Ubiquitous presence of the hammerhead ribozyme motif along the tree of life

Abstract

Examples of small self-cleaving RNAs embedded in noncoding regions already have been found to be involved in the control of gene expression, although their origin remains uncertain. In this work, we show the widespread occurrence of the hammerhead ribozyme (HHR) motif among genomes from the Bacteria, Chromalveolata, Plantae, and Metazoa kingdoms. Intergenic HHRs were detected in three different bacterial genomes, whereas metagenomic data from Galapagos Islands showed the occurrence of similar ribozymes that could be regarded as direct relics from the RNA world. Among eukaryotes, HHRs were detected in the genomes of three water molds as well as 20 plant species, ranging from unicellular algae to vascular plants. These HHRs were very similar to those previously described in small RNA plant pathogens and, in some cases, appeared as close tandem repetitions. A parallel situation of tandemly repeated HHR motifs was also detected in the genomes of lower metazoans from cnidarians to invertebrates, with special emphasis among hematophagous and parasitic organisms. Altogether, these findings unveil the HHR as a widespread motif in DNA genomes, which would be involved in new forms of retrotransposable elements.

FIGURE 1.

Small sequence strings of 22–32 nt were used for the HHR motif searches. (A) Schematic representation of seeds used for the bioinformatic searches. The conserved sequence motifs among all seeds (black boxes) corresponded to the region containing the U-turn (U Box) and a purine-rich motif (P Box). (B) Schematic representation of the I/II-type (left) and II/III-type (right) HHRs considered in this study, sharing Helix II, U, and P boxes (in black color). Consensus self-cleavage site (RUH box), Helix I, and Helix III domains not included in the searches, but required to form a catalytically active RNA, are depicted in gray color. Purine and pyrimidine nucleotides involved in most naturally occurring loop1–loop2 interactions are indicated as P and Y, respectively.

FIGURE 2.

Bacterial genomic data showing the presence of HHRs. (A) The HHR motifs obtained in the plant-associated bacteria Azorhizobium caulinodans, (left) and Gluconobacter oxydans (right). Positions of the ribozymes within each genome are shown in red with an arrow. The hypothetical ORFs coded in the sense and antisense strands are shown in red and blue, respectively. Nucleotides involved in the conserved loop–loop interaction are underlined. (B) The HHR motifs detected within the genome of the Desulfotomaculum reducens bacteria. Kinetic analysis of the in vitro self-cleavage capabilities of this ribozyme is shown at the right. (C) Some examples of HHR motifs detected from the Global Ocean metagenomics project (Rusch et al. 2007). A case showing a U insertion at the HHR catalytic core is shown in the right inset.

FIGURE 3.

HHRs in Chromalveolata and Plantae genomic data. (A) The HHR motifs detected in sequences of water molds from the Chromalveolata kingdom (Phytophtora infestans, Phytophtora sojae, and Hyaloperonospora parasitica). Some of the intraspecies nucleotide variability is highlighted with arrows. (B) Examples of genomic HHRs found in the algae Chlamydomonas reindhartii. The number of CAG repetitions is shown by subscripts. Monomer units are indicated by arrows. (C) HHR found in the club moss Selaginella moellendorfii associated with telomeric DNA sequences. (D) II/III-type HHR motifs found in ESTs from Nicotiana tabacum and Artemisia annua plants. (E) Examples of I/II-type HHR motifs found in sequences from grape vine Vitis vinifera (left). Sequence heterogeneity within HHRs is highlighted with boxed nucleotides) or the parasitic witchweed Striga asiatica (right). (F) Evidences of in vivo activity for HHR motifs mapping at the 5′ end of several plant ESTs.

FIGURE 4.

Examples of II/III-type HHR motifs associated to repetitive and telomeric DNA in metazoans. Examples of metazoan II/III-type HHR motifs detected in (A) Cnidaria, (B) Rotifera, (C) Annelida, and (D) Arthropoda species. Natural HHR heterogeneity for each entry is highlighted with boxed nucleotides.