Abstract
The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.
Full text
PDF











Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aebi M., Hornig H., Padgett R. A., Reiser J., Weissmann C. Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA. Cell. 1986 Nov 21;47(4):555–565. doi: 10.1016/0092-8674(86)90620-3. [DOI] [PubMed] [Google Scholar]
- Aroian R. V., Koga M., Mendel J. E., Ohshima Y., Sternberg P. W. The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature. 1990 Dec 20;348(6303):693–699. doi: 10.1038/348693a0. [DOI] [PubMed] [Google Scholar]
- Aroian R. V., Sternberg P. W. Multiple functions of let-23, a Caenorhabditis elegans receptor tyrosine kinase gene required for vulval induction. Genetics. 1991 Jun;128(2):251–267. doi: 10.1093/genetics/128.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Atweh G. F., Anagnou N. P., Shearin J., Forget B. G., Kaufman R. E. Beta-thalassemia resulting from a single nucleotide substitution in an acceptor splice site. Nucleic Acids Res. 1985 Feb 11;13(3):777–790. doi: 10.1093/nar/13.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blumenthal T., Thomas J. Cis and trans mRNA splicing in C. elegans. Trends Genet. 1988 Nov;4(11):305–308. doi: 10.1016/0168-9525(88)90107-2. [DOI] [PubMed] [Google Scholar]
- Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974 May;77(1):71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carstens R. P., Fenton W. A., Rosenberg L. R. Identification of RNA splicing errors resulting in human ornithine transcarbamylase deficiency. Am J Hum Genet. 1991 Jun;48(6):1105–1114. [PMC free article] [PubMed] [Google Scholar]
- Chen S. H., Zhang M., Thompson A. R., Bray G. L., Scott C. R. Splice junction mutations in factor IX gene resulting in severe hemophilia B. Nucleic Acids Res. 1991 Mar 11;19(5):1172–1172. doi: 10.1093/nar/19.5.1172. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cladaras C., Hadzopoulou-Cladaras M., Felber B. K., Pavlakis G., Zannis V. I. The molecular basis of a familial apoE deficiency. An acceptor splice site mutation in the third intron of the deficient apoE gene. J Biol Chem. 1987 Feb 15;262(5):2310–2315. [PubMed] [Google Scholar]
- Cox G. N., Laufer J. S., Kusch M., Edgar R. S. Genetic and Phenotypic Characterization of Roller Mutants of CAENORHABDITIS ELEGANS. Genetics. 1980 Jun;95(2):317–339. doi: 10.1093/genetics/95.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deshler J. O., Rossi J. J. Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron. Genes Dev. 1991 Jul;5(7):1252–1263. doi: 10.1101/gad.5.7.1252. [DOI] [PubMed] [Google Scholar]
- Ferguson E. L., Horvitz H. R. Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode Caenorhabditis elegans. Genetics. 1985 May;110(1):17–72. doi: 10.1093/genetics/110.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferguson E. L., Sternberg P. W., Horvitz H. R. A genetic pathway for the specification of the vulval cell lineages of Caenorhabditis elegans. Nature. 1987 Mar 19;326(6110):259–267. doi: 10.1038/326259a0. [DOI] [PubMed] [Google Scholar]
- Fields C. Information content of Caenorhabditis elegans splice site sequences varies with intron length. Nucleic Acids Res. 1990 Mar 25;18(6):1509–1512. doi: 10.1093/nar/18.6.1509. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fouser L. A., Friesen J. D. Effects on mRNA splicing of mutations in the 3' region of the Saccharomyces cerevisiae actin intron. Mol Cell Biol. 1987 Jan;7(1):225–230. doi: 10.1128/mcb.7.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gao F., Endo H., Yamamoto M. Length heterogeneity in rat salivary gland alpha 2 mu globulin mRNAs: multiple splice-acceptors and polyadenylation sites. Nucleic Acids Res. 1989 Jun 26;17(12):4629–4636. doi: 10.1093/nar/17.12.4629. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goguel V., Liao X. L., Rymond B. C., Rosbash M. U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection. Genes Dev. 1991 Aug;5(8):1430–1438. doi: 10.1101/gad.5.8.1430. [DOI] [PubMed] [Google Scholar]
- Guillermit H., Fanen P., Ferec C. A 3' splice site consensus sequence mutation in the cystic fibrosis gene. Hum Genet. 1990 Sep;85(4):450–453. doi: 10.1007/BF02428306. [DOI] [PubMed] [Google Scholar]
- Hanioka N., Kimura S., Meyer U. A., Gonzalez F. J. The human CYP2D locus associated with a common genetic defect in drug oxidation: a G1934----A base change in intron 3 of a mutant CYP2D6 allele results in an aberrant 3' splice recognition site. Am J Hum Genet. 1990 Dec;47(6):994–1001. [PMC free article] [PubMed] [Google Scholar]
- Hata A., Emi M., Luc G., Basdevant A., Gambert P., Iverius P. H., Lalouel J. M. Compound heterozygote for lipoprotein lipase deficiency: Ser----Thr244 and transition in 3' splice site of intron 2 (AG----AA) in the lipoprotein lipase gene. Am J Hum Genet. 1990 Oct;47(4):721–726. [PMC free article] [PubMed] [Google Scholar]
- Hawkins J. D. A survey on intron and exon lengths. Nucleic Acids Res. 1988 Nov 11;16(21):9893–9908. doi: 10.1093/nar/16.21.9893. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hedley M. L., Forman J., Tucker P. W. Mutation of 3' splice sites in two different class I genes results in different usage of cryptic splice sites. J Immunol. 1989 Aug 1;143(3):1018–1025. [PubMed] [Google Scholar]
- Higashi Y., Tanae A., Inoue H., Hiromasa T., Fujii-Kuriyama Y. Aberrant splicing and missense mutations cause steroid 21-hydroxylase [P-450(C21)] deficiency in humans: possible gene conversion products. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7486–7490. doi: 10.1073/pnas.85.20.7486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jackson I. J. A reappraisal of non-consensus mRNA splice sites. Nucleic Acids Res. 1991 Jul 25;19(14):3795–3798. doi: 10.1093/nar/19.14.3795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kay R. J., Russnak R. H., Jones D., Mathias C., Candido E. P. Expression of intron-containing C. elegans heat shock genes in mouse cells demonstrates divergence of 3' splice site recognition sequences between nematodes and vertebrates, and an inhibitory effect of heat shock on the mammalian splicing apparatus. Nucleic Acids Res. 1987 May 11;15(9):3723–3741. doi: 10.1093/nar/15.9.3723. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kobayashi K., Jackson M. J., Tick D. B., O'Brien W. E., Beaudet A. L. Heterogeneity of mutations in argininosuccinate synthetase causing human citrullinemia. J Biol Chem. 1990 Jul 5;265(19):11361–11367. [PubMed] [Google Scholar]
- Kramer J. M., Cox G. N., Hirsh D. Comparisons of the complete sequences of two collagen genes from Caenorhabditis elegans. Cell. 1982 Sep;30(2):599–606. doi: 10.1016/0092-8674(82)90256-2. [DOI] [PubMed] [Google Scholar]
- Kramer J. M., French R. P., Park E. C., Johnson J. J. The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen. Mol Cell Biol. 1990 May;10(5):2081–2089. doi: 10.1128/mcb.10.5.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kramer J. M., Johnson J. J., Edgar R. S., Basch C., Roberts S. The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis. Cell. 1988 Nov 18;55(4):555–565. doi: 10.1016/0092-8674(88)90214-0. [DOI] [PubMed] [Google Scholar]
- Kuiper M. T., Holtrop M., Vennema H., Lambowitz A. M., de Vries H. A 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa. J Biol Chem. 1988 Feb 25;263(6):2848–2852. [PubMed] [Google Scholar]
- Lamond A. I., Konarska M. M., Sharp P. A. A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev. 1987 Aug;1(6):532–543. doi: 10.1101/gad.1.6.532. [DOI] [PubMed] [Google Scholar]
- Metherall J. E., Collins F. S., Pan J., Weissman S. M., Forget B. G. Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron. EMBO J. 1986 Oct;5(10):2551–2557. doi: 10.1002/j.1460-2075.1986.tb04534.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miner J. H., Wold B. J. c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation. Mol Cell Biol. 1991 May;11(5):2842–2851. doi: 10.1128/mcb.11.5.2842. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitchell P. J., Urlaub G., Chasin L. Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells. Mol Cell Biol. 1986 Jun;6(6):1926–1935. doi: 10.1128/mcb.6.6.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mules E. H., Dowling C. E., Petersen M. B., Kazazian H. H., Jr, Thomas G. H. A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients. Am J Hum Genet. 1991 Jun;48(6):1181–1185. [PMC free article] [PubMed] [Google Scholar]
- Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
- Newman A. J., Norman C. U5 snRNA interacts with exon sequences at 5' and 3' splice sites. Cell. 1992 Feb 21;68(4):743–754. doi: 10.1016/0092-8674(92)90149-7. [DOI] [PubMed] [Google Scholar]
