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

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...

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Veröffentlicht in:Nucleic acids research Jg. 45; H. 12; S. e116
Hauptverfasser: Panda, Amaresh C., De, Supriyo, Grammatikakis, Ioannis, Munk, Rachel, Yang, Xiaoling, Piao, Yulan, Dudekula, Dawood B., Abdelmohsen, Kotb, Gorospe, Myriam
Format: Journal Article
Sprache:Englisch
Veröffentlicht: England Oxford University Press 07.07.2017
Schlagworte:
Animals
Base Sequence
Cell Line
Computational Biology
Exons
Exoribonucleases - chemistry
HeLa Cells
High-Throughput Nucleotide Sequencing
Humans
Introns
Methods Online
Mice
Molecular Sequence Annotation
Myoblasts - cytology
Myoblasts - metabolism
Poly A - genetics
Poly A - metabolism
Polyadenylation
RNA - genetics
RNA - isolation & purification
RNA - metabolism
RNA Cleavage
RNA, Messenger - chemistry
RNA, Messenger - genetics
RNA, Messenger - metabolism
ISSN:0305-1048, 1362-4962, 1362-4962
Online-Zugang:Volltext
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Zusammenfassung: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.
Bibliographie:ObjectType-Article-1
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ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkx297