Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
![Distribution of double-helical RNA segments in the STMV virion. The crystal structure of STMV [4] reveals 30 segments of double- helical RNA (blue). Each helix contains 9 base pairs, centered on a crystallographic two-fold axis. An icosahedral cage (pink) is shown for reference. Adopted from [8]. doi:10.1371/journal.pone.0054384.g001](profile/Nicholas-Hud/publication/235372228/figure/fig1/AS:299870159163399@1448506025983/Distribution-of-double-helical-RNA-segments-in-the-STMV-virion-The-crystal-structure-of.png)
Distribution of double-helical RNA segments in the STMV virion. The crystal structure of STMV [4] reveals 30 segments of double- helical RNA (blue). Each helix contains 9 base pairs, centered on a crystallographic two-fold axis. An icosahedral cage (pink) is shown for reference. Adopted from [8]. doi:10.1371/journal.pone.0054384.g001
Source publication
Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a pu...
Context in source publication
Context 1
... tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded, positive-sense RNA genome, 1058 nucleotides in length. A capsid of 60 identical protein subunits surrounds the genome in the STMV particle. Like other satellite viruses, STMV encodes its own capsid protein but requires a helper virus for replication. For a review on the general properties of STMV, see Dodds [1]. STMV has been studied extensively as a model for the assembly of other single-stranded RNA viruses [2], and as a vector for the delivery of foreign genes into tobacco plants [3]. Efforts to characterize the RNA and its role in assembly have produced mixed results. The virus crystal structure has been solved at 1.8 A ̊ resolution [4], although some of the protein and 41% of the RNA are not visible in the electron density maps. The RNA that is visible is revealed as 30 double-helical segments, each 9 base pairs in length and closely associated with dimers of coat protein ( Figure 1). The helical axes are perpendicular to the icosahedral 2- fold axes, forming part of the edges of an icosahedron. With this constraint on the structure, Larson and McPherson proposed that the RNA forms a series of stem-loop substructures, with only short-range (local) base pairing. They suggested that coat proteins bind to successive stem-loops as these form upon emerging from the replication complex [5]. The results of atomic force microscopy (AFM) experiments are consistent with this hypothesis [6]. Schroeder et al. used chemical probing to examine the RNA structure inside the virus. They combined these data with the assumption of co-replicational folding to produce an ensemble of models for the secondary structure [7]. Each of these contains a series of 30 stem loops, with local base pairing; it is important to emphasize that the absence of long-range base pairs is an assumption built into the model, not a hypothesis that was tested by the chemical probing. They reported a single ‘‘most representative’’ secondary structure from that ensemble. We recently used that secondary structure to develop an all-atom model for the mature virus [8], containing every single amino acid and every single nucleotide. (We believe this is the first such model for any virus.). The capsid-free form of STMV RNA has been relatively overlooked in structural studies, in part because the secondary structure of the encapsidated RNA is believed to be different than the free RNA [5]. A tRNA-like structure (TLS) has been predicted at the 3 9 end of the molecule [9,10], but there is no evidence in the crystallographic data for or against its existence in the encapsidated RNA. A feature seen in AFM images of phenol extracted RNA could be interpreted as the predicted TLS [6], but Schroeder et al. have concluded that the TLS is not compatible with their chemical modification data [7]. Larson et al. have argued that, if the tRNA-like structure and replication recognition site structure were maintained inside the virus, there would be insufficient RNA remaining to connect the stem-loop segments [4]. Here we report a secondary structure model for in vitro transcribed STMV RNA, based on chemical probing data obtained using selective 2 9 -hydroxyl acylation analyzed by primer extension (SHAPE) [11]. SHAPE provides information on local nucleotide dynamics [12], thus reflecting the extent to which a nucleotide is constrained by base pairing or other interactions [13]. The SHAPE signal is highly correlated with Watson-Crick base pairing [14], and is capable of significantly improving the accuracy of RNA secondary structure predictions [13]. Our primary motivation for this work is to establish the secondary structure for the free STMV RNA, in the absence of the capsid protein. We also compare our probing data to the secondary structure proposed by Schroeder et al. for the RNA in virio , [7], and to the predicted tRNA-like structure at the 3 9 end of the RNA [9,10]. SHAPE [11] involves treating the RNA with an electrophilic reagent that reacts selectively at the ribose 2 9 -OH position of conformationally flexible nucleotides to form 2 9 - O -adducts. Reverse transcription using fluorescently labeled primers gives cDNA fragments whose lengths are determined by locations of ...
