Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing
Top Cited Papers
Open Access
- 28 April 2006
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 34 (8), e63
- https://doi.org/10.1093/nar/gkl151
Abstract
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.Keywords
This publication has 42 references indexed in Scilit:
- Tag-based approaches for transcriptome research and genome annotationNature Methods, 2005
- SL trans-splicing: easy come or easy go?Trends in Genetics, 2005
- Delay in Synthesis of the 3′ Splice Site Promotes trans-Splicing of the Preceding 5′ Splice SiteMolecular Cell, 2005
- Finishing the euchromatic sequence of the human genomeNature, 2004
- Dynamic transcriptome of miceTrends in Biotechnology, 2004
- Alternativetrans-splicing of constant and variable exons of aDrosophilaaxon guidance gene,lolaPublished by Cold Spring Harbor Laboratory ,2003
- The Sequence of the Human GenomeScience, 2001
- Initial sequencing and analysis of the human genomeNature, 2001
- Mis-splicing yields circular RNA molecules.The FASEB Journal, 1993
- Scrambled exonsCell, 1991