1. Generation of Alternative Ultrabithorax Isoforms and Stepwise Removal of a Large Intron by Resplicing at Exon–Exon Junctions 
Allyson R Hatton, Vaidyanathan Subramaniam, A.Javier Lopez 
Molecular Cell 
Volume 2, Issue 6, Pages (December 1998)
DOI: /S (00)

2. Figure 1 Alternative Splicing of Ubx RNAs
The structure of the Ubx transcription unit is shown at the top, not drawn to scale. E5′, common exon at 5′ end; B, B element (27 nt), defined between two alternative 5′ splice sites (labeled “a” and “b”) for exon E5′; mI, microexon I (51 nt); mII, microexon II (51 nt); E3′, common exon at 3′ end; HD, homeodomain. The structures of the alternatively spliced Ubx isoforms and a summary of their expression are shown below (for detailed description, see Lopez et al. 1996). The b subtypes (which contain the B element) comprise a minor proportion of each major class (I, II, and IV). Class I is also expressed in the CNS but at very low levels. Class II is expressed at highest levels in the CNS but also in other tissues. Classes II and IV are expressed in distinct but overlapping stage- and neuron-specific patterns in the CNS.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2 Exon Skipping and Resplicing Hypotheses
(A) Under the exon skipping hypothesis, mI or mI plus mII are excluded from tissue-specific mRNAs by ignoring their 3′ and 5′ splicing signals; the upstream and downstream exons are spliced to each other directly, and the cassette exon is removed together with the flanking introns. A decreasing number of discrete introns is removed to produce isoforms Ia, IIa, and IVa, but their size increases. Only the production of isoforms lacking the B element is shown; to include the B element, the downstream 5′ splice site of exon E5′ (site “b” in Figure 1) would be used.
(B) mI and mII are flanked by potentially competing 5′ splice sites after joining to E5′. The diagram shows the intermediates produced when mI or mII are joined to E5′ at the “a” or “b” sites. Rectangles indicate exons. M = C or A; R = purine. The E5′a/mI and E5′a/mII junctions regenerate good potential 5′ splice sites based on similarity to the Drosophila consensus (Mount et al. 1992), whereas the junctions with E5′b should be weak sites.
(C) Under the resplicing hypothesis, mI and mII are spliced constitutively to the upstream exon in the nascent transcript; each regenerates a 5′ splice site at the junction with E5′, and this site is used to remove the cassette exon in specific cell types during splicing of the downstream intron. This allows each intron to be removed independently as soon as it is transcribed, avoiding the synthesis of a full-length intermediate regardless of the isoform to be produced. Inclusion of mI and mII is regulated by modulating the competition between the 5′ splice sites that come to flank them in the splicing intermediates. Only isoforms lacking the B element are shown; the B element would be included by using 5′ splice site “b” for exon E5′ and the 5′ splice sites that this regenerates with mI and mII (see [B]).
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3 Analysis of Partially Spliced RNAs Produced by UbxMX17
Reverse transcription was primed with an oligonucleotide targeting Ubx intron 2 downstream of exon mI and amplification was performed with primers targeting exon E5′ and exon mI. The identities of the E5′b/mI and E5′a/mI splicing products were verified by diagnostic digests with NotI (cleaves in E5′) and BglII (cleaves in mI).
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4 The Ubx Minigene System
The minigenes (not shown to scale) contain all four Ubx exons and encode the entire open reading frame; the introns have been shortened internally, and the minigenes end 288 bp downstream of the first Ubx cleavage/polyadenylation signal. The homeodomain has been replaced with an HA epitope tag fragment to allow distinction from endogenous Ubx RNAs. Restriction enzyme sites at the junctions of individual fragments are shown: R, EcoRI; H, HindIII; P, PstI; B, BamHI. Other symbols as in Figure 1. In Ubx.4F1, exons E5′ and mI have been fused as if spliced using the “a” 5′ splice site of E5′. In Ubx.4mI*, the indicated mutations within mI are expected to reduce the efficiency of the 5′ splice site regenerated at the E5′/mI junction. In Ubx.4a*, the indicated mutations are expected to reduce the efficiencies of the “a” 5′ splice site and of the site regenerated at the E5′a/mI junction.
