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Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads.

Carbonell A, De la Peña M, Flores R, Gago S - Nucleic Acids Res. (2006)

Bottom Line: Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (-) hammerhead.These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads.Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avenida de los Naranjos s/n, 46022 Valencia, Spain.

ABSTRACT
Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (+) and (-) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (-) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(+)-AUC approximately ELVd-(+)-GUC>ELVd-(+)-GUA> ELVd-(+)-AUA. However, the ELVd-(+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.

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(A) Rod-like secondary structure that the nucleotides involved in both hammerheads adopt in the predicted most stable conformation of the reference variant of ELVd (+) RNA (accession number AJ536613). Sequences forming the hammerheads are delimited by flags, motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. (B) Structure of the ELVd (+) and (−) hammerheads according to crystallographic data obtained for the Schistosoma mansoni hammerhead (16). Open square next to open triangle denotes Hoogsteen/sugar edge. Numbering of nucleotides and nomenclature of helices and loops is consistent with the standard criterion. Motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. The ovals represent the proposed tertiary interactions between loops 1 and 2 that increase the self-cleavage rates. The inset at the right displays the mutants derived from the ELVd (+) hammerhead used in the present work, with the changes introduced within boxes. (C) Structure of the (+) hammerheads of PLMVd, CChMVd and sTRSV.
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fig1: (A) Rod-like secondary structure that the nucleotides involved in both hammerheads adopt in the predicted most stable conformation of the reference variant of ELVd (+) RNA (accession number AJ536613). Sequences forming the hammerheads are delimited by flags, motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. (B) Structure of the ELVd (+) and (−) hammerheads according to crystallographic data obtained for the Schistosoma mansoni hammerhead (16). Open square next to open triangle denotes Hoogsteen/sugar edge. Numbering of nucleotides and nomenclature of helices and loops is consistent with the standard criterion. Motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. The ovals represent the proposed tertiary interactions between loops 1 and 2 that increase the self-cleavage rates. The inset at the right displays the mutants derived from the ELVd (+) hammerhead used in the present work, with the changes introduced within boxes. (C) Structure of the (+) hammerheads of PLMVd, CChMVd and sTRSV.

Mentions: The detailed morphology of both ELVd hammerheads has been reported previously (20) and, therefore, we will only summarize here their most salient features when compared with other natural hammerheads that have been used in this work. Both (+) and (−) ELVd strands contain the 11 residues that form the central conserved core of natural hammerheads (3), flanked by helix I/loop 1 (7 bp and 4 nt), helix II/loop 2 (4 bp and 4 nt forming a stable loop of the GNRA family) (23,24), and helix III (6 and 5 bp for + and − hammerhead, respectively) (Figure 1 and Table 1). This morphology is similar to that of both hammerheads of PLMVd (25), and to the (+) hammerheads of CChMVd (26) and sTRSV (2) (Figure 1), although helices I of ELVd hammerheads are the longest reported in natural hammerheads (3), with that of the (+) hammerhead being particularly stable (5/7 bp are G–C). The sequence heterogeneity observed in natural ELVd variants is consistent with the existence in helix I of both ELVd hammerheads of a base pair between positions 1.7 and 2.7, because a U–G pair is substituted by a U–A pair or viceversa (20). In the (+) ELVd hammerhead, positions 15.2 and 16.2 form a U–A pair instead of the predominant C–G pair, a situation also found in the (+) hammerhead of satellite RNA of cereal yellow dwarf virus RPV (sCYDV-RPV) (27) and in the (−) hammerhead of two cherry small circular RNAs (csc RNAs) (28,29). In these three hammerheads the residue preceding the self-cleavage site is A and not C (Table 1) as in most natural hammerheads, suggesting some relationship between the three positions (20,28,29). The two ELVd hammerheads are more related to each other than to other hammerheads, as also occurs between the hammerheads of other viroid and viroid-like RNAs (5,25,26,28,29).


Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads.

