An intronic enhancer regulates splicing of the twintron of Drosophila melanogaster prospero pre-mRNA by two different spliceosomes

Abstract

We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5' splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.

FIG. 1.

Splicing of the prospero pre-mRNA is temporally regulated during Drosophila embryogenesis and early larval development. (A) Schematic of the alternatively spliced second intron of D. melanogaster prospero. The U2-type intron is 730 nucleotides long. The U12-type intron contains 59 and 28 nucleotides (dotted boxes) from the pros-L open reading frame, flanking the U2-type intron. The sequences encoding five amino acids of the homeodomain (HD) that are altered by alternative splicing are shown as horizontally lined and checkered boxes. The positions of primers for reverse transcription-PCR analysis are indicated. (B) Input RNA levels correlate with reverse transcription-PCR product levels. In vitro-transcribed RNAs, derived from pros-L and pros-S cDNA, were mixed at various ratios, reverse transcribed, and PCR amplified with the pros-specific primers. The DNA amplicons were then electrophoretically separated, and the quantities of pros-L and pros-S amplicons were measured by PhosphorImager analysis. (C) Stage-specific splicing of pros pre-mRNA during Drosophila embryogenesis and early larval stages. Total RNA was extracted from wild-type embryos collected at different times, reverse transcribed, and PCR amplified. The DNA amplicons were resolved on 12% denaturing polyacrylamide gels. (D) Quantification of the pros-L and pros-S mRNA products at various times throughout Drosophila embryogenesis and early larval stages. For each time point, the percentage of the pros-L (or pros-S) product was calculated by dividing the radioactivity in the pros-L (or pros-S) band by the radioactivity in the pros-L plus the pros-S band. pros-S is denoted by squares and pros-L by triangles.

FIG. 2.

RNA substrates used for in vitro and in vivo splicing assays. (A) Sequence of the wild-type pros minigene construct, which includes parts of exons 2 and 3 and a shortened intron 2. The sequence was derived from D. melanogaster genomic sequence nucleotides 4069 to 4296 and nucleotides 4798 to 4984 (accession AF190403) and is numbered relative to the U2-type 5′ splice site. The splice site consensus sequences are underlined; U12-type splice sites are in dark red; U2-type splice sites are in blue; the purine-rich element (PRE) is green. Residues mutated in R106-135 are shown in yellow; those in the R136-177 mutation are shown in purple. (B) Summary of deletion and replacement mutations used to delineate pros twintron splicing regulation in vitro and in vivo. The sequence of the purine-rich element of the wild-type pros substrate is shown on top in green. Sequences that were kept intact are shown in green, and those that were mutated are in purple. Heterologous SR protein binding sequences are shown in orange; dashes represent deletions. Minigenes are named by designating the nucleotides deleted (Δ) or replaced (R) within the intron.

FIG. 3.

In vitro splicing of prospero substrates in Drosophila Kc nuclear extract. (A) Reverse transcription-PCR analysis of unspliced (unspl; lane 1) and wild-type pros (lane 2) in Kc nuclear extract in the presence of the U12a or U2b 2′-O-methyl blocking oligonucleotides (lanes 3 and 4). Lanes 5 and 6 show in vitro-transcribed RNAs derived from pros-L and pros-S cDNAs, which were reverse transcribed with oligonucleotide PR324 followed by PCR amplification with primers PR514 and PR323. Amplified DNA fragments were resolved on 12% polyacrylamide gels. The pre-mRNA and the spliced products, pros-L and pros-S, are indicated. (B) Splicing of the pros twintron requires sequences within intron 2. The pros splicing substrates listed in Fig. 2B were spliced in vitro, reverse transcription-PCR amplified, and analyzed as in A. (C and D) Graphical representation of the data in B showing the percentage of spliced product for each pros substrate tested, calculated as percent pros-L/pre-mRNA (left-hand scale) and percent pros-S/pre-mRNA (right-hand scale). Measurements were made directly from the gels by PhosphorImager analysis. Each value is an average of at least three independent splicing reactions; error bars indicate the standard deviation associated with each average value. Black bars, pros-L products of U2-type splicing; gray bars, pros-S products of U12-type splicing.

FIG.4.

First 20 nucleotides of the purine-rich element are necessary for pros in vitro splicing. (A) Purine substitutions that substitute a heterologous sequence for the purine-rich element while preserving the spacing found in the parent substrate, wild-type pros. The wild-type purine-rich element sequence is underlined. (B) The substrates in A were spliced in Kc nuclear extract, followed by reverse transcription-PCR. The pre-mRNA and the spliced products (pros-L and pros-S) are indicated. (C) Graphic representation of pros-L and pros-S spliced products. Each percentage, calculated as described for Fig. 3C and D, is from a minimum of three independent experiments.

FIG. 5.

Inhibition of in vitro splicing by competition with purine-rich element RNA. (A) Kc nuclear extract was incubated with pre-mRNA and increasing amounts of an RNA containing the purine-rich element sequence (lanes 5 to 7) or a nonspecific sequence (Fig. 2B, R32-77; mPRE, lanes 2 to 4) that does not substitute for the purine-rich element in in vitro splicing assays. After splicing, the RNAs were analyzed by reverse transcription-PCR as above. The molar excess of competitor is indicated at the top of each lane. The wild-type pros pre-mRNA and products, pros-L and pros-S, are indicated. (B) Graphic representation of the data in A showing the percentage of pros-L and pros-S spliced products in the presence of RNA competitors. Each value was calculated as percent pros-L (or pros-S)/pre-mRNA; the value of wild-type pros with no competitor was set at 100%.

FIG.6.

Splicing of prospero substrates in Drosophila S2 cells. (A) Organization of the pros minigene used for transfection. The line underneath the construct shows the probe used in the RNase protection assay. Vector sequences are not drawn to scale. (B) Representative gels of RNase-protected fragments analyzing the products of transient transfection. The pros-S product is always detected as a doublet, often seen in RNase protection analyses. (C and D) The bar graphs show the levels of pros-L splicing for each substrate tested. Error bars indicate the standard deviation of the measurements from four to six in vivo splicing experiments. The percent of pros-L was defined as [(total counts in the pros-L product)/(total counts in the pre-mRNA)].