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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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RNase T1 mapping of the wild type and homopurine bp mutant ribozymes. (A) Autoradiogram of a 10% PAGE gel of T1 probing performed with 5′-end-labelled wild-type and mutated trans-acting ribozymes. Alkaline hydrolysis of the wild-type ribozyme was performed in order to determine the location of each position (lane OH). Lanes 1 and 2 are negative controls (no reaction and no substrate) performed with the wild-type and mutant RzG75C ribozymes, respectively. RNase T1 hydrolysis was performed on both the wild-type ribozyme (lane 3) and the RzG75C (lane 4) either in the absence or the presence of the SdA4 analogue as indicated by the symbols (−) and (+), respectively. The sites of RNase T1 hydrolyses are identified, and intensities of the hydrolyses correlate with the intensities of the arrow heads. The positions of the guanosines are indicated on the left. (B) to (D) are schematic representation of the nucleotide sequences and secondary structures of the ribozymes. (B) is the wild-type ribozyme. (C) and (D) are two secondary structures for the RzG75C mutant that differs for the J1/4 and J4/2 junctions.
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Figure 7: RNase T1 mapping of the wild type and homopurine bp mutant ribozymes. (A) Autoradiogram of a 10% PAGE gel of T1 probing performed with 5′-end-labelled wild-type and mutated trans-acting ribozymes. Alkaline hydrolysis of the wild-type ribozyme was performed in order to determine the location of each position (lane OH). Lanes 1 and 2 are negative controls (no reaction and no substrate) performed with the wild-type and mutant RzG75C ribozymes, respectively. RNase T1 hydrolysis was performed on both the wild-type ribozyme (lane 3) and the RzG75C (lane 4) either in the absence or the presence of the SdA4 analogue as indicated by the symbols (−) and (+), respectively. The sites of RNase T1 hydrolyses are identified, and intensities of the hydrolyses correlate with the intensities of the arrow heads. The positions of the guanosines are indicated on the left. (B) to (D) are schematic representation of the nucleotide sequences and secondary structures of the ribozymes. (B) is the wild-type ribozyme. (C) and (D) are two secondary structures for the RzG75C mutant that differs for the J1/4 and J4/2 junctions.

Mentions: In order to verify if the adoption of the homopurine bp is influenced by the binding of the substrate to the ribozyme, RNase T1 probing was performed. RNase T1 hydrolyses all guanosines located in single-stranded regions. In the ribozyme alone, the three consecutive guanosines, G40, G41 and G42, appeared as being single-stranded (Figure 7A). Upon addition of the SdA4 analogue these guanosines became inaccessible. The P1.1 pseudoknot, which includes G40 and G41, is formed in addition to the homopurine bp (Figure 7A). Thus, the binding of the substrate is required for the formation of these structural features. Several mutants were designed in order to probe the effect of homopurine bp formation on the secondary structure of the ribozyme, especially on the positioning of the catalytic C76. RNase T1 probing of a mutant ribozyme (RzG75C) including a G42–C75 Watson–Crick bp instead of a G42–G75 homopurine bp was performed (Figure 7A–C). This mutant is completely devoid of any cleavage activity (15). The resulting banding pattern showed several differences (Figure 7A, lanes 3 and 5), the most striking being at the level of the J1/4 junction: the three consecutives guanosines were not hydrolysed regardless of the presence or absence of the SdA4 analogue. This suggests that even in the absence of the substrate analogue, these residues were already engaged in the formation of base pairs. This observation received additional support from in-line probing data showing that the residues of both the J1/4 junction and the bottom of the J4/2 junction (i.e. C75, C76, U77 and A78) were not hydrolysed (data not shown). Together, these results suggest that the residues of these regions form a double-stranded structure that extends the P4 stem (Figure 7C and D). This suggests to us that a potential contribution of the homopurine bp might be to prevent the formation of such a non-productive structure (i.e. alternative inactive folding). The homopurine bp seems to interrupt an elongation of the P4 stem, and contributes to the conservation of the catalytic cytosine as a single-stranded residue. Results from mutagenesis of the homopurine bp have demonstrated that an A42A75 homopurine bp is more active than a G42G75 bp, as has been observed previously (15). The presence of an AA homopurine bp appears to limit the number of potential alternative structures that can be formed, which may explain the higher occurrence of A42A75, rather than G42G75, in the selection performed.Figure 7.


