Poster abstracts

Poster number 90 submitted by Christopher Wostenberg

dsRBDs: the workhorse of the miRNA maturation pathway

Christopher Wostenberg (Chemistry Department at Pennsylvania State University, University Park), Kaycee A. Quarles (Chemistry Department at Pennsylvania State University, University Park), Scott A. Showalter (Chemistry Department at Pennsylvania State University, University Park)

Abstract:
miRNAs are small non-coding ssRNAs, ~ 22 nucleotides, which are involved in gene-regulation by base-pairing with mRNA. At least 60% of human genes are regulated by one or more miRNAs, which leads to control of cellular homeostasis (1, 2), while misregulation leads to disease states (3). Maturation occurs in two independent and spatially separated steps. First, in the nucleus, the single-stranded tail of primary miRNA is cleaved by the “Microprocessor”, composed of Drosha, an RNase III enzyme, and its cofactor DGCR8, a dsRNA binding protein (dsRBP) (4). Next, the precursor miRNA is exported to the cytosol and the terminal loop is cleaved by the RNase III enzyme Dicer aided by the dsRBPs TRBP or PACT (4). The most common protein motif in the pathway is the dsRNA binding domain (dsRBD), which prefers to bind A-form dsRNA (4, 5). A total of ten dsRBDs are involved in miRNA maturation, one in Drosha, two in DGCR8, one in Dicer, three in TRBP and three in PACT.
NMR spin relaxation and MD simulations on two of the dsRBDs in the Microprocessor, Drosha-dsRBD and DGCR8-dsRBD1, were preformed to provide further information on the molecular mechanism of binding (6). The study was motivated by EMSAs, which showed that Drosha-dsRBD does not bind dsRNA but isolated DGCR8-dsRBD1 does (6). Our results show that while loop 2 in both dsRBDs is highly dynamic, it is the increased flexibility of loop 1 in Drosha-dsRBD that potentially explains the lack of dsRNA binding in by the dsRBD. We also investigated the interactions of the two tandem dsRBDs of DGCR8 with MD simulations (7). The crystal structure along with preliminary NMR data revealed that the two dsRBDs of DGCR8 are packed against a well-defined secondary structure formed from the linker and the C-terminal tail (8), which contrasts with the flexible linker seen in PKR (9). Recent work has focused on understanding the dsRBDs in the second step of miRNA maturation, especially Dicer-dsRBD due to its high binding affinity.

References:
1. Friedman, R. C. et al., Genome Res. 2009, 19 (1), 92-105.
2. Stanczyk, J. et al., Arthrit. Rheum. 2008, 58 (4), 1001-1009.
3. Wang, Y. et al., Genet. 2008, 74 (4), 307-315.
4. Jinek, M. and Doudna, J. A., Nature 2009, 457 (7228), 405-412.
5. Tian, B. et al., Nat. Rev. Mol. Cell Biol. 2004, 5 (12), 1013-1023.
6. Wostenberg, C.et al., Biochem. 2010, 49, 10728-10736.
7. Wostenberg, C. et al., Biophys. J. 2010, 99, 248-256.
8. Sohn, S. Y. et al., Nat. Struct. Mol. Biol. 2007, 14 (9), 847-853.
9. Nanduri, S. et al., EMBO J. 1998, 17 (18), 5458-5465.

Keywords: miRNA, dsRBD, Microprocessor