Carbonell A, De la Peña M, Flores R, Gago S - Nucleic Acids Res. (2006)

(A) Rod-like secondary structure that the nucleotides involved in both hammerheads adopt in the predicted most stable conformation of the reference variant of ELVd (+) RNA (accession number AJ536613). Sequences forming the hammerheads are delimited by flags, motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. (B) Structure of the ELVd (+) and (−) hammerheads according to crystallographic data obtained for the Schistosoma mansoni hammerhead (16). Open square next to open triangle denotes Hoogsteen/sugar edge. Numbering of nucleotides and nomenclature of helices and loops is consistent with the standard criterion. Motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. The ovals represent the proposed tertiary interactions between loops 1 and 2 that increase the self-cleavage rates. The inset at the right displays the mutants derived from the ELVd (+) hammerhead used in the present work, with the changes introduced within boxes. (C) Structure of the (+) hammerheads of PLMVd, CChMVd and sTRSV.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636495&req=5

fig1: (A) Rod-like secondary structure that the nucleotides involved in both hammerheads adopt in the predicted most stable conformation of the reference variant of ELVd (+) RNA (accession number AJ536613). Sequences forming the hammerheads are delimited by flags, motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. (B) Structure of the ELVd (+) and (−) hammerheads according to crystallographic data obtained for the Schistosoma mansoni hammerhead (16). Open square next to open triangle denotes Hoogsteen/sugar edge. Numbering of nucleotides and nomenclature of helices and loops is consistent with the standard criterion. Motifs conserved in most natural hammerheads are within boxes, and self-cleavage sites are marked by arrows. Black and white backgrounds refer to (+) and (−) polarities, respectively. The ovals represent the proposed tertiary interactions between loops 1 and 2 that increase the self-cleavage rates. The inset at the right displays the mutants derived from the ELVd (+) hammerhead used in the present work, with the changes introduced within boxes. (C) Structure of the (+) hammerheads of PLMVd, CChMVd and sTRSV.
Mentions: The detailed morphology of both ELVd hammerheads has been reported previously (20) and, therefore, we will only summarize here their most salient features when compared with other natural hammerheads that have been used in this work. Both (+) and (−) ELVd strands contain the 11 residues that form the central conserved core of natural hammerheads (3), flanked by helix I/loop 1 (7 bp and 4 nt), helix II/loop 2 (4 bp and 4 nt forming a stable loop of the GNRA family) (23,24), and helix III (6 and 5 bp for + and − hammerhead, respectively) (Figure 1 and Table 1). This morphology is similar to that of both hammerheads of PLMVd (25), and to the (+) hammerheads of CChMVd (26) and sTRSV (2) (Figure 1), although helices I of ELVd hammerheads are the longest reported in natural hammerheads (3), with that of the (+) hammerhead being particularly stable (5/7 bp are G–C). The sequence heterogeneity observed in natural ELVd variants is consistent with the existence in helix I of both ELVd hammerheads of a base pair between positions 1.7 and 2.7, because a U–G pair is substituted by a U–A pair or viceversa (20). In the (+) ELVd hammerhead, positions 15.2 and 16.2 form a U–A pair instead of the predominant C–G pair, a situation also found in the (+) hammerhead of satellite RNA of cereal yellow dwarf virus RPV (sCYDV-RPV) (27) and in the (−) hammerhead of two cherry small circular RNAs (csc RNAs) (28,29). In these three hammerheads the residue preceding the self-cleavage site is A and not C (Table 1) as in most natural hammerheads, suggesting some relationship between the three positions (20,28,29). The two ELVd hammerheads are more related to each other than to other hammerheads, as also occurs between the hammerheads of other viroid and viroid-like RNAs (5,25,26,28,29).

Bottom Line: Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (-) hammerhead.These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads.Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Avenida de los Naranjos s/n, 46022 Valencia, Spain.

ABSTRACT
Eggplant latent viroid (ELVd) can form stable hammerhead structures in its (+) and (-) strands. These ribozymes have the longest helices I reported in natural hammerheads, with that of the ELVd (+) hammerhead being particularly stable (5/7 bp are G-C). Moreover, the trinucleotide preceding the self-cleavage site of this hammerhead is AUA, which together with GUA also found in some natural hammerheads, deviate from the GUC present in most natural hammerheads including the ELVd (-) hammerhead. When the AUA trinucleotide preceding the self-cleavage site of the ELVd (+) hammerhead was substituted by GUA and GUC, as well as by AUC (essentially absent in natural hammerheads), the values of the self-cleavage rate constants at low magnesium of the purified hammerheads were: ELVd-(+)-AUC approximately ELVd-(+)-GUC>ELVd-(+)-GUA> ELVd-(+)-AUA. However, the ELVd-(+)-AUC hammerhead was the catalytically less efficient during in vitro transcription, most likely because of the transient adoption of catalytically-inactive metastable structures. These results suggest that natural hammerheads have been evolutionary selected to function co-transcriptionally, and provide a model explaining the lack of trinucleotide AUC preceding the self-cleavage site of most natural hammerheads. Comparisons with other natural hammerheads showed that the ELVd-(+)-GUC and ELVd-(+)-AUC hammerheads are the catalytically most active in a post-transcriptional context with low magnesium.

Show MeSH