon, S. Chul, Tuan Anh Nguyen, Yeon-Gil Choi, Myung Hyun Jo, Sungchil Hohng, V. Narry Kim, and Jae-Sung Woo. 2015. Structure of Human DROSHA. Cell. 164:81-90.

Nguyen, Tuan Anh, Myung Hyun Jo, Yeon-Gil Chol, Joha Park, S. Chil Kwon, Sungchul Hohng, V. Narry Kim, and Jae-Sung Woo. 2015. Functional Anatomy of Human Microprocessor. Cell. 161:1374-1387.

Watson, James D., Tania A. Baker, Stephen P. Bell, Alexander Gann, Michael Levine, Richard Losick, with Stephen C. Harrison. 2014. Molecular Biology of the Gene. Cold Spring Harbor Laboratory Press. 20:701-732.

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