Citations
... Couplings near the center of Fig. 3A provide a valuable point of comparison between our measurements and previous structural studies. Three different SHAPE chemical probing studies [38][39][40] , as well as direct measurements using atomic force microscopy 39 and cryo-electron microscopy 44 , suggest that the central region of STMV RNA adopts a T-shaped domain containing three long-range connections: a 90-nt-long hairpin and a 270-nt-long hairpin branching from a 50-nt-long central duplex that connects regions over 470 nt apart (highlighted by ellipses in Fig. 3B). ...
... Visualizing these couplings on top of a structural model of the T-domain further demonstrates the good agreement between our patch-probe measurements and the consensus structure (Fig. 3C, right). 38 shows connections in ellipse 4; SHAPE probing of RNA extracted from virus particles by Archer et al. 39 shows connections in ellipse 5; DMS, kethoxal, and CMCT chemical probing and crystallographic analysis of RNA packaged in virus particles by Schroeder et al. 41 shows connections in ellipse 6 and 7. See Extended Data Fig. 6 for dominant couplings corresponding to 24-nt probes and arc plot representations of the data. ...
... Here there is less consensus. Previous chemical probing studies by Athavale et al. 38 , Archer et al. 39 , and Schroeder et al. 41 show considerable differences in connectivity (Fig. 3D). Some of these may be due to differences in the chemical probing protocol or the source of the RNA, and others might reflect differences in the folding models used to interpret the data. ...
We describe a simple method to infer intramolecular connections in a population of long RNA molecules in vitro. First we add DNA oligonucleotide 'patches' that perturb the RNA connections, then we use a microarray containing a complete set of DNA oligonucleotide 'probes' to record where perturbations occur. The pattern of perturbations reveals couplings between different regions of the RNA sequence, from which we infer connections as well as their prevalences in the population. We validate this patch-probe method using the 1,058-nucleotide RNA genome of satellite tobacco mosaic virus (STMV), which has previously been shown to have multiple long-range connections. Our results not only indicate long duplexes that agree with previous structures but also reveal the prevalence of competing connections. Together, these results suggest that globally-folded and locally-folded structures coexist in solution. We show that the prevalence of connections changes when pseudouridine, an important component of natural and synthetic RNA molecules, is substituted for uridine in STMV RNA.
... to study viral genomes in a variety of both simplified and complex biologically relevant states. These states include RNA transcribed in vitro and refolded (referred to as in vitro RNA) (62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72), RNA gently extracted from virus particles (ex virion RNA) (37,58,64,(73)(74)(75)(76)(77)(78)(79)(80)(81) or from infected cells (cell-free RNA) (78), RNA in native virus particles (in virion RNA) (58,64,74,80), RNA in infected cell lysates (81), and RNA in infected cells (72,74) (Figure 2a). Comparisons of SHAPE reactivity profiles obtained for viral RNA molecules probed in different biological states reveal state-specific RNA conformations (31,58,64,72,74,(80)(81)(82)(83)(84)(85), and sites occupied by RNA-binding proteins (58,64,72,(80)(81)(82)(83) or small molecules (86,87) (Figure 2c). ...
... SHAPE structure probing has also been used to study several plant satellite viral genomes, which require coinfection with a helper virus to replicate (62,77). The 1,058-nucleotide STMV genome was investigated independently by two groups, yielding in vitro and ex virion SHAPEdirected genome structure models, with outstanding agreement between studies (62,77,80). ...
... SHAPE structure probing has also been used to study several plant satellite viral genomes, which require coinfection with a helper virus to replicate (62,77). The 1,058-nucleotide STMV genome was investigated independently by two groups, yielding in vitro and ex virion SHAPEdirected genome structure models, with outstanding agreement between studies (62,77,80). The SHAPE-based STMV structure models reveal a multidomain architecture supported by AFM and cryo-electron microscopy studies (77, 103) (previously reviewed and illustrated in 52). ...
