Poster abstracts

Poster number 56 submitted by Grace Miner

NMR and thermodynamic studies of the human oncogenic microRNA, human miR-34c

Grace Miner (Department of Chemistry and Biochemistry, Miami University), Blanton S. Tolbert (Department of Chemistry and Biochemistry, Miami University)

Abstract:

MicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional regulators of gene expression. Initially, miRNAs are synthesized as long hairpin precursors that undergo a series of enzymatic processes prior to becoming biologically active. The heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) was recently shown to stimulate the processing of a subset of primary miRNAs by forming direct interactions with their apical loops and other non-canonical sites [3]. Using a database search (mirbase.org), we compiled a list of miRNAs that contain the hnRNP A1 high affinity UAG binding motif located in the apical loops. Of these, we selected the human miR-34c (34c) to test the hypothesis that its apical loop is a binding site for hnRNP A1. MiR-34c has been implicated in some cancers and neurodegenerative diseases; thus, knowledge of its interaction with protein partners may have broad biomedical implications. Using electrophoretic mobility shift assays, we found that the UP1 domain of hnRNP A1 forms a stable 1:1 complex with a truncated version of human 34c. To further characterize the interaction, calorimetric titrations were performed to determine the complete thermodynamic profile at 298 K (Kd=265 nM, ΔG^o= -8.92 kcal/mol, ΔH^o=-39.6 kcal/mol, and -TΔS^o=30.68 kcal/mol). As a step towards identifying the structural basis of UP1-34c complex stability, we used 2D NOE experiments to show that 34c folds into a stable hairpin conformation. Taken together, these data clearly show that hnRNP A1 binds to miR-34c and given the high affinity, this complex may have biological relevance.

References:
1. He, X., L. He, and G. J. Hannon.

Keywords: miRNA, hnRNP A1