STRUCTURAL ANALYSIS OF RNA INTERACTIONS WITHIN THE MURINE MusD TRANSPORT ELEMENT USING NUCLEAR MAGNETIC RESONANCE
Cassiah J. Cox1, Stuart F.J. Le Grice2, and John P. Marino1
1University of Maryland, College Park, Institute of Bioscience and Biotechnology Research, Rockville, MD
1National Institute of Standards and Technology, Gaithersburg, MD
2National Cancer Institute-Frederick, Frederick, MD
In most retroviruses, a 3’ untranslated region of the viral genome functions as an RNA export element called the Constitutive Transport Element (CTE). The CTE has been shown to facilitate nuclear export via interactions with other viral and/or cellular factors. However, the mechanisms employed by retroviruses to export the unspliced genomic mRNA out of the nucleus are complex and remain unclear, creating a need for methods to characterize these systems. Endogenous retroviruses, such as the Type D murine LTR-retorotransposon (MusD) share ancestry and genomic organization with retroviruses and can provide valuable information about the replication and evolution of retroviruses. MusD contains an RNA export element with CTE-like activity similar to retroviruses. The secondary structure of the MusD RNA Transport Element (MTE) and two classes of possible tertiary interactions were reported by colleagues who used chemical probing strategies to characterize the MTE. The presumed interactions and structural motifs are believed to be essential to its function in mediating RNA transport. Although, chemical probing strategies have significantly advanced our ability to rapidly determine the secondary structure of large regulatory RNA elements, translating this information into an accurate three-dimensional RNA structure remains a difficult challenge. However, Nuclear Magnetic Resonance (NMR) structural analysis can provide unambiguous information that sheds light on how these elements structurally serve as recognition elements for cellular export machinery. Accordingly, we are utilizing high resolution NMR to measure the tertiary interactions predicted based on earlier chemical probing data reported: a kissing complex between hairpin loops IL8 and L3, and a pseudoknot. One-dimensional and two-dimensional NMR measurements collected on isolated RNA hairpins from the MTE, IL8 and L3, confirm the proposed kissing complex. Structurally, this interaction could be unique from others that have been characterized.