Phylogenetic footprinting of non-coding RNA: hammerhead ribozyme sequences in a satellite DNA family of Dolichopodacave crickets (Orthoptera, Rhaphidophoridae)

Abstract

BackgroundThe great variety in sequence, length, complexity, and abundance of satellite DNA has made it difficult to ascribe any function to this genome component. Recent studies have shown that satellite DNA can be transcribed and be involved in regulation of chromatin structure and gene expression. Some satellite DNAs, such as the pDo500 sequence family in Dolichopoda cave crickets, have a catalytic hammerhead (HH) ribozyme structure and activity embedded within each repeat.ResultsWe assessed the phylogenetic footprints of the HH ribozyme within the pDo500 sequences from 38 different populations representing 12 species of Dolichopoda. The HH region was significantly more conserved than the non-hammerhead (NHH) region of the pDo500 repeat. In addition, stems were more conserved than loops. In stems, several compensatory mutations were detected that maintain base pairing. The core region of the HH ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among 198 sequences. RNA folding of the HH sequences revealed that a potentially active HH ribozyme can be found in most of the Dolichopoda populations and species.ConclusionsThe phylogenetic footprints suggest that the HH region of the pDo500 sequence family is selected for function in Dolichopoda cave crickets. However, the functional role of HH ribozymes in eukaryotic organisms is unclear. The possible functions have been related to trans cleavage of an RNA target by a ribonucleoprotein and regulation of gene expression. Whether the HH ribozyme in Dolichopoda is involved in similar functions remains to be investigated. Future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead.

Conformational heterogeneity and the determinants of tertiary stabilization in the hammerhead ribozyme from Dolichopoda cave crickets

“…Similar substitutions at positions 2, 3 and 4 strongly reduced the observed rate, as did the A-to-G substitution at position 6. Each of these observations is consistent with the loosely-defined motif proposed previously (L 2 = RN n A), 25 and with the presence of adenosines at both these positions in five other Dolichopoda species, 40 including D. schiavazzi (L 2 = AGG GAA). In the schistosome and sTRSV HHRz, the terminal A forms a reverse Hoogsteen pair with IL 1 U1 or L 1 U1, respectively, 17 and it has been modeled as making similar interactions in the CChMVd hammerhead.…”

“…A recent study identified 135 additional HHRz sequences in repetitive DNA from 10 of 12 Dolichopoda species. Importantly, this study also presented evidence that the ribozyme nucleotides are more conserved than the flanking sequences and that they are under positive selection pressure to retain nucleolytic function, 40 again supporting a biological role for HHRz activity. When transcribed in vitro, the HHRz from D. baccettii displayed a higher rate of self-cleavage in vitro (0.01 min -1 in 5 mM Mg 2+ at 25°C) than did HHRz from other Dolichopoda species, 33 but these rates were 100-to 10,000-fold lower than what has been observed for other extended HHRz.…”

“…The pDo500 satellite DNA is a repetitive element comprising roughly 5% of the Dolichopoda cave cricket genome. 45 The HHRz within this element exhibits self-cleavage in adult crickets 33 and appears to be under positive selection to retain activity (non-random retention of base pairing potential and fewer inactivating mutations than expected by chance); 40 thus, it may be important for the normal biology of these insects. The fact that it is active in vivo also suggests that it could potentially be exploited as a tool for biotechnology and for artificial gene regulation.…”

“…IL 1 is positioned to interact with a hexanucleotide element, 5'-GGG GGA-3', within Loop II (L 2 ). D. baccettii and D. schiavazzii are closely related phylogenetically, 41 and their ribozyme sequences are nearly identical, 40 including the corresponding putative interacting elements, which in D. schiavazzii are 5'-UCC CUC U:UA UA-3' in IL 1 and 5'-AGGGAA-3' in L 2 (differences underlined). To test the significance of this juxtaposition, two ribozyme variants with different lengths of stem I were designed, denoted DbP2 (76 nt) (Dolichopoda baccettii Pst3 clone with 2 helical segments in stem I) and DbP1 (51 nt) ( Fig.…”

High‐pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self‐cleaving molecule

“…It was first discovered in subviral plant pathogens, viroids [1], and satellite RNAs [2], where it is involved in their replication through a rolling‐circle mechanism [3]. HHRs have subsequently been identified in the genomes of amphibians [4], schistosomes [5], cave crickets [6,7], plants [8], and mammals [9]. With a simple bioinformatic analysis, the ubiquitous presence of this ribozyme was detected among most kingdoms of life, suggesting a conserved role for these ribozymes in mRNA biogenesis, in the transfer of retrotransposable elements, and in the hypothesized ancient RNA world [10,11].…”