STRUCTURAL AND FUNCTIONAL ANALYSIS OF NON CODING RNAS: MMLV READTHROUGH ELEMENTS AND HIV-1 PACKAGING SIGNAL.

Non-protein coding RNA (ncRNA) is transcribed from genomic DNA, but rather than becoming a template for protein synthesis, ncRNA's role is dependent on its catalytic activity, regulatory ability, or structural arrangement. ncRNAs were once thought to be evolutionary debris but are now frequently referred to as functional RNAs as they have been linked to many essential life processes. Unlike mRNA, the nucleotide sequences of ncRNA cannot immediately reveal their physiological function. Therefore, structure/function studies aimed at illuminating the molecular basis of action of ncRNAs are becoming increasingly important to the scientific community. Mass spectrometry (MS) is able to provide a universal platform for uncovering information on both structure and function of RNA. MS3D (Mass Spectrometry for 3-Dimensional Analysis) combines RNA structure probing with Fourier transform ion cyclotron resonance (FT-ICR) MS to investigate RNA structures. Solvent accessible chemical probes are utilized to uncover information regarding base pairing, stacking, and ligand binding, while bifunctional crosslinkers are used to reveal the positions of nucleobases in the global fold of the RNA molecule, providing a 2D protection map. Together with fundamental information pertaining to RNA structure, these constraints can be translated into 3D models of the target substrates. MS3D models of the Moloney murine leukemia virus (MMLV) readthrough elements, which may be part of a riboswitch, as well as the Human Immunodeficiency Virus (HIV)-1 cis packaging signal (Ψ-RNA) monomer, were obtained using the constraints provided by MS mapping and a combination of simulated annealing and energy minimization. Finally, MS-based technologies were applied to the functional studies of non-coding RNA. Site directed mutation revealed a critical GNRA (where R stands for a purine and N for any base) interaction between two domains of Ψ-RNA. Several approaches were used to determine functional aspects of the putative MMLV riboswitch, including diagnostic RNA crosslinking using mechlorethamine (nitrogen mustard), locked nucleic acid (LNA) probes, MS-amenable readthrough assays, and a MS-based assay for the identification of a putative riboswitch actuator