Numerous changes in alternatively spliced mRNA isoforms induced by insulin and wingless. (a) Features of microarray design. The array contains 36-mer probes complementary to each exon and splice junction (sjnc) for all annotated Drosophila genes for which there is evidence of alternative splicing. The number of genes, mRNAs and probes present in the array are indicated. (b) Summary of regulated junctions and genes detected upon activation of insulin and wingless pathways. (c) Distribution of classes of alternative splicing events for all Drosophila genes (left) and for those regulated by insulin (middle) and wingless signaling (right). AFE, alternative first exon; ATE, alternative terminal exon; alt3(5)'ss, alternative 3(5)'splice site.

Overrepresented sequence motifs present at the 5' end of intronic regions associated with splice junctions regulated by the (a) wingless and (b) insulin pathways. Motifs were derived from a dataset of sequences corresponding to the 50 nucleotides of introns flanking splice junctions that change upon activation of a signaling pathway, as well as the corresponding regions in the same intron of the other 11 Drosophila species. Motifs were identified using MEME and PHYLOGIBBS software and the specificity of the enrichment assessed with a set of control sequences derived from constitutive and alternative splice junctions that do not change upon activation of the signaling pathway. A detailed account of motifs and statistical assessment of their significance can be found in [63]. Represented are the relative frequencies of each nucleotide at each position in the nine nucleotide motifs. Genes containing the junctions included in each of the motifs are as follow. Insulin motif (44): sbb, cg15611, graf, cg7995, cg13213, cul-2, cher, ald, cg6265, cg7950, cg1021, cg7059, tomosyn, cg8036, cg1141, wdb, cg3168, cg8789, cg32425, cg16833, cg13499, cg4502, cg31732, cg32103, cg33085, sesB, scb, sdc, nemy, Ef2b, keap1, drpr, cg15105, : cg5059, spi, cg6231, cg14869, cpx, spri, cg16758, dom, Ca-P60A, ptp99A, cg33130. Wingless motif (10): stat92E, trx, cg2747, smi35A, hph, ced-6, cg33130, slo, cg4502, cg5794.

Activation of insulin and wingless signaling pathways in Drosophila S2 cells. (a) Schematic representation of the insulin and wingless signal transduction cascades and controls of their activation in our experimental system. Key protein components and their interactions for each pathway are schematized. Dashed lines represent cell and nuclear membranes. C and N indicate cytoplasm and nucleus, respectively. Stimulation of insulin signaling from 0-8 h was monitored by western blotting using an anti-phospho-Akt antibody (left panel). Activation of the wingless pathway, achieved through RNA interference (RNAi)-mediated depletion of axin (axn), resulted in the nuclear accumulation of Armadillo (Arm) as assessed by western blot analysis and activation of a known target gene, naked cuticle (nkd) monitored by RT-PCR (right lower panel). Amplification of tubulin (tub) transcripts served as loading control. The arrow indicates the time-point used for our microarray analysis. (b) Distribution of genes showing transcriptional up- and down-regulation upon activation of insulin and wingless. (c) Validation of microarray predictions by quantitative RT-PCR. Three genes are shown for each pathway. Results are presented as log2 ratio of signals obtained under conditions of pathway activation and controls. Z-scores predicted by microarray data analysis are indicated below the graphs.

Validation of microarray-predicted changes in splice junctions using quantitative RT-PCR. Examples of alternative splicing patterns regulated by (a) insulin and (b) wingless signaling are shown. For each gene, a primer pair was designed to amplify a constitutive part of the transcript, thus monitoring general changes in transcription (exp). In addition, primer pair(s) in which one of the primers covers a splice junction were used to amplify and monitor changes in expression of particular isoforms, as indicated. Changes in splice junctions were evaluated relative to the change in gene transcription. RT-PCR results are presented as log2 ratio of eCp values obtained under conditions of pathway activation and controls. The corresponding Z-score values from the microarray prediction are indicated below the graphs for each event. Various classes of alternative splicing events are detected, including alternative first exons, alternative 5' or 3' splice sites, cassette and mutually exclusive exons and more complex patterns. In some cases, expression changes are not significant and alternative splicing changes are detected in the absence of significant changes in expression (for example, wdb, cg2201, trx, stat92E). In others, changes in splice junctions are clearly distinct from changes in expression (for example, cg14207) or even occur in the opposite direction (for example, babo). In some instances, changes in one splice junction probe monitoring a particular spliced isoform are not reciprocated by converse changes in probes monitoring the alternatively spliced product. This suggests the existence of additional processing pathways. Indeed, semi-quantitative RT-PCR using primers external to some of the alternatively spliced regions frequently detects the existence of additional, non-annotated isoforms (data not shown).