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A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

Nehdi A, Perreault J, Beaudoin JD, Perreault JP - Nucleic Acids Res. (2007)

Bottom Line: As a result of this finding, the secondary structure of this ribozyme has been redrawn.The formation of the C19-G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs.Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

ABSTRACT
A bioinformatic covariation analysis of a collection of 119 novel variants of the antigenomic, self-cleaving hepatitis delta virus (HDV) RNA motif supported the formation of all of the Watson-Crick base pairs (bp) of the catalytic centre except the C19-G81 pair located at the bottom of the P2 stem. In fact, a novel Watson-Crick bp between C19 and G80 is suggested by the data. Both chemical and enzymatic probing demonstrated that initially the C19-G81 pair is formed in the ribozyme (Rz), but upon substrate (S) binding and the formation of the P1.1 pseudoknot C19 switches its base-pairing partner from G81 to G80. As a result of this finding, the secondary structure of this ribozyme has been redrawn. The formation of the C19-G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs. Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.

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Chemical and enzymatic probing of the bottom of the P2 stem. (A) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC24U,C25U,G40U,G41U (3, 4) and the RzA78U,A79U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. (B) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C19G81G80) and the mutants RzC19,G81A,G80A, RzC19,G81AG80 and RzC19G81G80A, respectively. The positions of the C19 and C24 (used to establish the relative level of hydrolysis) are indicated on the right. (C) Histogram of the relative levels of RNase V1 hydrolysis of C19 for each ribozyme.
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Figure 4: Chemical and enzymatic probing of the bottom of the P2 stem. (A) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC24U,C25U,G40U,G41U (3, 4) and the RzA78U,A79U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. (B) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C19G81G80) and the mutants RzC19,G81A,G80A, RzC19,G81AG80 and RzC19G81G80A, respectively. The positions of the C19 and C24 (used to establish the relative level of hydrolysis) are indicated on the right. (C) Histogram of the relative levels of RNase V1 hydrolysis of C19 for each ribozyme.

Mentions: In-line probing is a method that relies on the fact that there is a natural rate of spontaneous cleavage within RNAs (16). Cleavage occurs when a phosphodiester linkage is subjected to an internal nucleophilic attack by the 2′ oxygen adjacent to and in-line with it. Structured regions of RNA, such as those in the base-paired stems, are less susceptible to spontaneous cleavage than are non-structured regions (17). In-line probing experiments were performed with the trans-acting version of the HDV ribozyme for which the kinetic behaviour has been extensively characterized under both single- and multiple-turnover conditions (18). The use of a trans-acting version permits the monitoring of the binding of the substrate to the ribozyme, a step which has been shown to be essential for many conformational transitions to take place (5,19,20). Probing experiments were performed using a trace amount (<1 nM) of 32P 5′-end-labelled ribozyme in either the presence, or the absence, of an excess of uncleavable substrate (SdA4; [S] ≫ [Rz]). The use of a substrate analogue that includes a deoxyriboadenine adjacent to the cleavage site prevents the cleavage from occurring. In the absence of SdA4, RNA degradation was observed in all single-stranded regions, including the nucleotides of both the P1 region (positions 33–39) and the J1/4 junction (G40 and G41) that are not base paired under these conditions (Figure 4A, lane 1; see also Supplementary Figure S1A). The nucleotides of the J4/2 junction, including G80, appear to be single-stranded. Because the residue G81 does not appear to be hydrolysed, this indicates that it is base paired with C19. Upon the addition of SdA4, several modifications in the banding pattern were observed (Figure 4A, lane 2; see also Supplementary Figure 1A). In general, the intensities of the majority of the bands were weaker, indicating that the presence of the substrate led to a more compact structure. Specifically, the nucleotides of the P1, P1.1 and P4 stems appeared to be less susceptible to hydrolysis, supporting the stacking of these stems into one helix (3). Moreover, the nucleotides of both the L3 loop and J4/2 junction were less hydrolysed, indicating the rearrangement of these single-stranded domains into a more compact catalytic core. However, the most obvious difference in the presence of the substrate was observed at the bottom of the P2 stem: the residue G81 became highly susceptible to the in-line attack, indicating that if it is indeed base paired in the absence of the substrate, it switches into a single-stranded conformation in its presence. Similar observations were made when the in-line probing was performed in the presence of the 3′ cleavage product that bound to the P1 region (data not shown). Moreover, terbium-mediated footprinting results also support the occurrence of a rearrangement of the bottom of the P2 stem (21).Figure 4.


