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

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.

Figure 1

(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.

Figure 2

(A) Positions of the ELVd (+) hammerhead covered by two antisense oligodeoxyribonucleotides (in color). Other details as in the legend to Figure 1. (B) Self-cleavage during transcription of the ELVd (+) hammerhead in the presence and absence of the antisense oligodeoxyribonucleotides (lanes 1 and 2, respectively), and of the mutant ELVd-(+)-GUC hammerhead (in which the wild-type AUA trinucleotide preceding the self-cleavage site has been substituted by GUC) in the presence of the antisense oligodeoxyribonucleotides (lane 3). Lane 4, RNA markers of 65 and 55 nt. Aliquots of the reaction mixtures were analyzed by PAGE in 15% gels containing 8 M urea that were stained with ethidium bromide. Positions of the complete (C) primary transcript (60 nt) and of the resulting self-cleavage 3′ fragment (3′F) (51 nt) are indicated with arrows. The self-cleavage 5′ fragment (9 nt) migrated out of the gel.

Figure 3

Representative self-cleavage kinetics at 10 μM (A) and 50 μM (B) magnesium of the purified wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. The fraction of product at different times (F_t) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation F_t = F_∞ (1−e^−kt), where F_∞ is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (k_cat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F_∞ (between parenthesis) are shown below. The concentration of Mg was ∼1000-fold higher than that of hammerheads.

Figure 4

(A) Representative self-cleavage kinetics during transcription of the wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. Transcriptions (at pH 8 and 2 mM MgCl₂) were made in the absence of the antisense oligodeoxyribonucleotides. Reaction aliquots were removed at different time intervals, quenched with an excess of stop solution, separated by PAGE in 15% gels containing 8 M urea, and revealed and quantitated by a bioimage analyzer. Positions of the complete (C) primary transcript and of the resulting self-cleavage 3′ fragment (3′F), with their sizes in nt are indicated on the left. The smaller self-cleavage 5′ fragment (9 nt) has migrated out of the gel. (B) Quantitative data were fitted to the equation derived previously (22) by a least square method. The inset displays the first 1.5 min for each experiment.

Figure 5

Representative self-cleavage kinetics at 10 μM (A) and 50 μM (B) magnesium of the purified ELVd-(+)-GUC mutant hammerhead and of three other natural hammerheads with the trinucleotide GUC preceding the self-cleavage site. The fraction of product at different times (F_t) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation F_t = F_∞(1−e^−kt), where F_∞ is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (k_cat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F_∞ (between parenthesis) are shown below. ND, not detectable self-cleavage. The concentration of Mg was ∼1000-fold higher than that of hammerheads.

Figure 6

Model for the transient folding of four hammerheads with different trinucleotides preceding the self-cleavage site. During transcription, nucleotides forming part of the hammerhead catalytic core (in dark background) can be transiently involved in catalytically-inactive metastable structures. The higher stability of the structure with the trinucleotide AUC preceding the self-cleavage site can specifically delay the adoption of the catalytically-active hammerhead. In the scheme of the catalytically-active hammerhead (right) the pair X–X represents C–G or U–A, and the Y represents C or A.