Structural and functional characterization of nuclease-resistant viral RNAs
Biochemistry
Final Report Abstract
Folded viral RNA elements (called xrRNAs) that block cellular exonucleases to produce biologically active degradation products have been well studied in mosquito-borne flaviviruses (e.g., Dengue, West Nile and Zika viruses), yet it remained unknown whether RNA-structure based exonuclease-resistance could be found elsewhere in biology. As a DFG-funded postdoctoral fellow at the University of Colorado School of Medicine, I identified a new class of xrRNAs in unrelated plant-infecting RNA viruses. I solved the structure of several members of this new xrRNA class using x-ray crystallography, revealing a complex fold that is very different from that of the previously described xrRNAs. Using single-molecule FRET studies, I uncovered a unique dynamic folding pathway that co-opts the exonuclease’s helicase activity for codegradational remodeling of the RNA structure. Through computational tools and biochemical assays, I showed that members of this new xrRNA class pervade 2 large families of plantinfecting RNA viruses (Tombusviridae and Luteoviridae), demonstrating the importance and widespread utility of this RNA element. With a graduate student I supervised, I furthermore discovered the smallest flavivirus xrRNA element to date; the structure of this “minimal” xrRNA shines light on xrRNA evolution. Together, my work uncovered the widespread use of viral RNA structures that inhibit the cellular RNA degradation machinery, established xrRNAs as an authentic functional class of structured RNA, and highlighted the diversity of RNA sequences that can fold into nuclease-resistant structures.
Publications
- "A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure." Proc Natl Acad Sci USA 2018 Jun 19;115(25):6404-6409
Steckelberg AL, Akiyama BM, Costantino DA, Sit TL, Nix JC, Kieft JS
(See online at https://doi.org/10.1073/pnas.1802429115) - “Exoribonuclease-Resistant RNAs Exist within both Coding and Noncoding Subgenomic RNAs.” mBio. 2018 Dec 18;9(6):e02461-18
Steckelberg AL, Vicens Q, Kieft JS
(See online at https://doi.org/10.1128/mbio.02461-18) - “Different tertiary interactions create the same important 3-D features in a distinct flavivirus xrRNA.” RNA 2020 Oct 1;rna.077065.120
Jones RA, Steckelberg AL, Vicens Q, Szucs MJ, Akiyama BM, Kieft JS
(See online at https://doi.org/10.1261/rna.077065.120) - “The crystal structure of a Polerovirus exoribonuclease-resistant RNA shows how diverse sequences are integrated into a conserved fold.” RNA 2020 Aug 26;rna.076224.120
Steckelberg AL, Vicens Q, Costantino DA, Nix JC, Kieft JS
(See online at https://doi.org/10.1261/rna.076224.120)
