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Nucleic Acids Research 2006 34(8):e63; doi:10.1093/nar/gkl151
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Published online 8 May 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

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Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Hitoshi Suzuki, Yuhong Zuo, Jinhua Wang1, Michael Q. Zhang1, Arun Malhotra and Akila Mayeda*

Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine 1011 NW 15th Street, Miami, FL 33136, USA 1 Cold Spring Harbor Laboratory 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA

*To whom correspondence should be addressed. Tel: +1 305 243 4621; Fax: +1 305 243 3065; Email: mayeda{at}miami.edu

Received December 23, 2005. Revised February 1, 2006. Accepted March 17, 2006.

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3' to 5' exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.

Present address: Jinhua Wang, Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA


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