Hammerhead ribozymes are small self-cleaving RNAs that promote strand scission by internal phosphoester transfer. Comparative sequence analysis was used to identify numerous additional representatives of this ribozyme class than were previously known, including the first representatives in fungi and archaea. Moreover, we have uncovered the first natural examples of “type II” hammerheads, and our findings reveal that this permuted form occurs in bacteria as frequently as type I and III architectures. We also identified a commonly occurring pseudoknot that forms a tertiary interaction critical for high-speed ribozyme activity. Genomic contexts of many hammerhead ribozymes indicate that they perform biological functions different from their known role in generating unit-length RNA transcripts of multimeric viroid and satellite virus genomes. In rare instances, nucleotide variation occurs at positions within the catalytic core that are otherwise strictly conserved, suggesting that core mutations are occasionally tolerated or preferred.

Estimates of the total number of bacterial species1-3 suggest that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins4 and RNAs5. Bioinformatics searches6-10 of genomic DNA from bacteria commonly identify novel noncoding RNAs (ncRNAs)10-12 such as riboswitches13,14. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered15,16. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline17 to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, suggesting that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.

The hammerhead ribozyme is a small catalytic RNA motif capable of endonucleolytic (self-) cleavage. It is composed of a catalytic core of conserved nucleotides flanked by three helices, two of which form essential tertiary interactions for fast selfscission under physiological conditions. Originally discovered in subviral plant pathogens, its presence in several eukaryotic genomes has been reported since. More recently, this catalytic RNA motif has been shown to reside in a large number of genomes. We review the different approaches in discovering these new hammerhead ribozyme sequences and discuss possible biological functions of the genomic motifs.

The Y genes encode small noncoding RNAs whose functions remain elusive, whose numbers vary between species, and whose major property is to be bound by the Ro60 protein (or its ortholog in other species). To better understand the evolution of the Y gene family, we performed a homology search in 27 different genomes along with a structural search using Y RNA specific motifs. These searches confirmed that Y RNAs are well conserved in the animal kingdom and resulted in the detection of several new Y RNA genes, including the first Y RNAs in insects and a second Y RNA detected in Caenorhabditis elegans. Unexpectedly, Y5 genes were retrieved almost as frequently as Y1 and Y3 genes, and, consequently are not the result of a relatively recent apparition as is generally believed. Investigation of the organization of the Y genes demonstrated that the synteny was conserved among species. Interestingly, it revealed the presence of six putative "fossil" Y genes, all of which were Y4 and Y5 related. Sequence analysis led to inference of the ancestral sequences for all Y RNAs. In addition, the evolution of existing Y RNAs was deduced for many families, orders and classes. Moreover, a consensus sequence and secondary structure for each Y species was determined. Further evolutionary insight was obtained from the analysis of several thousand Y retropseudogenes among various species. Taken together, these results confirm the rich and diversified evolution history of Y RNAs.

We have investigated the secondary structure of peach latent mosaic viroid (PLMVd) in solution, and we present here the first description of the structure of a branched viroid in solution. Different PLMVd transcripts of plus polarity were produced by using the circularly permuted RNA method and the exploitation of RNA internal secondary structure to position the 5 and 3 termini and studied by nuclease mapping and binding shift assays using DNA and RNA oligonucleotides. We show that PLMVd folds into a complex, branched secondary structure. In general, this structure is similar to that reported previously, which was based on sequence comparison and computer modelling. The structural microheterogeneity is apparently limited to only some small domains. More importantly, this structure includes a novel pseudoknot that is conserved in all PLMVd isolates and seems to allow folding into a very compact form. This pseudoknot is also found in chrysanthemum chlorotic mottle viroid, suggesting that it is a unique feature of the viroid members of the PLMVd subgroup.Viroids are small (ϳ300 nucleotides), single-stranded, circular RNAs that infect higher plants, causing significant losses in the agricultural industry (see references 7 and 13 for reviews). Viroids have been classified in two groups (groups A and B) based primarily on whether or not they possess five typical structural domains found in the group B viroids (7). Further division among the group B members depends on the sequence and length of the conserved central region. Viroids that do not possess any kind of sequence or structural similarity with the group B viroids have been classified as belonging to group A. The viroids from this group possess self-cleaving hammerhead motifs that are crucial for their replication via a rolling circle mechanism.The group A viroids include the avocado sunblotch viroid (ASBVd), the peach latent mosaic viroid (PLMVd), and the chrysanthemum chlorotic mottle viroid (CChMVd) (10). Both PLMVd and CChMVd have been proposed to adopt branched secondary structures (Fig. 1A) instead of the rod-like ones proposed for most viroids, including ASBVd (10). The unusual conformations of PLMVd and CChMVd are supported by their insolubility in 2 M lithium chloride, whereas ASBVd and a number of non-self-cleaving viroids (i.e., the group B viroids) are soluble in this high salt solution (10). In general, secondary structures of viroids are predicted using computer software and are useful for the formulation of hypotheses on the structure-function relationships of these RNA molecules (4). Characterization of biological structures in vitro as well as in vivo is obviously more accurate for elucidating the structure-function relationship. The only secondary structure of a viroid that has been extensively studied in solution is that of the potato spindle tuber viroid (8). This group B species, which was shown to adopt a rod-like shape in solution, is responsible for most of our knowledge of the biology of viroids.In order to determine the secondary struct...