- Ogg S. C., Anderson P., Wickens M. P. Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract. Nucleic Acids Res. 1990 Jan 11;18(1):143–149. doi: 10.1093/nar/18.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Patterson B., Guthrie C. A U-rich tract enhances usage of an alternative 3' splice site in yeast. Cell. 1991 Jan 11;64(1):181–187. doi: 10.1016/0092-8674(91)90219-o. [DOI] [PubMed] [Google Scholar]
- Reed R., Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. doi: 10.1016/0092-8674(85)90064-9. [DOI] [PubMed] [Google Scholar]
- Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
- Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruffner D. E., Dugaiczyk A. Splicing mutation in human hereditary analbuminemia. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2125–2129. doi: 10.1073/pnas.85.7.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruskin B., Green M. R. Role of the 3' splice site consensus sequence in mammalian pre-mRNA splicing. Nature. 1985 Oct 24;317(6039):732–734. doi: 10.1038/317732a0. [DOI] [PubMed] [Google Scholar]
- Satokata I., Tanaka K., Miura N., Miyamoto I., Satoh Y., Kondo S., Okada Y. Characterization of a splicing mutation in group A xeroderma pigmentosum. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9908–9912. doi: 10.1073/pnas.87.24.9908. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Senapathy P., Shapiro M. B., Harris N. L. Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project. Methods Enzymol. 1990;183:252–278. doi: 10.1016/0076-6879(90)83018-5. [DOI] [PubMed] [Google Scholar]
- Smith C. W., Porro E. B., Patton J. G., Nadal-Ginard B. Scanning from an independently specified branch point defines the 3' splice site of mammalian introns. Nature. 1989 Nov 16;342(6247):243–247. doi: 10.1038/342243a0. [DOI] [PubMed] [Google Scholar]
- Su T. S., Lin L. H. Analysis of a splice acceptor site mutation which produces multiple splicing abnormalities in the human argininosuccinate synthetase locus. J Biol Chem. 1990 Nov 15;265(32):19716–19720. [PubMed] [Google Scholar]
- Sulston J. E., Brenner S. The DNA of Caenorhabditis elegans. Genetics. 1974 May;77(1):95–104. doi: 10.1093/genetics/77.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas J. D., Conrad R. C., Blumenthal T. The C. elegans trans-spliced leader RNA is bound to Sm and has a trimethylguanosine cap. Cell. 1988 Aug 12;54(4):533–539. doi: 10.1016/0092-8674(88)90075-x. [DOI] [PubMed] [Google Scholar]
- Thomas J., Lea K., Zucker-Aprison E., Blumenthal T. The spliceosomal snRNAs of Caenorhabditis elegans. Nucleic Acids Res. 1990 May 11;18(9):2633–2642. doi: 10.1093/nar/18.9.2633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Treisman R., Orkin S. H., Maniatis T. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes. Nature. 1983 Apr 14;302(5909):591–596. doi: 10.1038/302591a0. [DOI] [PubMed] [Google Scholar]
- Tromp G., Prockop D. J. Single base mutation in the pro alpha 2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame pro alpha 2(I) chain. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5254–5258. doi: 10.1073/pnas.85.14.5254. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vijayraghavan U., Parker R., Tamm J., Iimura Y., Rossi J., Abelson J., Guthrie C. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 1986 Jul;5(7):1683–1695. doi: 10.1002/j.1460-2075.1986.tb04412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wenger R. H., Nielsen P. J. Reannealing of artificial heteroduplexes generated during PCR-mediated isotyping. Trends Genet. 1991 Jun;7(6):178–178. doi: 10.1016/0168-9525(91)90430-x. [DOI] [PubMed] [Google Scholar]
- Wiebauer K., Herrero J. J., Filipowicz W. Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals. Mol Cell Biol. 1988 May;8(5):2042–2051. doi: 10.1128/mcb.8.5.2042. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wieringa B., Hofer E., Weissmann C. A minimal intron length but no specific internal sequence is required for splicing the large rabbit beta-globin intron. Cell. 1984 Jul;37(3):915–925. doi: 10.1016/0092-8674(84)90426-4. [DOI] [PubMed] [Google Scholar]
- Zorn A. M., Krieg P. A. PCR analysis of alternative splicing pathways: identification of artifacts generated by heteroduplex formation. Biotechniques. 1991 Aug;11(2):180–184. [PubMed] [Google Scholar]
- von Mende N., Bird D. M., Albert P. S., Riddle D. L. dpy-13: a nematode collagen gene that affects body shape. Cell. 1988 Nov 18;55(4):567–576. doi: 10.1016/0092-8674(88)90215-2. [DOI] [PubMed] [Google Scholar]


