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Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs.

Flores R, Serra P, Minoia S, Di Serio F, Navarro B - Front Microbiol (2012)

Bottom Line: As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers.Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae.Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC) Valencia, Spain.

ABSTRACT
As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunviroidae adopt multibranched conformations occasionally stabilized by kissing-loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

No MeSH data available.


Related in: MedlinePlus

Secondary structure and tridimensional model proposed for most natural hammerhead ribozymes. (A) Schematic representation of a typical hammerhead structure (that of the plus strand of PLMVd) as originally formulated. Residues strictly or highly conserved in most natural hammerheads are on a black background. Arrow marks the self-cleavage site and dashes indicate Watson–Crick (and wobble) base-pairs. (B) Schematic representation of the same hammerhead structure according to X-ray crystallography and NMR data. The proposed tertiary interaction between loops 1 and 2 that facilitates catalytic activity in vivo, is denoted with a gray oval. Dashes indicate Watson–Crick (and wobble) base-pairs and dots non-canonical interactions. (C) Detailed 3D model of this hammerhead structure showing the interactions between loops 1 and 2 (in magenta). Residues in yellow form a local element of higher-order structure (the uridine turn).
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Figure 6: Secondary structure and tridimensional model proposed for most natural hammerhead ribozymes. (A) Schematic representation of a typical hammerhead structure (that of the plus strand of PLMVd) as originally formulated. Residues strictly or highly conserved in most natural hammerheads are on a black background. Arrow marks the self-cleavage site and dashes indicate Watson–Crick (and wobble) base-pairs. (B) Schematic representation of the same hammerhead structure according to X-ray crystallography and NMR data. The proposed tertiary interaction between loops 1 and 2 that facilitates catalytic activity in vivo, is denoted with a gray oval. Dashes indicate Watson–Crick (and wobble) base-pairs and dots non-canonical interactions. (C) Detailed 3D model of this hammerhead structure showing the interactions between loops 1 and 2 (in magenta). Residues in yellow form a local element of higher-order structure (the uridine turn).

Mentions: As indicated above, the finding that members of the family Avsunviroidae are catalytic RNAs is one of the most striking features of viroids. The catalytic activity of these RNAs resides in the ability of their both polarity strands to fold into hammerhead structures, which facilitate splitting of a specific phosphodiester bond through a transesterification that produces 5′-hydroxyl and 2′,3′ cyclic phosphodiester termini (Hutchins et al., 1986; Prody et al., 1986). An account of the data supporting the functional role of hammerhead ribozymes in promoting self-excision into unit-length strands of the multimeric intermediates, generated in replication through a rolling-circle mechanism, has been presented previously (Flores et al., 2000, 2001). From a structural perspective, X-ray crystallography (and to a lower extent NMR, see below), have provided key insights on the actual shape of two of these ribozymes in their natural (full-length) version, and reconciled conflicting interpretations of previous structural and biochemical data obtained with minimal (incomplete) versions thereof. Rather than being composed of a central conserved core flanked by three double-stranded helices (I, II, and III) usually capped by short loops (1, 2, and 3), altogether looking like a hammerhead, the actual shape instead resembles a misshapen Y in which helices II and III are virtually colinear (Martick and Scott, 2006; Chi et al., 2008; Figures 6A,B). Importantly, these studies confirmed physical contacts between loops 1 and 2 predicted by in vitro and in vivo analyses of natural hammerheads showing that this tertiary interaction is critical for catalysis at the low physiological levels of Mg2+ (De la Peña et al., 2003; Khvorova et al., 2003), most likely because it facilitates sampling of the active conformation. Dissection by NMR spectroscopy, site-directed mutagenesis, and kinetic and infectivity analyses of another two natural hammerheads (those of CChMVd) has resulted in a detailed picture of the loop–loop contacts (Figure 6C), the key aspects of which – base-pairing between the pyrimidine at the 5′ side of loop 1 and the purine at the 3′ side of loop 2, and interaction of an extra-helical pyrimidine of loop 1 with loop 2 – appear to exist in other natural hammerheads as well (Dufour et al., 2009). Incorporation of these tertiary stabilization motifs has resulted in a second generation of hammerheads catalyzing trans-cleavage of specific RNAs at the low concentrations of Mg2+ existing in vivo (Carbonell et al., 2011 and references therein). Comparative studies on the effects of mutations in co- and post-transcriptional self-cleavage have revealed an additional feature of natural hammerheads: they have been evolutionarily selected to operate co-transcriptionally, in other words, concurrently with RNA folding during replication (Carbonell et al., 2006). This finding, together with the existence of alternative non-self-cleaving conformations that involve the nucleotides conserved in hammerheads (Hernández and Flores, 1992; Flores et al., 2000), explain why a fraction of the viroid molecules remains uncleaved and can thus serve as templates for replication through a rolling-circle mechanism. Analysis of ELVd RNAs expressed in chloroplasts of Chlamydomonas reinhardtii has shown that deletion mutants able to self-cleave efficiently display ligation defects, indicating that additional nucleotides – apart those forming the hammerhead – are involved in the conformation promoting ligation (Martínez et al., 2009). Moreover, this conformation should favor the physical proximity and positioning of the 5′-hydroxyl and 2′,3′ cyclic phosphodiester termini (resulting from self-cleavage) for their joining most likely catalyzed by a chloroplastic tRNA ligase (Nohales et al., 2012).


Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs.

Flores R, Serra P, Minoia S, Di Serio F, Navarro B - Front Microbiol (2012)

Secondary structure and tridimensional model proposed for most natural hammerhead ribozymes. (A) Schematic representation of a typical hammerhead structure (that of the plus strand of PLMVd) as originally formulated. Residues strictly or highly conserved in most natural hammerheads are on a black background. Arrow marks the self-cleavage site and dashes indicate Watson–Crick (and wobble) base-pairs. (B) Schematic representation of the same hammerhead structure according to X-ray crystallography and NMR data. The proposed tertiary interaction between loops 1 and 2 that facilitates catalytic activity in vivo, is denoted with a gray oval. Dashes indicate Watson–Crick (and wobble) base-pairs and dots non-canonical interactions. (C) Detailed 3D model of this hammerhead structure showing the interactions between loops 1 and 2 (in magenta). Residues in yellow form a local element of higher-order structure (the uridine turn).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Secondary structure and tridimensional model proposed for most natural hammerhead ribozymes. (A) Schematic representation of a typical hammerhead structure (that of the plus strand of PLMVd) as originally formulated. Residues strictly or highly conserved in most natural hammerheads are on a black background. Arrow marks the self-cleavage site and dashes indicate Watson–Crick (and wobble) base-pairs. (B) Schematic representation of the same hammerhead structure according to X-ray crystallography and NMR data. The proposed tertiary interaction between loops 1 and 2 that facilitates catalytic activity in vivo, is denoted with a gray oval. Dashes indicate Watson–Crick (and wobble) base-pairs and dots non-canonical interactions. (C) Detailed 3D model of this hammerhead structure showing the interactions between loops 1 and 2 (in magenta). Residues in yellow form a local element of higher-order structure (the uridine turn).
Mentions: As indicated above, the finding that members of the family Avsunviroidae are catalytic RNAs is one of the most striking features of viroids. The catalytic activity of these RNAs resides in the ability of their both polarity strands to fold into hammerhead structures, which facilitate splitting of a specific phosphodiester bond through a transesterification that produces 5′-hydroxyl and 2′,3′ cyclic phosphodiester termini (Hutchins et al., 1986; Prody et al., 1986). An account of the data supporting the functional role of hammerhead ribozymes in promoting self-excision into unit-length strands of the multimeric intermediates, generated in replication through a rolling-circle mechanism, has been presented previously (Flores et al., 2000, 2001). From a structural perspective, X-ray crystallography (and to a lower extent NMR, see below), have provided key insights on the actual shape of two of these ribozymes in their natural (full-length) version, and reconciled conflicting interpretations of previous structural and biochemical data obtained with minimal (incomplete) versions thereof. Rather than being composed of a central conserved core flanked by three double-stranded helices (I, II, and III) usually capped by short loops (1, 2, and 3), altogether looking like a hammerhead, the actual shape instead resembles a misshapen Y in which helices II and III are virtually colinear (Martick and Scott, 2006; Chi et al., 2008; Figures 6A,B). Importantly, these studies confirmed physical contacts between loops 1 and 2 predicted by in vitro and in vivo analyses of natural hammerheads showing that this tertiary interaction is critical for catalysis at the low physiological levels of Mg2+ (De la Peña et al., 2003; Khvorova et al., 2003), most likely because it facilitates sampling of the active conformation. Dissection by NMR spectroscopy, site-directed mutagenesis, and kinetic and infectivity analyses of another two natural hammerheads (those of CChMVd) has resulted in a detailed picture of the loop–loop contacts (Figure 6C), the key aspects of which – base-pairing between the pyrimidine at the 5′ side of loop 1 and the purine at the 3′ side of loop 2, and interaction of an extra-helical pyrimidine of loop 1 with loop 2 – appear to exist in other natural hammerheads as well (Dufour et al., 2009). Incorporation of these tertiary stabilization motifs has resulted in a second generation of hammerheads catalyzing trans-cleavage of specific RNAs at the low concentrations of Mg2+ existing in vivo (Carbonell et al., 2011 and references therein). Comparative studies on the effects of mutations in co- and post-transcriptional self-cleavage have revealed an additional feature of natural hammerheads: they have been evolutionarily selected to operate co-transcriptionally, in other words, concurrently with RNA folding during replication (Carbonell et al., 2006). This finding, together with the existence of alternative non-self-cleaving conformations that involve the nucleotides conserved in hammerheads (Hernández and Flores, 1992; Flores et al., 2000), explain why a fraction of the viroid molecules remains uncleaved and can thus serve as templates for replication through a rolling-circle mechanism. Analysis of ELVd RNAs expressed in chloroplasts of Chlamydomonas reinhardtii has shown that deletion mutants able to self-cleave efficiently display ligation defects, indicating that additional nucleotides – apart those forming the hammerhead – are involved in the conformation promoting ligation (Martínez et al., 2009). Moreover, this conformation should favor the physical proximity and positioning of the 5′-hydroxyl and 2′,3′ cyclic phosphodiester termini (resulting from self-cleavage) for their joining most likely catalyzed by a chloroplastic tRNA ligase (Nohales et al., 2012).

Bottom Line: As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers.Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae.Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC) Valencia, Spain.

ABSTRACT
As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunviroidae adopt multibranched conformations occasionally stabilized by kissing-loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

No MeSH data available.


Related in: MedlinePlus