Discovery And Characterization Of A Novel Class Of Ribozyme-Hovlinc

Ribozymes are RNA molecules able to catalyze specific biochemical reactions,such as RNA splicing,t RNA maturation and peptide bond formation.The first discovery of ribozyme in the early 1980 s demonstrates that RNA,while could carry genetic information like DNA,could also act as biological catalyst like protein enzymes.The discovery of ribozymes eventually led to the formulation of the “RNA world”hypothesis.Hitherto,a variety of ribozymes have been identified or artificially designed.Among the diversity of ribozymes,the small,self-cleaving ribozymes comprised of <200nucleotides have attracted extensive research attentions because of their potential applications in broad fields.This type of ribozyme catalyzes self-cleavage of phosphodiester RNA backbone at one specific site.Based on this catalytic characteristic,ribozymes could be designed to cleave the target RNA sequences at the specific site in cis or trans,which provide a powerful tool for manipulation of gene expression and clinical gene therapy.However,the absence of experimental highthroughput method for identification of such ribozyme limits this study.So far,only 11 classes of naturally-occurring small self-cleaving ribozymes have been identified,especially,only 4 ribozymes of this type(belonging to three classes)have been found in the human genome.In this study,we developed a novel experimental genome-wide screen for the identification of self-cleaving ribozymes.The screen is based on the characteristic of the self-cleaving reactions resulting in 2 products: an upstream one with 2′-3′-cyclic phosphate at 3′ end and a downstream one with 5′-OH termini.In the screen,the genomic DNA was isolated,fragmented,and in vitro transcribed(IVT).The RNA products,after a cleavage step,were treated by Rpp H(5′ pyrophosphohydrolase)and XRN-1 exonuclease successively.Rpp H converts 5′-triphosphate termini of RNAs to5′-monophosphate,and XRN-1 selectively degrades RNA molecules with 5′-monophosphate.Thus,RNAs with 5′-OH termini would be enriched in the treatment products.Finally,high-throughput sequencing was employed to screen the enriched RNA molecules with 5′-OH termini which represents the downstream products of selfcleaving ribozymes.Thus,the genomic coordinate of self-cleaving ribozymes could be identified.By devising the screen on the human genome,we detected one known naturallyoccurring self-cleaving ribozyme — CPEB3,and discovered one novel self-cleaving ribozyme,named hovlinc ribozyme by us.The hovlinc ribozyme shows unique RNA secondary structure and biochemical properties different from all the other known classes of small self-cleaving ribozyme,indicating that it belongs to a novel class of such ribozyme.Evolutionary analysis of the sequence of hovlinc ribozyme revealed a clear evolutionary path with appearance between ～130 and ～65 million years ago(Ma)and gain of self-cleavage activity very recently,～13–10 Ma,in the common ancestor of humans,chimpanzees and gorillas.This ribozyme is embedded within the 3′ end of a very long intergenic non-coding(vlinc)RNAs in the human genome,and possesses the self-cleavage activity in vivo.In summary,this study developed a novel genome-wide approach to identify selfcleaving ribozymes,and discovered a new ribozyme — hovlinc ribozyme — belonging to a novel class of self-cleaving ribozymes and locating in a vlinc RNA of the human genome.We further studied the evolutionary path,biochemical properties,RNA secondary structures and in vivo activity of this ribozyme.All in all,this work provides a novel method for identification of self-cleaving ribozymes,and a new candidate structure for biological application based on self-cleaving ribozymes.Moreover,this study proposes the possibility that lnc RNA could function by carrying catalytic RNA domains,which opens a tantalizing new possibility for the research of lnc RNA.