Abstract
Viral RNAs were selected by evolution to possess maximum functionality in a minimal sequence. Depending on the classification of the virus and the type of RNA in question, viral RNAs must alternately be replicated, spliced, transcribed, transported from the nucleus into the cytoplasm, translated and/or packaged into nascent virions, and in most cases, provide the sequence and structural determinants to facilitate these processes. One consequence of this compact multifunctionality is that viral RNA structures can be exquisitely complex, often involving intermolecular interactions with RNA or protein, intramolecular interactions between sequence segments separated by several thousands of nucleotides, or specialized motifs such as pseudoknots or kissing loops. The fluidity of viral RNA structure can also present a challenge when attempting to characterize it, as genomic RNAs especially are likely to sample numerous conformations at various stages of the virus life cycle. Here we review advances in chemoenzymatic structure probing that have made it possible to address such challenges with respect to cis-acting elements, full-length viral genomes and long non-coding RNAs that play a major role in regulating viral gene expression.
Highlights
As a consequence of constant improvements in screening technology (Connelly et al, 2017), recent years have witnessed a resurgence in efforts to target RNA with small molecules, evidenced by the discovery of ligands capable of stimulating exon skipping (Luo and Disney, 2014) and others directed against a bacterial riboswitch (Howe et al, 2015), micro RNA linked to hepatocellular carcinoma (Childs-Disney and Disney, 2016) and RNA repeats associated with spinocerebellar ataxia type 10 (Yang et al, 2016)
The resulting structural model was later refined by SHAPE and mutational profiling (SHAPEMaP), a more sensitive, higher throughput variant of the technique that exploits the power of generation sequencing (Siegfried et al, 2014)
In the context of long RNAs, discrete structural motifs such as hairpins, kissing loops and pseudoknots can be verified and analyzed by antisense-interfered SHAPE, a technique in which short oligonucleotides are hybridized to part of a putative motif while effects of this hybridization on the remainder of the motif are characterized by SHAPE (Legiewicz et al, 2010)
Summary
As a consequence of constant improvements in screening technology (Connelly et al, 2017), recent years have witnessed a resurgence in efforts to target RNA with small molecules, evidenced by the discovery of ligands capable of stimulating exon skipping (Luo and Disney, 2014) and others directed against a bacterial riboswitch (Howe et al, 2015), micro RNA (miRNA) linked to hepatocellular carcinoma (Childs-Disney and Disney, 2016) and RNA repeats associated with spinocerebellar ataxia type 10 (Yang et al, 2016). The capacity of a regulatory RNA to assume alternative configurations in the absence of protein factors presents a unique challenge in SHAPE-based structural studies, since a mixed population is being sampled This problem can be solved by using native gel electrophoresis to fractionate RNA conformers, which can be probed separately in situ, extracted, and subjected to subsequent processing as in conventional CE-SHAPE. In the context of long RNAs, discrete structural motifs such as hairpins, kissing loops and pseudoknots can be verified and analyzed by antisense-interfered SHAPE (aiSHAPE), a technique in which short oligonucleotides are hybridized to part of a putative motif while effects of this hybridization on the remainder of the motif are characterized by SHAPE (Legiewicz et al, 2010) These technologies, together with structural features of select viral RNA elements they were used to characterize, are reviewed here
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