Circular RNA (CircRNA) is yet another vital addition to the noncoding RNA family. They are mainly derived by fusion of downstream 3' splice donor with upstream 5' splice acceptor by a noncanonical form of alternative splicing mechanism called backsplicing. An array of functional aspects of these circRNAs has been reported in animal systems. However, functional investigation of circRNA in plants is very limited. In this chapter, we described a methodological outline to study the circRNA biogenesis and to characterize its function(s). Sequence of a newly identified Oryza sativa Indica circRNA flanked by complementary repeat sequences of a rice intron was assembled to yield a circRNA expression cassette. This cassette can be cloned into any plant expression vector which has a suitable promoter (CaMV 35S or ubiquitin promoter) and terminator, and can be used for any circRNA-mediated functional studies. Subsequent agroinfection of rice calli with this cassette yielded circRNA expressing transgenic plants. These transgenic plants were used to establish a correlation between the expressing circRNA, parental gene, and interacting miRNAs. Moreover, effect of circRNA overexpression on plant phenotype under various stress conditions can be studied using these transgenic plants. Also, RNA pull-down assay can be performed to identify the circRNA interacting proteins and the expression of these RBPs can also be studied from these transgenic plants.
Keywords: Binary vector; CircRNA; CircRNA expression cassette; Divergent RT-PCR; Plant tissue culture.