Strong Correlation between SHAPE Chemistry and the Generalized NMR Order Parameter ( S 2 ) in RNA

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190

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used highthroughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cisacting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.RNA structure | evolution | motif discovery | functional validation H epatitis C virus (HCV) currently infects over 170 million people. There is no vaccine, and therapy, generally involving treatment with IFN and ribavirin, is often ineffective (1). Efficacious anti-HCV therapeutics are becoming available (2), but the extent to which they will mitigate the hepatitis C disease burden remains to be seen. Roughly 70% of acutely infected individuals fail to clear the virus and become lifelong HCV carriers, at risk for progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (3).HCV genomes are single-stranded, ∼9,650-nt, messengersense RNA molecules (4). The naked RNA initiates autonomous replication when transfected into cells and establishes chronic HCV infection in chimpanzees (5, 6). The HCV genomic RNA carries genetic information at two levels: a single large ORF encodes viral proteins and complex RNA structural elements regulate the viral replication cycle (4). Viral replication begins when conserved RNA elements in the 5′ UTR bind the 40S ribosome subunit and recruit essential translation factors (7). Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8-17).The HCV genomic RNA is physically compact (18) and highly structured (19). These features likely facilitate persistent HCV infections in humans by protecting the genome from degradation by innate antiviral defenses (20,21). Two elements of this defense are RNase L, which cleaves in single-stranded regions (22), and diverse double...

Section: Resultsmentioning

confidence: 99%

Functionally conserved architecture of hepatitis C virus RNA genomes

Mauger¹,

Golden

Yamane

et al. 2015

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128

190

“…1A) (33). SHAPE uses a chemical reaction at the RNA 2′-hydroxyl position to measure local nucleotide flexibility (34,35); flexibility can be reduced either by base pairing or by bound protein. We found that the two RNA strands in the dimer are held together by intermolecular base pairs in two palindromic stretches, termed PAL1 and PAL2, and by G-C base-pairing interactions in a highly conserved double stem-loop motif (SL1-SL2) (31,33,(36)(37)(38).…”

Section: Resultsmentioning

confidence: 99%

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

Gherghe

Lombo

Leonard

et al. 2010

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All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs-only four nucleotides per genomic RNA-reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.retrovirus | RNA recognition code | RNA SHAPE chemistry E xpression of a single viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. If present in the cell, the viral genomic RNA (vRNA) is selectively packaged into nascent particles; this selectivity is due to a cisacting packaging signal in the RNA, termed Ψ (1, 2). Remarkably, when no Ψ-containing RNA is present, Gag still assembles efficiently, encapsidating cellular mRNAs nonselectively in place of the vRNA (3-5).There are many indications that Ψ represents a high-affinity binding site for the Gag protein both in HIV-1 and in simpler retroviruses (6-14). However, the molecular mechanisms underlying selective encapsidation of vRNAs are incompletely understood, as are the features that enable Gag to bind preferentially to vRNA rather than to other cellular RNAs. Gag proteins contain several distinct domains, always including matrix (MA), capsid, and nucleocapsid (NC). vRNA packaging is mediated by the multidomain Gag protein, but Gag is cleaved following release of the virus from the cell. The NC domain plays a principal role in interactions with nucleic acids and is largely responsible for the specific interaction between Gag and its cognate viral RNA (12,13). This domain of Gag is highly basic and contains one or more "zinc knuckles" with a conserved spacing of Zn 2þ -coordinating cysteine and histidine residues. Mutations that abolish Zn 2þ coordination impair selective encapsidation of vRNA during virus assembly (6, 15). In addition, MA domains of many retroviral Gag proteins interact with nucleic acids (16-21) and may also contribute to specific i...

“…SHAPE reagents selectively form 2Ј-O-adducts at flexible nucleotides in RNA (26,27). Sites of 2Ј-O-adduct formation are identified as stops to primer extension using fluorescently labeled DNA primers, resolved by capillary electrophoresis.…”

Section: A130 Has Distinct Local Dynamics and A Critical Role In Rnase Pmentioning

confidence: 99%

C2′-endo nucleotides as molecular timers suggested by the folding of an RNA domain

Mortimer

Weeks

2009

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A striking and widespread observation is that higher-order folding for many RNAs is very slow, often requiring minutes. In some cases, slow folding reflects the need to disrupt stable, but incorrect, interactions. However, a molecular explanation for slow folding in most RNAs is unknown. The specificity domain of the Bacillus subtilis RNase P ribozyme undergoes a rate-limiting folding step on the minute time-scale. This RNA also contains a C2 -endo nucleotide at A130 that exhibits extremely slow local conformational dynamics. This nucleotide is evolutionarily conserved and essential for tRNA recognition by RNase P. Here we show that deleting this single nucleotide accelerates folding by an order of magnitude even though this mutation does not change the global fold of the RNA. These results demonstrate that formation of a single stacking interaction at a C2 -endo nucleotide comprises the rate-determining step for folding an entire 154 nucleotide RNA. C2 -endo nucleotides exhibit slow local dynamics in structures spanning isolated helices to complex tertiary interactions. Because the motif is both simple and ubiquitous, C2 -endo nucleotides may function as molecular timers in many RNA folding and ligand recognition reactions.RNA folding ͉ RNA SHAPE chemistry T o function properly inside the cell, RNA molecules undergo complex folding transitions to form specific, biologically active, three-dimensional structures (1). These essential structures involve both local base pairing and also complex higherorder tertiary interactions. A persistent and incompletely explained observation is that many RNAs fold very slowly, on timescales requiring minutes or longer. RNAs that fold slowly span all sizes including small riboswitch and catalytic RNAs, medium-sized catalytic introns, and the large RNAs in ribosomes (2-6).Slow folding has important consequences both because correct folding ultimately governs the rate at which an RNA can perform its biological function and because bottlenecks in assembly potentially require cellular mechanisms to overcome slow folding. In some cases, slow folding results from formation of stable, non-native, and kinetically trapped states (7,8). Such misfolding is remedied, in part, by cellular chaperone activities (9-11). In many cases, the molecular basis for slow folding is unknown.The intricate three-dimensional structures formed by RNA molecules ultimately reflect the underlying contributions of individual nucleotides. Most nucleotides in an RNA molecule exist in the C3Ј-endo conformation. Although less frequent, the C2Ј-endo conformation is highly overrepresented in catalytic active sites and in critical tertiary structures (12, 13). At the static structure level, the C2Ј-endo conformation induces greater helical twist in an A-form helix (14, 15) and spans a much longer 5Ј-to-3Ј distance (12,16). At the level of local motion, some C2Ј-endo nucleotides exhibit extraordinarily slow conformational dynamics with half-lives on the 10-100-s timescale (17).Here we show that the slow conformational ...