Multi-level Regulation On Translational Of Rice Black-streaked Dwarf Virus

Rice black-streaked dwarf virus?RBSDV?,a member of the second group of the genus Fijivirus in the family Reoviridae,can cause rice black-streaked dwarf disease and maize rough dwarf disease.Genome of the RBSDV is composed of ten double-stranded RNAs?dsRNA?and each segment of them contains a cap structure?m7GPPPN?at the 5'end but lacks 3'poly?A?tail,10 dsRNAs?S1 to S10?totally encode 13 proteins.S5,S7 and S9 are bicistron RNAs,respectively encoding two proteins,while other 7 segments are monocistronic RNAs.The 5'and 3'terminal sequences of the 10 dsRNAs of RBSDV are conserved respectively,and adjacent sequences of the conserved 5'and 3'ends potentially form base pairs.In this paper,effect and mechanism of the untranslational regions of monocistronic RNAs in RBSDV genome on translation were identified.In addition,the expression characteristics of the bicistron RNAs in RBSDV genome and expression regulation of their second ORF were preliminarily analyzed.S3 and S10 of RBSDV genome were used to study the translation regulation of single cistronic RNA through firefly luciferase?Fluc?reporter gene vector and mutagenesis.Following results were identified as:?1?the 5'UTR of S3 and S10 presented the activity of internal entry site?IRES?and positively regulate the translation of reporter gene Fluc in the absence or presence of a 5'cap.5'conserved sequences and adjacent sequences in the 5'UTR were involved in the IRES activity.?2?3'UTR inhibited the positive effect of 5'UTR on translation.The molecular basis is the RNA-RNA interactions between 5'UTR and 3'UTR.Moreover,this RNA-RNA interaction also inhibited the efficiency of cap-dependent translation.RBSDV S7 and S9 are non-overlapped bicistron RNA,whose expression characteristic of two proteins was directly analyzed by in vitro translation with ³⁵S labelled amino aicd.In addition,cis-elements involved in the expression of the second protein were preliminarily mapped via firefly luciferase?Fluc?reporter vector and mutagenesis.Following results were identified as:?1?Two proteins encoded by S7 or S9 can directly expressed from genomic RNA,and it is suggested that the second protein of S7 or S9 was expressed in the manner of internal initiation.Expression level of the second protein P7b in S7 was about 4.7%of that of the first protein P7a while expression level of the second protein P9b in S9 was about 18.8%of that of the first protein P9a.?2?Two small ORFs in the gene interval region?IGR?between P9a and P9b in S9 inhibited protein expression efficiency of the P9b,because disappearance of these two small ORFs increased expression level of the P9b by 73.4%.?3?IGR of S7 or S9has weak IRES activity,and the corresponding 3'UTR can synergistically improve the IRES activity of IGR.Taken together,3'UTR inhibited the translation of proteins in monocistronic RNA such as S3 and S10,while 3'UTR enhanced the translation of second proteins in bicistronic RNAs via the potential interaction with IGR.It is suggested that 3'UTR may be a key region in which bicistronic RNAs regulates the expression ratio of two proteins.