The role of dsRBDs in the miRNA maturation pathway

Open Access
Author:
Wostenberg, Christopher William
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
May 21, 2012
Committee Members:
  • Scott A Showalter, Dissertation Advisor
  • Scott A Showalter, Committee Chair
  • Philip C. Bevilacqua, Committee Member
  • David D Boehr, Committee Member
  • Andrey S Krasilnikov, Committee Member
Keywords:
  • dsRBD
  • miRNA
  • NMR
  • MD
  • DGCR8
  • Dicer
  • Drosha
  • IDP
  • FCP1
  • RAP74
Abstract:
Macromolecular interactions are involved in important biological processes like transcription, translation, signaling, and defense. In order to understand these biochemical processes it is vital to comprehend the mechanism of macromolecular interactions via two key characteristics: structure and dynamics. I chose to investigate fast dynamics on two systems which were already structurally characterized: the processing of microRNAs (miRNAs) precursors by dsRNA binding domains (dsRBDs) in humans and the intrinsically disordered protein FCP1 binding to RAP74, which is involved in termination of transcription and recycling of RNA polymerase II. miRNAs, a large class of small ssRNAs, are involved in gene regulation by base-pairing with messenger RNA (mRNA). Maturation of miRNAs occurs in two independent and spatially separated steps. In the nucleus, the ssRNA tail of primary miRNA is cleaved by the “microprocessor”, which is composed of a RNase III enzyme (Drosha) and its cofactor (DGCR8). Next, the precursor miRNA is exported to the cytosol, and the terminal loop is cleaved by another RNase III enzyme (Dicer), which is aided by a cofactor (either TRBP or PACT). Afterwards, the miRNA is loaded into the argonaute (Ago) protein generating the RNA-induced silencing complex (RISC). Maturation of miRNA is accomplished by total of ten dsRBDs over five proteins in the pathway. This dissertation specifically focuses on understanding the fast dynamics of four of them: DGCR8-dsRBD1, DGCR8-dsRBD2, Drosha-dsRBD and Dicer-dsRBD. The dsRBD is one of the most common RNA-binding motifs, found in all organisms and in both the cytoplasm and the nucleus. The dsRBD forms an αβββα topology, where the two α-helices lie on one face of three anti-parallel β-strands, with the other surface of the sheet being solvent exposed. The preferred binding partner of dsRBDs is A-form double-helix RNA, where a minimum of 11 base pairs of dsRNA interact with a dsRBD generally without sequence specific RNA interactions. A dynamic profile was obtained for the dsRBDs through MD simulations and NMR spin relaxation along with binding assays to determine functionality of the dsRBDs. To start, the two dsRBDs in tandem from DGCR8 were explored through MD simulations (chapter 2), motivated by the pre-organization observed in crystal and verified by preliminary NMR data. The results demonstrate that correlated motions impacting the conformation of the RNA-binding surface are mediated by a few interfacial interactions. Next, MD simulations in connection with NMR spin relaxation experiments were performed on Drosha-dsRBD, DGCR8-dsRBD1, and Dicer-dsRBD (chapter 3 and 4) revealing a dynamic profile where loop 2 is dynamic in all dsRBDS. Electrophoretic mobility shift assays (EMSAs) of the dsRBDs showed that isolated Drosha-dsRBD is the only dsRBD studied that does not bind dsRNA, which is attributed to Drosha-dsRBD having increased flexibility in loop 1. Together, the data illustrates that flexibility in loop 2 of the dsRBDs allows for binding of dsRNA, but flexibility in loop 1 hinders binding. To further study macromolecular interactions via MD simulations, the intrinsically disordered C-terminal domain of FCP1 bound to the winged helix domain of RAP74 was investigated. Unlike ordered protein complexes, which involve polar interactions over a small fraction of the protein surface, disordered proteins complexes like the RAP74-FCP1 complex involve large hydrophobic interactions. Additionally, FCP1 in the complex retains significant flexibility throughout the simulation.