Schistosome Satellite DNA Encodes Active Hammerhead Ribozymes

Abstract

Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Sm␣ repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Sm␣ ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.Schistosomes are a family of digenetic trematodes that parasitize many animal species; three members of this group, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum, infect over 200 million people worldwide. These blood flukes possess a complex life cycle initiated by the release of eggs from a human host. Fluke eggs produce larvae, called miracidia, which infect snails, a secondary host. Snails in turn shed cercariae, another larval form, which are able to penetrate human skin, transform into schistosomula, and, after a complex migration and differentiation process, develop into sexual adults. Adults produce eggs to complete the cycle. Hopefully, the study of the genomic structure of these species could provide key information for the more effective control of these devastating parasites.Most eukaryotic genomes contain families of interspersed repetitive DNA called SINEs (37), the sequences of which are generally related to tRNAs or 7SL RNA (1,3,10,41). Their repetitive nature is thought to be due to an amplification process involving reverse transcription of RNA transcripts which are subsequently integrated into host DNA (21, 40). The species S. mansoni contains a family of SINEs, the 335-bp Sm␣ repeats, which occur over 10,000 times in the haploid genome. Many copies of this repeat are clustered on the W female chromosome, while others are dispersed throughout the genome (38). In spite of this ability to amplify themselves, no function has been ascribed to SINEs.Transcripts of the highly conserved family of satellite DNAs (Sat2) found in the newt do, however, possess a self-processing activity typical of the hammerhead domain found in plant viroids and their satellite RNAs (5,7,13,39). Tandem arrays of the Sat2 repeats are dispersed throughout the genome of Notophthalmus viridescens and other newt species. Their transcripts have tissue-specific 5Ј ends, suggesting that transcription and/or self-cleavage are regulated in vivo (14). Although suggestive, no cellular role has yet been assigned to these self-cleaving transcri...

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.…”

“…115 RNAMOT was used as a scaffold to develop additional structure-based search programs, and it and its successors have been repeatedly used to identify novel tRNAs, group I self-splicing introns, hammerhead ribozymes, and ligand-binding RNAs at new genomic loci. 108,111,116,117 Comparable searches for HDV-like ribozymes were not performed until after the first crystal structure was solved and the motif was identified outside of the virus in the mammalian CPEB3 gene.…”

Intronic hammerhead ribozymes in mRNA biogenesis

“…Motifs from the three groups share sequence and structural homology between them, but also with the HHRs previously described in the satellite DNA of trematodes (Ferbeyre et al , 1998 ;Martick et al , 2008 ; De la Pe ñ a and Garc í a-Robles, 2010a ), suggesting either a phylogenetic or convergence relationship among them.…”

“…Almost simultaneously, a type I HHR was reported in the satellite DNA of the newt genome (Epstein and Gall , 1987 ), which is transcribed in tandem repeats of ~ 330 nt (Epstein and Coats , 1991 ) that form part of a small ribonucleoprotein complex with a yet unknown function (Luzi et al , 1997 ). Similar to the amphibian motifs, other genomic HHRs have been found to reside in DNA tandem repeats of carnation plants (Daros and Flores , 1995 ), schistosomes (Ferbeyre et al , 1998 ) and cave crickets (Rojas et al , 2000 ), suggesting a similar role for these genomic HHRs in the biology of such tandem-repetitive DNA. More recently, different bioinformatic approaches have uncovered a widespread occurrence of the HHR motif among all life kingdoms (De la Pe ñ a and Garc í a- Robles, 2010a,b ;Jimenez et al , 2011 ;Perreault et al , 2011 ;Seehafer et al , 2011 ; for a review see Hammann et al , 2012 ).…”

Gene expression control: Harnessing RNA switches

“…Finding an appropriate self-cleaving ribozyme, and a place to embed it within the mRNA so that ribozyme activity results in substantial reduction of gene expression, is expected to be difficult, as Mulligan's team has now confirmed. Of more than 100 natural and engineered selfcleaving ribozymes tested, only two ribozymes (both of the hammerhead class 5,6 ) caused more than a 10% reduction in gene expression. A number of issues likely conspire to make the success rate less than 2%.…”