RNA viruses encode the information required to usurp cellular metabolism and gene regulation and to enable their own replication in two ways: in the linear sequence of their RNA genomes and in higher-order structures that form when the genomic RNA strand folds back on itself. Application of high-resolution SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) structure probing to viral RNA genomes has identified numerous new regulatory elements, defined new principles by which viral RNAs interact with the cellular host and evade host immune responses, and revealed relationships between virus evolution and RNA structure. This review summarizes our current understanding of genome structure-function interrelationships for RNA viruses, as informed by SHAPE structure probing, and outlines opportunities for future studies.
... The parallel development of computational methods for comparative and integrative analysis of probing data has made it possible to recover biological insights from data generated in these experiments (32,33). To date, large-scale structural information exists for three ssRNA positive sense viral genomes: hepatitis C virus (HCV) (34), human immunodeficiency virus (35), and tomato bushy stunt virus (TBSV) (36) as well as the satellite tobacco mosaic virus (37,38). By structurally characterizing large regions of viral genomes at once, potentially interesting structures can be discovered much more rapidly, and in the proper context. ...
... At the moment, large-scale RNA structural maps exist for three ssRNA (+)sense viral genomes: hepatitis C virus (34), human immunodeficiency virus (35), and tomato bushy stunt virus (36). In addition, the structure of satellite tobacco mosaic virus has also recently been reported (37,38). The identification of SEs within ORFs has also recently been explored in HCV using SHAPE reactivity data and covariation analyses. ...
In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3 untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites.
... 9 A fully independent SHAPE analysis performed using in vitro transcribed RNA proposed a closely related model, also featuring long-range interactions. 10 These SHAPE-directed models were further supported by direct visualization of the genomic RNA using atomic force microscopy 9 and cryoelectron microscopy. 11 In contrast, multiple models have been proposed for the conformation of the in virio RNA that have emphasized short helices, without long-range base-pairing interactions, based on computational-only secondary structure modeling. ...
... This is a key finding as early models proposed only local stem loop structures, 4,12,14,22 but current models specifically support these long-range interactions. [9][10][11] We discuss our predicted secondary structures largely in terms of probability arcs, as these best represent the structural variability of some regions and their contrast to the single structures observed in other regions. In order to facilitate conventional visualization of the two conformations, we also constructed traditional minimum free energy secondary structure models from the SHAPE data (Figure 4; Supplemental Figure 1). ...
The RNA genomes of viruses likely undergo multiple functionally important conformational changes during their replication cycles, changes that are poorly understood at present. We used two complementary in-solution RNA structure probing strategies (SHAPE-MaP and RING-MaP) to examine the structure of the RNA genome of satellite tobacco mosaic virus inside authentic virions and in a capsid-free state. Both RNA states feature similar three-domain architectures in which each major replicative function - translation, capsid coding, and genome synthesis - fall into distinct domains. There are, however, large conformational differences between the in-virion and capsid-free states, primarily in one arm of the central T domain. These data support a model in which the packaged capsid-bound RNA is constrained in a local high-energy conformation by the native capsid shell. The removal of the viral capsid then allows the RNA genome to relax into a more thermodynamically stable conformation. These data support a model in which the RNA architecture of the central T domain changes during capsid assembly and disassembly and may play a role in genome packaging.
... Using SHAPE analysis, long-range secondary basepairing was observed, with a region delimited by nt positions 169À646 being reminiscent of viroid RNA. A tRNA-like 3 0 -end structure was also observed, emphasizing its role in replication and RNA stability-possibly preventing degradation or abrogating silencing, akin to viroids (Athavale et al., 2013). Using cryo-electron microscopy and SHAPE analyses, the viroid-like secondary structure has been realized in solution (Garmann et al., 2015). ...
Satellite viruses (SVs) are subviral pathogens that are entirely dependent upon the replication machinery of their helper viruses. There are only four known plant SVs: satellite tobacco necrosis virus, satellite tobacco mosaic virus, satellite panicum mosaic virus, and satellite maize white line mosaic virus. These SVs have positive-sense single-stranded RNA genomes of 800-1400. nt that are encapsidated within ~17-nm. T=1 icosahedral virions. SVs, in contrast to satellite-RNA or -DNA, encode a capsid protein for independent genome packaging of the cognate SV RNA. The unusually small and compact nature of these subviral agents has resulted in their use as models for fundamental virology studies, including gene expression, genome packaging, and virion structure.
