Catalyzing RNA

However, the discovery in 2004 of a ribozyme that could regulate genes in response to metabolic signals hinted at an expanded biological role for RNA catalysts, and reinvigorated the search for new RNA enzymes.² Surprisingly that ribozyme, located within an enzyme involved in bacterial cell wall synthesis, bound to both the enzyme’s product and to its transcript, cleaving the enzyme’s mRNA in a negative feedback loop.

Two years later, Jack Szostak and colleagues at the Howard Hughes Medical Institute in Boston,³ devised a clever selection approach to identify self-cleaving ribozymes in the human genome. They generated a library of single-stranded, closed circular genomic DNA fragments approximately 150 nucleotides long, and transcribed them in vitro. Then they purified sequences that could self-cleave, and that retained one copy of the intact ribozyme so that active sequences could be separated and used as a template in subsequent rounds of amplification. After 12 rounds, the authors cloned and sequenced a total of four naturally occurring human ribozymes, demonstrating that ribozymes were more common than initially believed.

In 2005, another approach was pioneered by Christian Hammann and colleagues of Kassel University in Germany, who took advantage of the greater homology in ribozyme shape than its sequence. He searched the EMBL sequence database for key residues in a motif that might predict a self-cleaving hammerhead ribozyme.⁴ Previously, no hammerhead ribozymes had been found in plants, so he searched the Arabidopsis thaliana genome, a common model organism, and identified two hammerhead ribozymes. They shared conserved flanking sequences, were apparently differentially transcribed in a variety of plant tissues, and were capable of self-cleavage, suggesting that they had genuine, but as yet unknown, biological functions in plants and were not merely remnants of viral genomic incorporation.

In 2008, Monika Martick at the University of California, Santa Cruz and colleagues expanded Hammann’s approach and searched mammalian mRNA sequence databases for hammerhead ribozymes with large sequence insertions in loop regions—a feature that confounds standard search algorithms.⁵ They found three hammerhead-like ribozymes in the mice lectin family transcripts, coding proteins required for normal bone physiology. Intriguingly, they also found homologs in other mammals—including rat, horse, and platypus—suggesting a conserved function. Cloning the ribozyme sequence into a reporter gene reduced its expression by 80%, suggesting that this ribozyme might reduce expression of genes involved in bone homeostasis.

Similarly, Andrej Lupták and colleagues the University of California, Irvine⁶ used a structural pattern resembling hepatitis delta virus (HDV) ribozymes to search sequenced genomes and identified a remarkable number of catalytically active HDV-like ribozymes in eukaryotic genomes across diverse phyla from insects to primitive vertebrates. Amazingly, most species contained more than one ribozyme or had multiple copies within their genome, most notably the nematodes: Pristionchus pacificus has 32 apparent HDV-like ribozymes, and Caenorhabditis japonicus has 122 apparent ribozymes. Some of these were found in or near predicted genes, and are expressed at different time points during development, again suggesting a genuine biological function.

However, important questions remain. In most cases, the biological function of ribozymes is unknown. Identifying new ribozymes in genetically tractable model organisms should be a priority, as this will make it possible to determine their function. The in vitro selection strategy and computational tools are now in place to quickly identify these ribozymes.

The existence of functional hammerhead ribozymes that have parts of their sequence dispersed throughout the genome suggests the intriguing possibility that functional ribozymes may be built from multiple RNA sequences. There are many noncoding regulatory RNAs found in all kingdoms of life. Perhaps some of them anneal to targets to form RNA enzymes, leading to self-cleavage. If so, they could possibly be the evolutionary precursor of modern small RNA silencing pathways. One thing is clear: RNA-guided RNA cleavage, whether catalyzed by protein or by RNA, is more than an evolutionary vestige and serves important functions in regulating gene expression. Precisely defining those functions is the next challenge.

Sean P. Ryder is an F1000 Faculty Member at the University of Massachusetts Medical School.

This article is an adaptation of an article published in F1000 Biology Reports, a publication of the Faculty of 1000. For the full-length version, click here. F1000 Biology consists of more than 2,000 leading biologists (Faculty Members) who select and review the most important published papers in their respective fields (Faculties). The next two pages describe recent selections from various Faculties.