Abstract
Lariat intermediates of a group II intron were investigated via RT-PCR. Several reverse transcriptases appeared capable of reading through a branched nucleotide. A new method has been established that yields precise information about the location of the branch point within an intron. As an extension of our approach, antisense transcripts of the previously cloned PCR products were successfully used in RNase Protection Assays, providing a tool for quantification of splicing intermediates. Application of the method presented to other self-splicing introns as well as introns in nuclear pre-mRNAs is envisaged.
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- Copertino D. W., Hall E. T., Van Hook F. W., Jenkins K. P., Hallick R. B. A group III twintron encoding a maturase-like gene excises through lariat intermediates. Nucleic Acids Res. 1994 Mar 25;22(6):1029–1036. doi: 10.1093/nar/22.6.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
- Hübschmann T., Hess W. R., Börner T. Impaired splicing of the rps12 transcript in ribosome-deficient plastids. Plant Mol Biol. 1996 Jan;30(1):109–123. doi: 10.1007/BF00017806. [DOI] [PubMed] [Google Scholar]
- Kim J. K., Hollingsworth M. J. Splicing of group II introns in spinach chloroplasts (in vivo): analysis of lariat formation. Curr Genet. 1993 Feb;23(2):175–180. doi: 10.1007/BF00352018. [DOI] [PubMed] [Google Scholar]
- Lorsch J. R., Bartel D. P., Szostak J. W. Reverse transcriptase reads through a 2'-5'linkage and a 2'-thiophosphate in a template. Nucleic Acids Res. 1995 Aug 11;23(15):2811–2814. doi: 10.1093/nar/23.15.2811. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michel F., Umesono K., Ozeki H. Comparative and functional anatomy of group II catalytic introns--a review. Gene. 1989 Oct 15;82(1):5–30. doi: 10.1016/0378-1119(89)90026-7. [DOI] [PubMed] [Google Scholar]
- Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
- Padgett R. A., Konarska M. M., Aebi M., Hornig H., Weissmann C., Sharp P. A. Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8349–8353. doi: 10.1073/pnas.82.24.8349. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhee Y., Valentine M. R., Termini J. Oxidative base damage in RNA detected by reverse transcriptase. Nucleic Acids Res. 1995 Aug 25;23(16):3275–3282. doi: 10.1093/nar/23.16.3275. [DOI] [PMC free article] [PubMed] [Google Scholar]