... Furthermore, a vast number of important pathogenic viruses including HIV, SARS coronavirus, poliovirus, Dengue fever virus, and many others utilize long RNAs as genetic material, which also play structural roles during virus assembly and genome packaging (13)(14)(15)(16)(17)(18)(19)(20). Previous studies have established the importance of local secondary and three-dimensional structure in the biological function of RNA (21,22). However, the effects of the secondary structure on the large-scale properties (e.g., size) of long RNAs remain poorly understood, even while its importance for virus assembly has been demonstrated (13)(14)(15)(16)(17)(18)(19)(20). ...
... in bp units (42,46). The MLD is estimated from RNA secondary structure predictions and can be further refined using structure probing experiments (21). Because there is heterogeneity among the many structures whose energies lie within a thermally available range (k B T), we use the Boltzmann-averaged MLD (denoted hMLDi), derived from an ensemble of RNA structures generated by prediction algorithms implemented in RNAfold (48). ...
... However, there are several notable discrepancies between the predicted and measured sizes. One limitation of our approach is that computational predictions may yield an incorrect structure and hence an MLD that differs from that of the experimentally determined secondary structure, as in the case of STMV RNA (21,55). Such failures of the computational approach are more likely (Table 1) and coloring is according to the class (black, single-stranded precursors of dsRNA viral genomes; red, genomes of ssRNA viruses; blue, cellular mRNAs; green, ribosomal RNA; and cyan, long noncoding RNAs. ...
Long RNA molecules are at the core of gene regulation across all kingdoms of life, whilst also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with non-repeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. In order to test whether a branched polymer model can estimate the overall sizes of large RNAs we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long non-coding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation – one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of “maximum ladder distance”. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low-ionic-strength sizes. These results suggest that sizes of long RNAmolecules are determined by the branching pattern of their secondary structures. They also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs.
... Such strategies have been observed in different classes of RNA viruses and play different roles in virus propagation (Nicholson and White, 2014). In fact, the entire genomes of some RNA viruses are highly structured and have complex global architecture (Athavale et al., 2013;Wu et al., 2013). ...
ELife digest
Flaviviruses include a large family of viruses that are harmful to human health, such as dengue virus, West Nile virus and Zika virus. Understanding the details of the life cycle of these viruses is important for better controlling and treating the diseases that they cause.
The genetic information of flaviviruses is stored in single-stranded molecules of RNA. To form new copies of a virus, the RNA must be replicated in a process that involves two critical steps. First, an enzyme called viral RNA polymerase NS5 must be recruited to a specific end of the RNA strand (known as the 5′ end). Then, the ends of the RNA strand bind together to form a circular loop. However, little is known about whether these two processes are linked, or how they are regulated.
Using bioinformatics, biochemical and reverse genetics approaches, Liu et al. have now identified a new section of RNA in the 5′ end of the flavivirus RNA, named the 5′-UAR-flanking stem (or UFS for short), which is critical for viral replication. The UFS plays an important role in efficiently recruiting the NS5 viral RNA polymerase to the 5′ end of the flavivirus RNA.
After the RNA forms a circle, the UFS unwinds. This makes the NS5 polymerase less likely to bind to the 5′ end of the RNA. Stabilizing the structure of the UFS impairs the ability of the RNA strand to form a circle, and hence reduces the ability of the RNA to replicate. Thus, the UFS links and enables communication between the processes that form the flavivirus RNA into a circle and that recruit the viral RNA polymerase to the RNA.
The structural basis of the interaction between the flavivirus RNA 5′ end, including the UFS element, and the viral RNA polymerase now deserves further investigation. It will be also important to explore whether other types of viruses regulate their replication via a similar mechanism.
DOI: http://dx.doi.org/10.7554/eLife.17636.002
... The Mg 2+ dependence of SHAPE reactivities is consistent with the formation of a Mg 2+ microcluster15 in both isolated Domain III and DIII core . We have previously determined the Mg 2+ dependence of SHAPE reactivity on a variety of RNAs, including a viral genome,29 Domain III of the LSU,16 the central core of the small ribosomal subunit, 3 the P4P6 domain of the Tetrahymena ribozyme,30 and the 23S rRNA (unpublished). The results show that SHAPE is a useful probe of binding of Mg 2+ to RNA and a suitable assay for formation of tertiary interactions. ...
