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.
Bibliographical noteFunding Information:
We are grateful to Dr M. P. Deutscher for valuable comments, encouragement and financial support. We thank Dr K. E. Rudd for critical reading of the manuscript, Dr K. Ohe for helpful suggestions, and G. Kozloski and T. Venkataraman for technical assistance. A. Mayeda was supported by a new investigator developmental grant from the Muscular Dystrophy Association, and an institutional research grant from the Sylvester Braman Family Breast Cancer Research Institute. A. Malhotra is supported by a grant from National Institute of Health (GM69972). J.W. and M.Q.Z. are supported by a grant from National Institute of Health (HG001696). Y.Z. is supported in part by a postdoctoral fellowship from the American Heart Association Florida/Puerto Rico Affiliate (0325296B). A. Mayeda and A. Malhotra are research members of the Sylvester Comprehensive Cancer Center. The Open Access publication charges for this article were waived by Oxford University Press