Poster abstracts

Poster number 66 submitted by Kaycee Quarles

Pri-miRNAs: the imperfect targets of the “microprocessor”

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

Abstract:
Found in multicellular organisms and encoded by viruses (1,2), microRNAs (miRNAs) are ~22-nucleotide-long single-stranded non-coding RNAs that participate in various developmental and differentiation processes via post-transcriptional regulation of gene expression (3-5). Despite high interest in these RNAs, the molecular mechanism of miRNA maturation by the “Microprocessor” complex—consisting of the double-stranded RNA binding protein DGCR8 in complex with the RNase III enzyme Drosha—is poorly understood. Mechanistic proposals emphasize a role of primary-miRNA (pri-miRNA) structural heterogeneity in Microprocessor binding based on the variety of bulges and internal loops present in pri-miRNA stemloops. However, atomistic structural biology has not shed insight into these mechanistic proposals due to the lack of determined pri-miRNA structures.
To begin evaluating the structural features of pri-miRNAs in solution, structure mapping using SHAPE chemistry (6) was performed on a panel of miRNAs. SHAPE reactivities indicate that many of the small imperfections predicted by mFold do not deform the helix significantly enough for the SHAPE reagent to access, suggesting that the processing proteins may not be able to recognize these imperfections either. On the other hand, electrophoretic mobility shift assays performed on a model pri-miRNA containing native imperfections binding DGCR8 showed tighter binding in comparison to binding a perfect duplex of similar length. The results suggest that DGCR8 may bind the pri-miRNA tighter than the perfect duplex due to the presence of the flanking single-strands and terminal loop; however, the interactions of DGCR8 with these pri-miRNA features are poorly defined. Defining the thermodynamics of DGCR8 binding pri-miRNA and the extent to which DGCR8 can tolerate helix imperfections will provide a framework for how the Microprocessor selects miRNA targets for processing and positions the Drosha cut site within them.

References:
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Keywords: miRNA, Microprocessor, structure