Poster abstracts
Poster number 58 submitted by Cade Harkner
Identification and characterization of proteins that interact with the oncomiR-1 NPSL2 hairpin which may regulate miR-92a biogenesis through directing RNA conformational change
Cade T. Harkner (Department of Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109, USA), Sarah C. Keane (Department of Chemistry and Program in Biophysics, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109, USA)
Abstract:
MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. To maintain proper microRNA expression levels, the enzymatic processing of primary (pri-) and precursor (pre-) microRNA elements must be strictly controlled. However, the molecular determinants underlying this strict regulation of microRNA biogenesis are not fully understood. RNA-binding proteins (RBPs) play important regulatory functions in the microRNA biogenesis pathway. We sought to identify proteins that could modulate RNA structures within the oncogenic polycistronic microRNA cluster miR-17~92a (oncomiR-1). The 3ʹ-domain of oncomiR-1 contains a four-way junction which is comprised of pri-miR-19b, pri-miR-92a, the non-precursor stem-loop element NPSL2, and helix50, a short helix linking back to the 5ʹ-domain. In the 3ʹ-domain, formation of the NPSL2 stem-loop structure sequesters the Drosha cleavage sites for pri-miR-92a within the junction, preventing formation of a pri-miR-92a basal helix, a structure essential for Drosha binding and processing. However, if the NPSL2 stem loop were unfolded and base paired with downstream sequence elements, the pri-miR-92a Drosha cleavage sites would be liberated and a basal helix could form. This structural rearrangement may function to regulate biogenesis of pre-miR-92a. We hypothesize that such rearrangement is protein-mediated, and thus performed proteomics-based screening to identify protein binding partners for the NPSL2 hairpin. Among our top hits are the DEAD-box ATPase DDX24 and hnRNPLL. In this work, we focused on hnRNPLL, a RBP with four RNA-recognition motifs (RRMs) in a 2x2 arrangement. We show that hnRNPLL does bind the NPSL2 stem-loop in vitro. We show that isolated dual RRMs bind NPSL2 with low affinity, but a construct containing all four RRMs shows moderate (low micromolar) binding. Follow-up work will investigate whether hnRNPLL can induce NPSL2 remodeling in vitro. This work continues to highlight the importance of RNA-protein interactions on the biogenesis of microRNAs, particularly from structurally complex polycistronic clusters.
Keywords: miRNA, hnRNP, RBPs