RNA functions as the central conduit for information transfer in biology - in simple replicating entities like RNA viruses and in complex multi-cellular organisms. RNA is uniquely able to play this role because it encodes information at two levels: in its linear primary sequence and in its ability to form functionally critical higher-order folds. Work to date, largely focused on intensive study of a few specific regulatory motifs, has revealed that RNA secondary and tertiary structures regulate splicing, translation and protein folding, binding by small ligands and drugs and proteins, and collapse into large-scale structural domains. There are only a small number of examples in which genome-scale RNA structures have been characterized. However, numerous new biological insights were uncovered in each case. RNA viruses are especially informative systems because their compact genomes feature a dense array of functionally important secondary and tertiary structure elements. Moreover, every identification of a new regulatory motif in a pathogenic virus presents a unique target for anti-virus therapeutic design. Guided by several years of preliminary and exploratory studies, we are poised to make very high- throughput and high-content RNA secondary and tertiary structure analysis straightforward. We will apply newly invented massively parallel secondary and tertiary structure constraint-generation technologies coupled with novel molecular dynamics-driven structural refinement to understand the biological roles of higher-order structure in the hepatitis C virus (HCV) RNA genome.
Our Specific Aims are designed to reveal numerous new roles for RNA structure in the replication cycle of HCV, to do so in a way likely to inform many fields of biology, to make possible new therapeutic strategies for inhibiting viral replication, and to create tools that can be widely adopted by non-expert laboratories for analysis of complex, biologically authentic RNAs.
Aim 1 : Analyze the structure of three representative HCV RNA genomes using SHAPE, detected by massively parallel sequencing, to identify conserved base pairing and tertiary structure motifs.
Aim 2 : Create and validate an accurate and scalable approach for using experimental base pairing and through-space tertiary constraints to drive three-dimensional fold refinement for large RNAs.
Aim 3 : Integrate the technologies developed in this work to refine three-dimensional structure models and to discover new regulatory motifs for plus-sense HCV RNA genomes.

Public Health Relevance

The hepatitis C virus (HCV) is a major worldwide health threat that causes fatal liver diseases and leads to two-thirds of all liver transplants and more than 50% of all liver cancers. This work will determine structures for complete HCV RNA genomes using innovative technologies created in the two collaborating project laboratories. This work is significant because it will create tools that can be widely adopted by non-expert laboratories for analysis of complex, biologically authentic RNAs;will reveal numerous new roles for RNA structure in HCV pathogenesis;and will identify novel frameworks for designing new antiviral therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM064803-10S1
Application #
8927816
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Sakalian, Michael
Project Start
2002-05-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
10
Fiscal Year
2014
Total Cost
$50,000
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Smola, Matthew J; Christy, Thomas W; Inoue, Kaoru et al. (2016) SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells. Proc Natl Acad Sci U S A 113:10322-7
Smola, Matthew J; Calabrese, J Mauro; Weeks, Kevin M (2015) Detection of RNA-Protein Interactions in Living Cells with SHAPE. Biochemistry 54:6867-75
Lu, Yi-Fan; Mauger, David M; Goldstein, David B et al. (2015) IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance. Sci Rep 5:16037
Weeks, Kevin M (2015) RNA clubs. RNA 21:760-1
Weeks, Kevin M (2015) Review toward all RNA structures, concisely. Biopolymers 103:438-48
Tyrrell, Jillian; Weeks, Kevin M; Pielak, Gary J (2015) Challenge of mimicking the influences of the cellular environment on RNA structure by PEG-induced macromolecular crowding. Biochemistry 54:6447-53
Kutchko, Katrina M; Sanders, Wes; Ziehr, Ben et al. (2015) Multiple conformations are a conserved and regulatory feature of the RB1 5' UTR. RNA 21:1274-85
Smola, Matthew J; Rice, Greggory M; Busan, Steven et al. (2015) Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10:1643-69
Krokhotin, Andrey; Houlihan, Kevin; Dokholyan, Nikolay V (2015) iFoldRNA v2: folding RNA with constraints. Bioinformatics 31:2891-3

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