The function of twister ribozyme variants in non-LTR retrotransposition in Schistosoma mansoni

Abstract

The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5′ end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5′ end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition.

Direct testing of natural twister ribozymes from over a thousand organisms reveals a broad tolerance for structural imperfections

“…Of these, however, only 14 (<1%) were experimentally validated (17), and subsequent HT mutational studies have focused on just two representative twister ribozymes (27,28,31). Since 2014, additional twisters have been predicted (24,38,50,65), suggesting that there may be large numbers of undiscovered variants that could aid in defining the structural and catalytic properties of twister ribozymes. Utilizing sequence similarity (Figure S1), we discovered 1,012 new twister ribozyme candidates.…”

“…Upon their discovery in 2014, the Breaker laboratory predicted twister ribozymes in 23 different organisms including anaerobic bacteria, cnidarians, insects, and plants as well as a fungus, flatworm, fish, and a virus (Figure S23A, inset) (17). Additional candidates were unearthed in plants, fish, flatworms, gastropods, and reptiles by Rfam (50) and Liu et al (65), while Lee et al detected over 3,500 twister ribozymes from metatranscriptomes (24). In total, twister ribozymes have been predicted in 78 different organisms, as well as in environmental samples and metatranscriptomes (17,24,50,65).…”

“…Additional candidates were unearthed in plants, fish, flatworms, gastropods, and reptiles by Rfam (50) and Liu et al (65), while Lee et al detected over 3,500 twister ribozymes from metatranscriptomes (24). In total, twister ribozymes have been predicted in 78 different organisms, as well as in environmental samples and metatranscriptomes (17,24,50,65).…”

Self-cleaving ribozymes conserved in RNA viruses unveil a new role in protein translation

“…In the last decade, these motifs have been discovered to be widespread in the DNA genomes of phages, bacteria, or eukaryotes [16][17][18] , including the human genome 19,20 . The precise biological functions of many of these genomic ribozymes is not well understood, but numerous hhrbz, dvrbz and twrbz motifs strongly associate with autonomous and non-autonomous DNA retrotransposons across plant and metazoan genomes [21][22][23][24][25][26] .…”

Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity

“…Twister ribozymes are prevalent across much of life, including bacteria, fish, insects, plants, and reptiles. , The genome of the staple crop rice ( Oryza sativa ) harbors eight sequences encoding twister ribozymes, named Osa 1–1 through Osa 1–8 . Rice is responsible for feeding over half of the world’s population, making it particularly important to study .…”