In a model describing the origin and evolution of the translation system, ribosomal RNA (rRNA) grew in size by accretion [Petrov et al., (2015) "History of the Ribosome and the Origin of Translation", Proc. Natl. Acad. Sci. U.S.A. 112, 15396-15401]. Large rRNAs were built up by iterative incorporation and encasement of small folded RNAs. In this model, rRNA robustness in folding arises from inherited autonomy of local folding. We propose that rRNAs can be decomposed at various granularities, retaining folding mechanism and folding competence. To test these predictions, we disassembled Domain III from the large ribosomal subunit (LSU). We determined whether local rRNA structure, stability and folding pathways are autonomous. Thermal melting, chemical footprinting and circular dichroism were used to infer rules that govern folding of rRNA. We deconstructed Domain III of the LSU rRNA by mapping out its complex multi-step melting pathway. We studied Domain III and two equal-size 'sub-Domains' of Domain III. The combined results are consistent with a model in which melting transitions of Domain III are conserved upon cleavage into sub-Domains. Each of the eight melting transitions of Domain III corresponds in Tm and ΔH with a transition observed in one of the two isolated sub-Domains. The results support a model in which structure, stability and folding mechanisms are dominated by local interactions, and are unaffected by separation of the sub-Domains. Domain III rRNA is distinct from RNAs that form long-range cooperative interaction networks at early stages of folding or that do not fold reversibly.
... For circular satRNAs, the structures and catalytic activities of their self-cleaving ribozymes have been extensively characterized (5) and global RNA structure-function analyses of the satRNA of Cereal yellow dwarf virus allowed for the identification of domains corresponding to promoters, ribozymes, and a packaging signal (6). In linear satRNAs and satRNA viruses, promoters for synthesis of minus-and plus-strands are located at terminal positions (4), while the packaging signals for satRNA viruses can exist as multiple dispersed secondary structures (7)(8)(9)(10)(11). Determinants of encapsidation in linear satRNAs have not yet been reported. ...
... Notably, studying the entire molecule, as opposed to fragments, is important because structural components may fold and/or function differently when positioned in a complete context. The global structure of the RNA of satellite Tobacco mosaic virus (STMV) has been studied by both atomic force microscopy (9,32) and cryo-electron microscopy (33), and the results are consistent with biochemical data suggesting a branched configuration (9,10). Secondary structure models based on chemical probing data of full-length RNAs have also been generated for STMV (9)(10)(11), however, biological activity has been reported for only a few substructures (8). ...
... The global structure of the RNA of satellite Tobacco mosaic virus (STMV) has been studied by both atomic force microscopy (9,32) and cryo-electron microscopy (33), and the results are consistent with biochemical data suggesting a branched configuration (9,10). Secondary structure models based on chemical probing data of full-length RNAs have also been generated for STMV (9)(10)(11), however, biological activity has been reported for only a few substructures (8). Similarly, complete structure models based on solution probing have been reported for satC of TCV and satRNA of Cucumber mosaic virus (CMV satRNA), but not all of the predicted substructures have been assessed for biological relevance (24,34). ...
Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand
RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these
proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences
and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons,
and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for
a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified
throughout the length of the satRNA. Notably, a 3′-terminal segment was found to adopt two mutually-exclusive RNA secondary
structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely
function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction
with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small
parasitic RNA and provide a valuable structural framework for detailed mechanistic studies.
... SV-AUC and analytical SEC allow one to monitor global compaction of RNA preparations in the presence of divalent ions, under equilibrium conditions (Cole, Lary, Moody, & Laue, 2008;Mitra, 2014), and the equipment required is commonly available. Chemical probing facilitates the determination of lncRNA secondary structure (Athavale et al., 2013;Novikova, Hennelly, & Sanbonmatsu, 2012), and it also utilizes reagents that are available to most investigators. In this review, we describe protocols for selective 2 0 -hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing, as they have been applied for studying lncRNAs (Novikova et al., 2012;Watts et al., 2009). ...
The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation-renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2'-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.
© 2015 Elsevier Inc. All rights reserved.
