Abstract
The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cultured cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.
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References
Abel S, Kiss T, Solysmoy F: Molecular analysis of eight U1 RNA gene candidates from tomato that could potentially be transcribed into U1 RNA sequence variants differing from each other in similar regions of secondary structure. Nucl Acids Res 17: 6319–6337 (1989).
Blumenthal T, Thomas J: Cis and trans mRNA splicing in C. elegans. Trends Genet 4: 305–308 (1988).
Bodeau JP, Walbot V: Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products. Mol Gen Genet 233: 379–387 (1992).
Goodall GJ, Filipowicz W: The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing. Cell 58: 473–483 (1989).
Goodall GJ, Filipowicz W: Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants. EMBO J 10: 2635–2644 (1991).
Goodall GJ, Kiss T, Filipowicz W: Nuclear RNA splicing and small nuclear RNAs and their genes in higher plants. Oxford Surv Plant Mol Cell Biol 7: 255–296 (1991).
Green MR: Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol 7: 559–599 (1991).
Guthrie C: Messenger RNA splicing in yeast: Clues to why the spliceosome is a ribonucleoprotein. Science 253: 157–163 (1991).
Kozak M: Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266: 19867–19870 (1991).
Legrain P, Rosbash M: Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell 57: 573–583 (1989).
Lou H, McCullough AJ, Schuler MA: 3′ splice site selection in dicot plant nuclei is position dependent. Mol Cell Biol 13: 4485–4493 (1993).
Luehrsen K, Walbot V: The impact of AUG start codon context on maize gene expression in vivo. Plant Cell Rep 13: 454–458 (1994).
Luehrsen KR, de Wet JR, Walbot V: Transient expression analysis in plants using firefly luciferase reporter gene. Meth Enzymol 216: 397–414 (1992).
Luehrsen KR, Taha S, Walbot V: Nuclear pre-mRNA processing in higher plants. Prog Nucl Acid Res Mol Biol 47: 149–193 (1993).
Luehrsen KR, Walbot V: Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol Gen Genet 225: 81–93 (1991).
Luehrsen KR, Walbot V: Addition of A- and U-rich sequence increases the splicing efficiency of a deleted form of a maize intron. Plant Mol Biol 24: 449–463 (1994).
Luehrsen KR, Walbot V: Intron creation and polyadenylation in maize are directed by AU-rich RNA. Genes Devel 8: 1117–1130 (1994).
Martinez-Zapater JM, Finkelstein R, Somerville CR: Drosophila P-element transcripts are incorrectly processed in tobacco. Plant Mol Biol 11: 601–607 (1988).
McCullough AJ, Lou H, Schuler MA. In vivo analysis of plant pre-mRNA splicing using an autonomously replicating vector. Nucl Acids Res 19: 3001–3009 (1991).
McCullough AJ, Lou H, Schuler MA: Factors affecting authentic 5′ splice site selection plant nuclei. Mol Cell Biol 13: 1323–1331 (1993).
Nash J: Bronze-2 gene of maize: Analysis of transcription and splicing patterns. Ph.D. thesis, Stanford University, Stanford, California (1992).
Nash J, Luehrsen KR, Walbot V: Bronze-2 gene of maize: reconstruction of a wild-type allele and analysis of transcription and splicing. Plant Cell 2: 1039–1049 (1990).
Nash J, Walbot V: Bronze-2 gene expression and intron splicing patterns in cells and tissues of Zea mays L. Plant Physiol 100: 464 (1992).
Patterson B, Guthrie C: A U-tract enhances usage of an alternative 3′ splice site in yeast. Cell 64: 181–187 (1991).
Pautot V, Brzezinski R, Tepfer M: Expression of a mouse metallothionein gene in transgenic plant tissues. Gene 77: 133–140 (1989).
Robberson BL, Cote GJ, Berget SM: Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol 10: 84–94 (1990).
Rosbash M, Seraphin B: Who's on first? The U1 snRNP-5′ splice site interaction and splicing. Trends Biochem 16: 187–190 (1991).
Sharp PA: Splicing of messenger RNA precursors. Science 235: 766–771 (1987).
Steitz JA: Splicing takes a Holliday. Science 257: 888–889 (1992).
Talerico M, Berget SM: Effect of 5′ splice site mutations on splicing of the preceding intron. Mol Cell Biol 10: 6299–6305 (1990).
Van Santen VL, Spritz RA: Splicing of plant pre-mRNAs in animal systems and vice versa. Gene 56: 253–265 (1987).
Van Santen VL, Spritz RA: Nucleotide sequence of a bean (Phaseolus vulgaris) U1 small nuclear RNA gene: implications for plant pre-mRNA splicing. Proc Natl Acad Sci USA 84: 9094–9098 (1987).
Van Santen VL, Swain W, Spritz RA: Nucleotide sequence of two soybean U1 snRNA genes. Nucl Acids Res 16: 4176–4180 (1988).
White O, Soderlund C, Shanmugan P, Fields C: Information contents and dinucleotide compositions of plant intron sequences vary with evolutionary origin. Plant Mol Biol 19: 1057–1064 (1992).
Wiebauer K, Herrero JJ, Filipowicz W: Nuclear pre-mRNA processing in plants: distinct modes of 3′-splicesite selection in plants and animals. Mol Cell Biol 8: 2042–2051 (1988).
Yost HJ, Lindquist S: Translation of unspliced transcripts after heat shock. Science 242: 1544–1548 (1988).
Yost HJ, Lindquist S: Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae. Mol Cell Biol 11: 1062–1068 (1991).
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Carle-Urioste, J.C., Ko, C.H., Benito, MI. et al. In vivo analysis of intron processing using splicing-dependent reporter gene assays. Plant Mol Biol 26, 1785–1795 (1994). https://doi.org/10.1007/BF00019492
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DOI: https://doi.org/10.1007/BF00019492