Functional Hammerhead Ribozymes Naturally Encoded in the Genome of Arabidopsis thaliana  

Abstract

The hammerhead ribozyme (HHRz) is an autocatalytic RNA motif found in subviral plant pathogens and transcripts of repetitive DNA sequences in animals. Here, we report the discovery and characterization of unique HHRzs encoded in a plant genome. Two novel sequences were identified on chromosome IV of Arabidopsis thaliana in a database search, which took into account recently defined structural requirements. The HHRzs are expressed in several tissues and coexist in vivo as both cleaved and noncleaved species. In vitro, both sequences cleave efficiently at physiological Mg 2þ concentrations, indicative of functional loop-loop interactions. Kinetic analysis of loop nucleotide variants was used to determine a threedimensional model of these tertiary interactions. Based on these results, on the lack of infectivity of hammerhead-carrying viroids in Arabidopsis, and on extensive sequence comparisons, we propose that the ribozyme sequences did not invade this plant by horizontal transfer but have evolved independently to perform a specific, yet unidentified, biological function.

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes

“…Ligation is the reverse of this reaction (26)(27)(28)(29)(30), and occurs if the cleavage product RNA is held in place by the structure of the ribozyme. The hammerhead (31,32), hepatitis delta virus (HDV) (33,34) and twister (12) …”

HDV Family of Self-Cleaving Ribozymes

“…2,3 Other ribozyme motifs, specifically hammerhead ribozymes, have been readily detected in numerous organisms following their initial identification. 1,5,6,[107][108][109][110] This contrast is likely due to several factors. First, the hammerhead motif is much less informationally complex than that of the HDV ribozymes.…”

Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

“…A few more catalytically active self-cleaving sequences have been found in humans 13 and plants 94 . Homology searches in various genomes have also identified several hammerheadlike motifs, the activities of which have not been demonstrated 95,96 .…”

“…A priority for future studies is to characterize the self-cleavage activities of recently identified genome-encoded ribozymes 13,94 , as well as candidate ribozyme and RNA sensor sequences 46,58,95,96,103 , that might reveal new aspects of genetic regulation by RNA elements. As a pivotal example, we cite the recent and long-awaited dissection of the functional role of eukaryotic riboswitches, which participate in gene expression control by alternative splicing of their pre-mRNAs 43 .…”

“…These ribozymes have been identified in only some mammalian species, and further study is needed to determine whether similar RNA elements exist, providing a more generally applicable mechanism for their function. By contrast, small self-cleaving ribozymes (hammerhead, hairpin, VS and HDV ribozymes) have specialized functions in replication, and are therefore unlikely to influence expression of the host genes specifically and directly.A few more catalytically active self-cleaving sequences have been found in humans 13 and plants 94 . Homology searches in various genomes have also identified several hammerheadlike motifs, the activities of which have not been demonstrated 95,96 .…”