Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
MS-based approaches for the structural determination of retroviral ribonucleoproteins
The interactions between the nucleocapsid (NC) domain of the Gag polyprotein and the 5'-untraslated region (5’-UTR) of viral RNA play multifaceted roles in the lifecycle of HIV-1. Owing to the well-known chaperone activity of NC, such interactions may induce remodeling of RNA structure, which results in either exposing or concealing RNA signals responsible for different viral functions. For this reason, mapping the sequences bound by NC must be followed by the characterization of the underlying RNA structure to fully understand the biological significance of the specific interactions.
We have developed complementary approaches based on mass spectrometry (MS), which enable the identification of protein binding sites and the investigation of their structural context. One of them involves the application of bifunctional alkylating reagents to form irreversible crosslinks between protein and RNA moieties that are placed within mutual striking distance by the ribonucleoprotein fold. The specific protein-RNA and RNA-RNA contacts are characterized by protease/nuclease digestion to isolate the conjugated products, followed by mass mapping and sequencing. The analysis of NC5’-UTR complexes in samples containing up to 10:1 protein to RNA ratios revealed the presence of six high-affinity sites, while a few others became detectable only after ionic strength was decreased to strengthen protein-RNA binding. The major sites mapped to the trans-activation response element (TAR), the primer binding site (PBS), the primer activation signal (PAS), and the packaging signal (Ψ-RNA).
In some instances, the RNA-RNA crosslinks detected in the same experiments revealed discrepancies with the leading secondary structures proposed for 5’-UTR, consistent with possible structure remodeling mediated by NC. The significance of these findings is currently being explored by utilizing a series of relatively short (i.e., 8-12 nt) antisense oligonucleotides that are complementary to the affected regions. Their specific binding is being monitored directly by MS and ion mobility spectrometry (IMS)-MS, which enables one to observe the effects of ligands on the overall topology and conformation of biomolecular complexes. The results are expected to provide the boundaries of the specific NC sites and to address possible long-range effects of binding on the entire fold of 5’-UTR. Further, this information will guide the design of mutants lacking determinant features necessary to foster NC binding. Supported by MS detection, the combination of crosslinking and antisense probing promises to offer the new insights necessary to understand the structural rearrangements involved with 5’-UTR processes, which could provide the keys for developing new antiviral strategies.
Acknowledgements: Research supported by The RNA Institute of UAlbany and the National Institutes of Health (GM064328-12).
The RNA Institute