Abstract
Measurements of the in vitro splicing efficiency of deletion mutant RNA precursors containing the small intron of the rabbit beta-globin gene, which are truncated in the first or in the second exon, revealed that no more than approximately 20 nucleotides of either exon are necessary for efficient splicing. At least for the second exon, this minimal length requirement is globin sequence-independent. Reduction of the exon-2 length to 14 nucleotides resulted in very inefficient splicing, whereas further reduction to 5 nucleotides apparently abolished the second splicing step (3' cutting and ligation), whereas the first step (5' cutting and branching) still occurred. The splicing efficiency of a double-mutant substrate retaining approximately 20 nucleotides of each exon was reduced to 50%. A kinetic study indicated that in the reaction of this double-mutant substrate the second, but not the first, splicing step was delayed, in contrast to the reaction of the wild-type precursor. Duplication or triplication of the entire sequence of exon-1 did not affect the splicing efficiency, whereas elongation of this exon with approximately 100 nucleotides of 5'-flanking (nontranscribed) beta-globin sequence diminished the level of correct splicing with the simultaneous appearance of aberrant lariat forms. We conclude that for mono-intronic precursors in which there is only one choice of splice sites, most of the exon sequences are not mechanistically involved in the splicing process.
Highlights
Annette Parent$, Scott Zeitlin, and Argiris Efstratiadis From the Departmentof Genetics and Development,Columbia University, New York, New York 10032
Measurements of the in vitro splicing efficiency of deletion mutant RNA precursors containing the small intron of the rabbit B-globingene, which are truncated in the first or in the second exon, revealed that no more than approximately 20 nucleotides of either exon are necessary for efficient splicing.At least for the second exon, this minimal length requirement is globin sequence-independent.Reduction of the exon-2 length to 14 nucleotides resulted in veryinefficientsplicing, whereas further reduction to 5 nucleotides apparently abolished the second splicing step (3’ cutting and ligation), whereas the first step(5‘ cutting and branching) still occurred
These results indicated that shortening of E l down to 24 In contrast to threesults obtainedwith the deletion mutant nucieotides, of which only 10 nucleotides is globin sequence, substrates, an addition mutant precursor (E1-260), with a had no dramatic effect on the relative efficiency of in vitro length of exon-1 increased by 95 nucleotides in comparison splicing (87% for El-91 and 80% for El-24)
Summary
Annette Parent$, Scott Zeitlin, and Argiris Efstratiadis From the Departmentof Genetics and Development,Columbia University, New York, New York 10032. I n vitro and in vivo assays of in vitro generated deletion and point mutant substrates (see e.g. Refs.12-17), as well asthe study of molecular defects in several forms of thalassemia (reviewed in Ref. 18),indicate that the minimum intron sequence requirements(in addition to a minimum length of approximately 80 nucleotides) are thesplice junctions and the lariat branching signal. If such natural sequences are deleted or modified by mutation theycan often be substituted by cryptic splice [12] or branchpointsites [16, 17]. The two double-mutant templates (E1-24/E2-21 and E1-34/E2-5) are combinations of the corresponding single-mutants
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