High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs

Abstract

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.

Detection and Analysis of Circular RNAs by RT-PCR

“…The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014).…”

“…However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA.…”

Identifying intronic circRNAs: progress and challenges

“…In an insightful editorial (1), Bogard and colleagues discussed a recent publication from our laboratory describing the method ‘RNase R treatment followed by Polyadenylation and poly(A) + RNA Depletion’ (RPAD) to isolate highly enriched circular RNAs (circRNAs) from heterogeneous total RNA pools (2). With accumulating evidence that circRNAs influence gene expression by modulating the function of regulatory noncoding RNAs (e.g., microRNAs) and RNA-binding proteins (3–5), interest in circRNAs has escalated in recent years.…”

“…In the RPAD procedure, after RNase R digestion, most of the remaining RNAs with a free 3’OH end were removed by introducing an additional step of polyadenylation followed by depletion of poly(A)-containing transcripts using oligo-d(T) beads and rRNA depletion. The actual full-length circRNA sequences could then be generated by aligning the RPAD- generated RNA-sequencing reads between the back-splice junction coordinates, instead of relying on bioinformatic predictions (2). This procedure effectively removed highly structured and non-polyadenylated RNAs and led to the identification of known and novel exonic circRNA (EcircRNA) isoforms as well as many novel intronic circRNAs (IcircRNAs) (2).…”

“…The actual full-length circRNA sequences could then be generated by aligning the RPAD- generated RNA-sequencing reads between the back-splice junction coordinates, instead of relying on bioinformatic predictions (2). This procedure effectively removed highly structured and non-polyadenylated RNAs and led to the identification of known and novel exonic circRNA (EcircRNA) isoforms as well as many novel intronic circRNAs (IcircRNAs) (2). …”