Abstract
Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges.
Highlights
Cis-acting sequences within the (+) strand RNA genomes of retroviruses and long terminal repeat (LTR) containing retrotransposons control several critical events in their life cycle, including transcription [1], translation [2], dimerization [3], packaging [4], RNA export [5], and DNA synthesis [6]
The structure of the MusD pseudoknot was likewise confirmed by ai-SHAPE, while a genetic analysis indicated that the ability to assume a pseudoknot configuration was a more critical determinant of function than absolute nucleotide sequence
An inconclusive acylation pattern in our recent SHAPE study of the 5 UTR of the feline immunodeficiency virus (FIV) genome [31] led us to postulation that certain regions were metastable, allowing them to adopt alternative structures, a notion strengthened by the observation of two closely-migrating RNA species following fractionation by nondenaturing polyacrylamide gel electrophoresis
Summary
Cis-acting sequences within the (+) strand RNA genomes of retroviruses and long terminal repeat (LTR) containing retrotransposons control several critical events in their life cycle, including transcription [1], translation [2], dimerization [3], packaging [4], RNA export [5], and DNA synthesis [6]. In almost all instances, enzymatic and chemical RNA footprinting has been performed on short RNAs prepared by in vitro transcription and labeled with 32P, eliminating any positional context, that is, regulatory roles that might be mediated by long-range, tertiary interactions This challenge has in part been addressed by Paillart et al via ex vivo footprinting of virion-associated RNA with dimethyl sulfate [24], a more “user-friendly” approach capable of providing information on RNA structure both in vitro and ex virio, and with fewer base-specific reagents, would clearly be advantageous. Future challenges of SHAPE, including increasing sensitivity where the amount of biological material is limiting, and studying interconverting RNA structures, are discussed
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