A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

Nehdi A, Perreault J, Beaudoin JD, Perreault JP - Nucleic Acids Res. (2007)

Chemical and enzymatic probing of the bottom of the P2 stem. (A) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC24U,C25U,G40U,G41U (3, 4) and the RzA78U,A79U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. (B) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C19G81G80) and the mutants RzC19,G81A,G80A, RzC19,G81AG80 and RzC19G81G80A, respectively. The positions of the C19 and C24 (used to establish the relative level of hydrolysis) are indicated on the right. (C) Histogram of the relative levels of RNase V1 hydrolysis of C19 for each ribozyme.
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Figure 4: Chemical and enzymatic probing of the bottom of the P2 stem. (A) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC24U,C25U,G40U,G41U (3, 4) and the RzA78U,A79U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. (B) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C19G81G80) and the mutants RzC19,G81A,G80A, RzC19,G81AG80 and RzC19G81G80A, respectively. The positions of the C19 and C24 (used to establish the relative level of hydrolysis) are indicated on the right. (C) Histogram of the relative levels of RNase V1 hydrolysis of C19 for each ribozyme.
Mentions: In-line probing is a method that relies on the fact that there is a natural rate of spontaneous cleavage within RNAs (16). Cleavage occurs when a phosphodiester linkage is subjected to an internal nucleophilic attack by the 2′ oxygen adjacent to and in-line with it. Structured regions of RNA, such as those in the base-paired stems, are less susceptible to spontaneous cleavage than are non-structured regions (17). In-line probing experiments were performed with the trans-acting version of the HDV ribozyme for which the kinetic behaviour has been extensively characterized under both single- and multiple-turnover conditions (18). The use of a trans-acting version permits the monitoring of the binding of the substrate to the ribozyme, a step which has been shown to be essential for many conformational transitions to take place (5,19,20). Probing experiments were performed using a trace amount (<1 nM) of 32P 5′-end-labelled ribozyme in either the presence, or the absence, of an excess of uncleavable substrate (SdA4; [S] ≫ [Rz]). The use of a substrate analogue that includes a deoxyriboadenine adjacent to the cleavage site prevents the cleavage from occurring. In the absence of SdA4, RNA degradation was observed in all single-stranded regions, including the nucleotides of both the P1 region (positions 33–39) and the J1/4 junction (G40 and G41) that are not base paired under these conditions (Figure 4A, lane 1; see also Supplementary Figure S1A). The nucleotides of the J4/2 junction, including G80, appear to be single-stranded. Because the residue G81 does not appear to be hydrolysed, this indicates that it is base paired with C19. Upon the addition of SdA4, several modifications in the banding pattern were observed (Figure 4A, lane 2; see also Supplementary Figure 1A). In general, the intensities of the majority of the bands were weaker, indicating that the presence of the substrate led to a more compact structure. Specifically, the nucleotides of the P1, P1.1 and P4 stems appeared to be less susceptible to hydrolysis, supporting the stacking of these stems into one helix (3). Moreover, the nucleotides of both the L3 loop and J4/2 junction were less hydrolysed, indicating the rearrangement of these single-stranded domains into a more compact catalytic core. However, the most obvious difference in the presence of the substrate was observed at the bottom of the P2 stem: the residue G81 became highly susceptible to the in-line attack, indicating that if it is indeed base paired in the absence of the substrate, it switches into a single-stranded conformation in its presence. Similar observations were made when the in-line probing was performed in the presence of the 3′ cleavage product that bound to the P1 region (data not shown). Moreover, terbium-mediated footprinting results also support the occurrence of a rearrangement of the bottom of the P2 stem (21).Figure 4.

Bottom Line: As a result of this finding, the secondary structure of this ribozyme has been redrawn.The formation of the C19-G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs.Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

ABSTRACT
A bioinformatic covariation analysis of a collection of 119 novel variants of the antigenomic, self-cleaving hepatitis delta virus (HDV) RNA motif supported the formation of all of the Watson-Crick base pairs (bp) of the catalytic centre except the C19-G81 pair located at the bottom of the P2 stem. In fact, a novel Watson-Crick bp between C19 and G80 is suggested by the data. Both chemical and enzymatic probing demonstrated that initially the C19-G81 pair is formed in the ribozyme (Rz), but upon substrate (S) binding and the formation of the P1.1 pseudoknot C19 switches its base-pairing partner from G81 to G80. As a result of this finding, the secondary structure of this ribozyme has been redrawn. The formation of the C19-G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs. Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.

Show MeSH
Related in: MedlinePlus