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

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.

Figures

Figure 1

(A) Digestions of the splicing products with RNase R, RNase II and PNPase. Human β-globin pre-mRNA was spliced with HeLa cell nuclear extract in vitro (lane 1) and the RNA products were further digested with each RNase for indicated incubation time (min; lanes 2–10). The RNAs were analyzed by denaturing 5.5% PAGE. The positions of splicing products and RNase-digested products are schematically indicated with their structures. DNA markers (New England Biolabs) are shown with their sizes (M). (B) Analyses of the lariat RNAs by debranching. In vitro spliced products and their RNase-digested RNA products [lanes 11–14; reactions are corresponding to lanes 1, 3, 7 and 9 in (A), respectively] were debranched with HeLa cell S100 extract (lanes 15–18). The RNAs were analyzed by denaturing 9.0% PAGE. The positions of RNA products are schematically represented with their structures. (C) Digestion pathway of the 3′ tail of lariat RNA by RNases and debranching of the products.

Figure 2

(A) Digestions of the branch RNA with RNase R, RNase II and PNPase. The loop portion of a lariat intron (lane 1; corresponding to Figure 1A, lane 3) was cleaved by oligonucleotide-targeted RNase H to prepare a branch RNA (lane 2). This branch RNA was digested with each RNase for 15 min (lanes 3–5). The RNAs were analyzed by denaturing 9.0% PAGE. The positions of loop RNA, branch RNA and RNase-digested products are schematically represented by their structures. (B) Cleavage of the loop RNA by RNase H and the degradation pathways by RNases are schematically shown.

Figure 3

RT–PCR detection of mRNA, intact lariat introns, and circular RNAs from either RNase R-digested (+) or mock-digested (−) human skeletal muscle total RNA. The RNA was directly analyzed by 1.0% agarose gel electrophoresis (lanes 1 and 2). The RNA was used for RT–PCR with primers to detect the dystrophin mRNA including exons 7 and 8 (lanes 3 and 4), three lariat introns as indicated (lanes 5–10), and the circular RNAs including multiple exons (lanes 11 and 12). The PCR products were analyzed by native 6.0% PAGE (lanes 3–12). The structures of the lariat intron and circular RNA are schematically represented and arrows indicate the positions of primers used for PCR.