Determing And Analizing The Cap-snatching Repertoire Of Rice Stripe Tenuivirus Based On An MRNA Enrichment Technology

Rice stripe tenuivirus(RSV)is the type species of the genus Tenuivirus,under the family Phenuiviridae.The genome of RSV comprises four single-stranded RNAs,which specify a total of seven proteins by employing a negtive or ambisense coding strategy.Similar to other segmented negative-sense RNA viruses(sNSV),RSV uses a mechanism called cap-snatching during gene expression.In cap-snatching,RSV snatches a capped-RNA leader 10-20 nt in length from host cellular mRNAs and uses the snatched leader as a primer of transcription,giving rise to viral mRNAs with host-derived heterogenous sequences at their 5' ends.Recently,our lab determined the capped-RNA leaders at the 5' ends RSV NP and NCP mRNAs with a high-throuput sequencing approach,the results of which provided many unprecedented insights into the cap-snatching mechanism of RSV.However,because of their low abundance relative to the genomic or anti-genomic RNAs,our attempts to sequence the 5' ends of other RSV mRNAs were unsuccessful..Therefor,we are not sure about the commonness of our findings to other RSV mRNAs.Especially,we observed a greater tendency of RSV to use the prime-and-realign mechanism in synthesising NP than in that of NCP,but we do not know whether or not there is a difference in prime-and-realign mechanism usage when RSV synthesizes other mRNAs.In the literature,there is report that eIF4E can bind to the cap structure of mRNAs.Several researchers have improved the affinity of eIF4E to the cap-structure by genetic manipulations.With the genetically modified eIF4E(eIF4EK119A),several researchers have enriched RNA species with a cap structure at their 5' ends.Based on these previous studies,this study was aimed to enrich RSV mRNAs to obtain a more completed "snatching spectrum" of RSV.Firstly,eIF4EK119A was cloned into the vector pGEX-4T-3 and,by optimizing experimental conditions(IPTG concentration is 0.25 mMol,16 ?,180 rpm and time is rang from 10 t0 13 h),a bacterially expressed fusion protein of eIF4EK119A with GST was obtained.The GST-eIF4EK119A was mixed with 60 microgram of total RNA extracted from RSV infected rice and about 3 microgram of mRNA was obtained by immuno-precipitation.Attempts were made to clone and sequence the 5' ends of RSV NS2,NSvc2,NS3,NP,NCP and NSvc4 mRNAs.The results showed that more than 90%of the sequences obtained contained an expected heterogenous sequence at their 5' ends,suggesting that we have succesfully enriched RSV mRNAs with eIF4EK119A.The 5' ends of RSV NS2,NSvc2,NS3,NP,NCP and NSvc4 mRNAs were determined by cap-seq.Analysis of the cap-snatching spectrum of RSV obtained in this way showed that:i)RSV uses a similar set of capped-RNA leaders to initiate the production of all the six kinds of mRNAs;ii)RSV has a greater tendency to use the prime-and-realign mechanism in transcribing genomic RNAs than that of antigenomic RNAs,the result of which is that transcripts derived from genomic mRNAs have a longer and more complex capped sequence that those derived from antigenomic RNAs.In all,RSV mRNAs was enriched by using a mutant eIF4E.Based on this,this study for the first time obtained a more completed "snatching spectrum" of RSV and for the first time revealed a greater tendency of RSV to use the prime-and-realign mechanism in transcribing genomic RNAs than that of antigenomic RNAs.This significantly advanced our understanding of the cap-snatching mechanism of RSV.