A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

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

RNase T1 mapping of the wild type and homopurine bp mutant ribozymes. (A) Autoradiogram of a 10% PAGE gel of T1 probing performed with 5′-end-labelled wild-type and mutated trans-acting ribozymes. Alkaline hydrolysis of the wild-type ribozyme was performed in order to determine the location of each position (lane OH). Lanes 1 and 2 are negative controls (no reaction and no substrate) performed with the wild-type and mutant RzG75C ribozymes, respectively. RNase T1 hydrolysis was performed on both the wild-type ribozyme (lane 3) and the RzG75C (lane 4) either in the absence or the presence of the SdA4 analogue as indicated by the symbols (−) and (+), respectively. The sites of RNase T1 hydrolyses are identified, and intensities of the hydrolyses correlate with the intensities of the arrow heads. The positions of the guanosines are indicated on the left. (B) to (D) are schematic representation of the nucleotide sequences and secondary structures of the ribozymes. (B) is the wild-type ribozyme. (C) and (D) are two secondary structures for the RzG75C mutant that differs for the J1/4 and J4/2 junctions.
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Related In: Results  -  Collection

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Figure 7: RNase T1 mapping of the wild type and homopurine bp mutant ribozymes. (A) Autoradiogram of a 10% PAGE gel of T1 probing performed with 5′-end-labelled wild-type and mutated trans-acting ribozymes. Alkaline hydrolysis of the wild-type ribozyme was performed in order to determine the location of each position (lane OH). Lanes 1 and 2 are negative controls (no reaction and no substrate) performed with the wild-type and mutant RzG75C ribozymes, respectively. RNase T1 hydrolysis was performed on both the wild-type ribozyme (lane 3) and the RzG75C (lane 4) either in the absence or the presence of the SdA4 analogue as indicated by the symbols (−) and (+), respectively. The sites of RNase T1 hydrolyses are identified, and intensities of the hydrolyses correlate with the intensities of the arrow heads. The positions of the guanosines are indicated on the left. (B) to (D) are schematic representation of the nucleotide sequences and secondary structures of the ribozymes. (B) is the wild-type ribozyme. (C) and (D) are two secondary structures for the RzG75C mutant that differs for the J1/4 and J4/2 junctions.
Mentions: In order to verify if the adoption of the homopurine bp is influenced by the binding of the substrate to the ribozyme, RNase T1 probing was performed. RNase T1 hydrolyses all guanosines located in single-stranded regions. In the ribozyme alone, the three consecutive guanosines, G40, G41 and G42, appeared as being single-stranded (Figure 7A). Upon addition of the SdA4 analogue these guanosines became inaccessible. The P1.1 pseudoknot, which includes G40 and G41, is formed in addition to the homopurine bp (Figure 7A). Thus, the binding of the substrate is required for the formation of these structural features. Several mutants were designed in order to probe the effect of homopurine bp formation on the secondary structure of the ribozyme, especially on the positioning of the catalytic C76. RNase T1 probing of a mutant ribozyme (RzG75C) including a G42–C75 Watson–Crick bp instead of a G42–G75 homopurine bp was performed (Figure 7A–C). This mutant is completely devoid of any cleavage activity (15). The resulting banding pattern showed several differences (Figure 7A, lanes 3 and 5), the most striking being at the level of the J1/4 junction: the three consecutives guanosines were not hydrolysed regardless of the presence or absence of the SdA4 analogue. This suggests that even in the absence of the substrate analogue, these residues were already engaged in the formation of base pairs. This observation received additional support from in-line probing data showing that the residues of both the J1/4 junction and the bottom of the J4/2 junction (i.e. C75, C76, U77 and A78) were not hydrolysed (data not shown). Together, these results suggest that the residues of these regions form a double-stranded structure that extends the P4 stem (Figure 7C and D). This suggests to us that a potential contribution of the homopurine bp might be to prevent the formation of such a non-productive structure (i.e. alternative inactive folding). The homopurine bp seems to interrupt an elongation of the P4 stem, and contributes to the conservation of the catalytic cytosine as a single-stranded residue. Results from mutagenesis of the homopurine bp have demonstrated that an A42A75 homopurine bp is more active than a G42G75 bp, as has been observed previously (15). The presence of an AA homopurine bp appears to limit the number of potential alternative structures that can be formed, which may explain the higher occurrence of A42A75, rather than G42G75, in the selection performed.Figure 7.

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