While the human transcriptome contains a large number of circular RNAs 
(circRNAs), the functions of most circRNAs remain unclear. Sequence annotation 
suggests that most circRNAs are generated from splicing in reversed orders 
across exons. However, the mechanisms of this backsplicing are largely unknown. 
Here we constructed a single exon minigene containing split GFP, and found that 
the pre-mRNA indeed produces circRNA through efficient backsplicing in human and 
Drosophila cells. The backsplicing is enhanced by complementary introns that 
form double-stranded RNA structure to bring splice sites in proximity, but such 
structure is not required. Moreover, backsplicing is regulated by general 
splicing factors and cis-elements, but with regulatory rules distinct from 
canonical splicing. The resulting circRNA can be translated to generate 
functional proteins. Unlike linear mRNA, poly-adenosine or poly-thymidine in 3' 
UTR can inhibit circular mRNA translation. This study revealed that backsplicing 
can occur efficiently in diverse eukaryotes to generate circular mRNAs.