Exploring RNA Flexibility Using Molecular Dynamics
Open Access
- Author:
- Penrod, Katheryn Anne
- Graduate Program:
- Chemistry
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 25, 2017
- Committee Members:
- Dr. Scott A. Showalter, Thesis Advisor/Co-Advisor
Dr. William G. Noid, Committee Member
Dr. Edward P. O'Brien, Committee Member
Dr. Paul S. Cremer, Committee Member
Dr. Costas D. Maranas, Committee Member - Keywords:
- RNA
Molecular Dynamics
MD
miRNA
microRNA - Abstract:
- Non-coding microRNAs (miRNAs) have been identified as powerful regulators of gene expression. Approximately 22 nucleotides in length, these RNAs are found in most eukaryotes, including humans. Through a process called RNA interference (RNAi), mature miRNA binds target mRNA molecules to accomplish gene silencing. Recently, miRNA has emerged as a potential therapeutic target for various disease states that have been linked to changes in miRNA expression. In humans, miRNA is produced in the form of a stem-loop pri-miRNA structure containing approximately 100 nucleotides. Through a series of binding and cleavage events collectively referred to as the miRNA maturation pathway, this primary miRNA (pri-miRNA) is converted to the single-stranded mature miRNA that participates in RNAi. The underlying molecular processes of the miRNA maturation pathway are not completely understood. Structural characterization of the proteins and nucleic acids along this pathway will contribute to a deeper understanding of miRNA that would be particularly beneficial in the pharmaceutical industry. Five proteins along the miRNA maturation pathway possess at least one double-stranded RNA binding domain (dsRBD) responsible for facilitating protein-RNA interactions. These evolutionarily-conserved domains form non-bonded interactions with the phosphodiester backbone and 2′-hydroxyl groups within the minor groove of dsRNA. Although the precise mechanism remains unclear, dsRBDs generally recognize their substrates in a shape-dependent and non sequence-specific manner. The TAR RNA binding protein (TRBP) is responsible for binding precursor miRNA (pre-miRNA) and presenting it to Dicer for cleavage. In a previous study, binding by TRBP was demonstrated to exclude sites of helical imperfections. The ubiquity of such imperfections in miRNA suggests that dsRBDs can sense these structural features in order ensure the proper orientation of their substrates for cleavage. The overarching aim of this work was to characterize the binding events along the miRNA maturation pathway with respect to RNA flexibility. Initially, we use circular dichroism (CD), isothermal titration calorimetry (ITC), and molecular dynamics (MD) simulations to investigate a simplified system containing TRBP-dsRBD2 and a perfect WC duplex of 20 GC base pairs. Strong protein-RNA contacts were observed in expected regions of the complex, supporting the previous notion that TRBP preferentially binds to perfect WC duplexes. Although cocrystal structures suggest that binding by TRBP and Xlrbpa-2 induces strong bends in coaxially-stacked GC10-mers, no large-scale conformational changes were detected in the TRBP / GC20 complex. We conclude that the bending observed in the cocrystal structures is most likely a result of the artificial double-stranded break between the oligomers. Having established a suitable method for performing and analyzing MD simulations of a simple dsRBD / dsRNA complex, we designed a more realistic system. We selected pri-mir-16-1 for this study based on the SHAPE-constrained MC-Pipeline structures previously determined by our laboratory. The three-dimensional structure model of pri-mir-16-1 reveals two deformable “hot spots.” The deformable region adjacent to the Drosha cut site was observed in other pri-miRNAs and was demonstrated to influence processing efficiency. A second region was identified for pri-mir-16-1 in the vicinity of its A37 / A76 mismatch and nearby U79 bulge. It has been suggested that structural distortions of this type promote DGCR8 binding by allowing the formation of strong axial bends. Toward complete characterization of this binding event, unbound pri-mir-16-1 was simulated using the same protocol as above. Preliminary results indicate that the procedures for trajectory analysis require modification to accommodate structural imperfections. Further investigation of this system will illuminate the effect of structural imperfections on the conformational flexibility of dsRNA and provide a reliable means of investigating similar interactions along the miRNA maturation pathway.