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 5 Alternative Splicing of Ubx Minigenes in Transgenic Embryos
(A) Minigenes were expressed in different tissues under control of the GAL4 system, and the mRNA splicing patterns were analyzed by quantitative RT-PCR. The minigene construct used in each set of three experiments is indicated at the top. The tissue in which the minigene was expressed under GAL4 control (in addition to salivary gland) is indicated above each lane: E, epidermis; M, mesoderm; N, neurons. The identities of the amplified bands (confirmed by diagnostic restriction digests) are indicated at the left. Similar results were obtained with two independent insertions of each minigene.
(B) Minigene Ubx.4 was expressed in neurons plus salivary gland and the splicing pattern was analyzed by quantitative RT-PCR using total RNA extracted from whole larvae or from dissected CNS. The identities of the amplified bands were confirmed by diagnostic restriction digests. The arrowhead marks isoform IVa, and the asterisk marks a band that is unrelated to Ubx and represents an endogenous cross-reacting RNA expressed specifically in the salivary gland of late larvae.
(C and D) Analysis of minigene expression and splicing in salivary gland and CNS by immunohistochemical staining. Minigene Ubx.4 was expressed under the control of the elavc155 GAL4 enhancer trap, and embryos were stained with monoclonal antibody 12CA5 (C), which recognizes the HA epitope in all isoforms produced from minigene mRNAs, or with monoclonal antibody 3.11F (D), which recognizes an epitope in mI (Lopez and Hogness 1991). Arrows indicate the salivary glands; arrowheads indicate a portion of the CNS (brain) in a different optical plane.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 6 Alternative Splicing of Ubx Minigenes in SL2 Cells
(A) Isoform II is produced by resplicing at the E5′a/mI junction. Minigenes under control of the polyubiquitin promoter were transfected into Drosophila SL2 cells, and the mRNA splicing patterns were analyzed by quantitative RT-PCR. The minigene used is indicated above each lane. WT is Ubx.4.
(B) Deletion of mII induces constitutive resplicing of mI.
Top: Structure of the ΔmII minigene. mII and part of the flanking intron sequences were deleted as a PstI fragment. P indicates the resulting junction between introns 2 and 3.
Bottom: Splicing patterns of ΔmII minigenes in SL2 cells. The identities of the mRNAs are indicated at the left for the wild-type minigene and at the right for the mutant minigenes. The identities of the isoforms and the accuracy of splice junctions were verified by diagnostic restriction digests and by direct sequencing of amplified products resolved on 2% agarose gels; the slower mobility of isoform IIIa (E5′a/mI/E3′) produced by the ΔmII mutants compared to isoform IIa (E5′a/mII/E3′) produced by the wild-type minigene is due to the difference in nucleotide sequence between these isoforms, which have identical lengths, and not by inaccurate splicing.
Molecular Cell 1998 2, DOI: ( /S (00) )

8. Figure 7
Conserved Features of mI Sequence Play a Role in Control of Resplicing
(A) Sequence of wild-type and mutant versions of mI. The complete sequence of wild-type mI is shown in the center. The identical sequence has been conserved among four Drosophila species spanning 60 million years (Bomze and Lopez 1994). The nucleotides that regenerate a 5′ splice site when juxtaposed to E5′ are underlined. The GC-rich element is overlined. The nucleotide changes in the mI1 and mI2 mutations are shown above and below the wild-type sequence.
(B) Splicing patterns of mutant minigenes. Minigenes under control of the polyubiquitin promoter were transfected into Drosophila SL2 cells, and the mRNA splicing patterns were analyzed by quantitative RT-PCR. The minigene used is indicated above each lane. WT is Ubx.4.
Molecular Cell 1998 2, DOI: ( /S (00) )