The Mechanism That RNA Splicing Enhances Nonsense-mediated MRNA Decay In Saccharomyces Cerevisiae

Nonsense-mediated m RNA decay(NMD)is a kind of important cellular mechanism that controls the quality of RNA.NMD can rapidly get rid of m RNA containing PTC(premature termination codon)and avoid the production of truncated proteins result from translation of aberrant m RNA,so that makes sure the normal state of organisms.NMD process depends on the translation of ribosomes,but recently the mechanism by which premature translation termination is distinguished from standard translation termination remains poorly understood.There are two models for explaining NMD,faux 3'UTR model believes the intensity of NMD has something to do with the length of 3'UTR,however EJC model thinks the EJC complex which deposits on m RNA after pre-m RNA splicing is the platform recruiting NMD factors and enhancing NMD.Faux 3'UTR model has been accepted by scientists in studies of Saccharomyces cerevisiae,the evidences of EJC model mainly comes from higher animal cells.Our lab's earlier study about NMD in fission yeast finds that the splicing of pre-m RNA can enhance NMD,but this enhancement does not depend on presence of EJC.To further investigate the relationship between pre-m RNA splicing and NMD,we use budding yeast as biological model and make the NMD reporter system based on GFP.Introducing a nonsense mutation close to AUG of GFP(PTC6)can induce the reduction of m RNA and rapid decay of m RNA,introducing a nonsense mutation far away to AUG of GFP(PTC140)moderately affect m RNA stability.These results indicate that the NMD reporter system based on GFP can reflect the strength and the weakness of NMD in Saccharomyces cerevisiae.Meanwhile,we introduce intron respectively from CYH2 and ACT1 into NMD reporter system based on GFP to examine the influence of m RNA splicing on NMD efficiency.The results show that the presence of intron remarkably enhances NMD: for the m RNA containing PTC140,both introns can induce m RNA reduction and promote decay.The contradiction against EJC model is that regardless of whether intron is located upstream or downstream of PTC,it enhances NMD.We make NMD reporter vectors from endogenous genes CYH2 and RPS11 B and observe the same phenomenon that intron enhances NMD and regardless of whether it is located upstream or downstream of PTC.At the same time,we find the distance between intron and PTC will influence NMD.The NMD is apparent when the distance is 90 nt,and NMD will be totally abolished when the distance is extended to 243 nt or 387 nt.These results indicate the distance between intron and PTC,instead of the position,have large effect on NMD.We guess that m RNA splicing factors affect NMD through association with m RNA because of lacking EJC and EJC components.At first,we screen all the non-essential m RNA splicing factors deletion library.We find PRP17? does not affect normal alternative splicing and have no effect on the degeneration of PTC6 m RNA through transforming vectors into wildtype yeast and the mutant yeast,but PRP17? inhibits splicing-dependent NMD(PTC140-ivs C).We think Prp17 p is specifically required for splicing-dependent NMD.We subsequently detect the translation efficiency in wildtype and mutant yeast.The result is that the presence of intron elevates the efficiency of translation,UPF1? and UPF2? decrease the efficiency of translation of m RNA with intron and do not affect the efficiency of translation of m RNA without intron.UPF3? has negative impact on all translation efficiency.PRP17? only inhibits translation of m RNA with intron.We believe Prp17 p participates in NMD by promoting translation.To sum up,our research is the first study reporting the phenomenon that intron can enhance NMD in Saccharomyces cerevisiae,the distance between of intron and PTC is essential for NMD.Also,we find Prp17 p is crucial for splicing-dependent NMD.Our results provide a reliable reference for clarifying the mechanism by which PTC is distinguished from NTC in Saccharomyces cerevisiae.