Abstract
High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function.
Highlights
CircularRNAs are a large family of covalently closed RNA molecules expressed ubiquitously in various organisms, including humans [1,2]
The majority of reported circRNAs are derived from exons of precursors of messenger RNAs and noncodingRNAs by a process called backsplicing [3,4,5,6,7,8]
More than one hundred thousand circRNAs have been identified to date, the functions of circRNAs have only been elucidated for a handful of them; these functions often include sequestration of microRNAs and RNA-binding proteins (RBPs), as well as competition with linear RNA splicing [7,8,13,14]
Summary
Circular (circ)RNAs are a large family of covalently closed RNA molecules expressed ubiquitously in various organisms, including humans [1,2]. Recent high-throughput RNA sequencing (RNA-seq) analyses have identified thousands of circRNAs, which can range in size from
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