Fig. 1. Assessing in vivo splicing rates by quantitative RT–PCR. Quantitation of unspliced introns from total cellular RNA containing pre-mRNAs of a hypothetical two intron-containing gene is schematized. In vitro-transcribed control RNA standards, truncated relative to the cellular RNA sequence by 10 nt, are added in equal amounts to separate reaction tubes containing a fixed amount of total cellular RNA. Reverse transcription is performed using short primers complementary to intronic sequences (1RT or 2RT). cDNAs are then PCR amplified using primer pairs that only amplify unspliced sequences (1F/1R or 2F/2R). The cellular and control RNA amplicons are then electrophoretically separated, and the quantity of each unspliced intron is inferred from the ratio of the amplicons produced.

Fig. 3. Plasmids that report the efficiency of U12-type versus U2-type splicing. (A) A segment of the D.melanogaster NHE3 gene including introns 4 (U2-type) and 5 (U12-type) and their flanking exons was fused, via a linker, to a yellow (Y) or cyan (C) fluorescent protein coding sequence containing destabilizing ‘PEST’ sequences to form U12Y or U12C. Intron 6 (U2-type) of NHE3 was inserted into the CFP or YFP coding sequences. A Gal4-responsive promoter and a Kozak consensus translation start site were directly upstream of the fusion protein. Exon and intron lengths are indicated above and below (not drawn to scale). To form U2Y (or U2C), consensus splicing sequences of the U12-type intron were mutated to U2-type sequences derived from an adenovirus intron, keeping the intron length constant. (B) The sequences of the protein-coding regions of the fusion constructs are shown, with exon sequences in uppercase and intron sequences in lowercase and alternative sequences indicated. Red, U12-type consensus sequences; green, converted U2-type sequences; gray, NHE3 exon sequences; brown, linker sequences; orange, PEST sequences. The mutations converting cyan to yellow fluorescence are highlighted in cyan and yellow, respectively. The mutations introduced to change hydrophobic amino acids in the predicted transmembrane domain of exon 5 are in bold. (C) A plasmid containing the U2C and U12Y constructs in tandem (named U2C–U12Y) was created to ensure equal transfection of both constructs. The reciprocal plasmid (U12C–U2Y) with a reversed color scheme controlled for inherent differences in CFP and YFP fluorescence yield.

Fig. 5. FACS analysis of transfected S2 cells. Density plots of yellow versus cyan fluorescence intensity for S2 cells co-transfected with metallothionein-Gal4 and various plasmids, obtained 12 h after cupric sulfate induction. The flow cytometer detection optics are inherently less sensitive for CFP than for YFP. 50 000 cells were observed on each plot. (A) Mock-transfected S2 cells show minimal cyan or yellow fluorescence. (B) U2Y-transfected cells show only yellow fluorescence. (C) U2C-transfected cells show only cyan fluorescence. (D) U2C–U12Y-transfected cells show greater cyan than yellow fluorescence (correcting for low CFP detection sensitivity). (E) U12C–U2Y-transfected cells show greater yellow than cyan fluorescence.

Fig. 2. U12-type introns are excised most slowly from three endogenous human pre-mRNAs. (A) The intron–exon structure is depicted for the three human genes, SmE, E2F2 and INSIG1. Shaded boxes represent exons, thin lines represent U2-type introns and thick lines represent U12-type introns, with intron lengths (in kb) indicated above. (B) Above are shown the gels from which the ratios of cellular to control amplicons (105–164 nt long with controls being 10 nt shorter) were measured. The amounts of each unspliced intron within the pre-mRNA population from growing HeLa cells (for SmE and E2F2) or SK Hep cells (for INSIG1) are shown graphically below. Solid bars represent U2-type introns and hatched bars represent U12-type introns, with error bars indicating the standard deviation of two experiments. 4.56 × 10^–19 moles, 1.01 × 10^–19 moles and 4.47 × 10^–18 moles of control RNA standards were added per microgram of total cellular RNA for the analysis of introns from SmE, E2F2 and INSIG1, respectively. (C) Control co-amplification of equal amounts of in vitro-transcribed full-length and 10-nt truncated RNAs shows approximately equal production of amplicons (ratio of 0.91 ± 0.08). 10^–19 moles of each RNA species were RT–PCR amplified.

Fig. 4. U2-dependent splicing produces brighter fluorescence than U12-dependent splicing in Drosophila S2 cells. (A–C) CFP and YFP fusion proteins are distinguishable. Drosophila S2 cells were separately co-transfected with either U2C or U2Y and metallothionein-Gal4, mixed and viewed 12 h after cupric sulfate induction using a YFP filter (A), phase contrast (B) or a CFP filter (C). (D–I) S2 cells co-transfected with U2C–U12Y (panels D–F) or U12C–U2Y (panels G–I) and metallothionein-Gal4 were similarly induced and viewed using a YFP filter (D and G), phase contrast (E and H) or a CFP filter (F and I). Similar results were obtained with constructs lacking ‘PEST’ sequences (data not shown).

Fig. 6. mRNA production is slowed by inefficient splicing of a U12-type intron. (A) Northern blot of total RNA isolated from S2 cells co-transfected with U2Y (lanes 1–5) or U12Y (lanes 6–10) and metallothionein-Gal4 at the indicated times after induction of expression. The blot was successively hybridized with random-primed DNA probes complementary to the YFP coding sequence (top panel) and to a loading control, ribosomal protein 49 (bottom panel). The identity of the slowly migrating band as partially spliced pre-mRNA, was confirmed by hybridization to a probe specific for the middle intron (data not shown). (B) Quantitative RT–PCR analysis of unspliced introns (similar to Figure 2B) from total RNA prepared 12 h after induction of U12Y-transfected cells and amplified in the presence of a single in vitro transcribed RNA standard containing three separate 10-nt deletions, allowing quantitation of all three introns. 1.33 × 10^–17 moles of control RNA standard was added per µg of total cellular RNA. Hatched bar, U12-type intron; solid bars, U2 type introns; error bars, standard deviation of two experiments. (C) Similar analysis of U2Y-transfected cells. Note that 10-fold less control RNA standard (1.33 × 10^–18 moles per µg of total RNA) was used. (D) Confirmation of equal amplification efficiency of in vitro transcribed full-length and 10-nt truncated RNAs (amplicon ratio of 1.02 ± 0.02) (as in Figure 2C). 10^–19 moles of each RNA species were RT–PCR amplified.