Optimization And Application Of Experimental Method For Identifying CircRNA In Rice

Circular RNAs(circRNAs)have been recently found in both plants and animals.CircRNA have been proved to function as miRNA sponges.It was also reported that circRNAs in human cells can be translated into proteins.Currently,there are many algorithms predicting circRNAs and a large number of circRNAs has been identified in a wide range of species.However,circRNAs with experimental validation are still limited.Particularly,it is difficult to validate circRNAs in plants compared as those in animals and humans.In this study,we tried to optimize the experimental method for validate circRNAs in rice.We have obtained more than 100 experimentally validated circRNAs,which is helpful for analyze the function and biological mechanisms of circRNAs.The results are as followed:(1)Compared with common RNase R(.an E.coli exoribonuclease that exhibits 3’ to 5’ exonuclease activity,efficiently digesting nearly all linear RNA species)treatment,it can increase the concentration of circRNAs when we combined RNase R treatment with RNA isolation kits.Meanwhile,our results showed that touch down PCR can improve specificity of circRNAs compared to traditional PCR.(2)128 rice circRNAs have been validated using our method including 70 full-length circRNAs.Alternative circularization of circRNAs has been revealed in rice through validation of 5 circRNA loci containing multiple circRNA isoforms.Our results also showed that there are a lot of circRNAs with non GT/AG splicing signal in rice.Meanwhile,we also did some research on identification of fusion circRNAs(circRNAs generated from different genes or even chromosomes)in rice and the we validated successfully two junctions within one circRNA.