Global microarray analysis. (A) Plot of total expression values versus log expression ratio of each positive control probe reveals a strong positive skew. Equal amounts of each positive control RNA were added to control and experimental samples. (B) The distribution of the net expression for each splice junction probe across all experiments is shown in the histogram. Cut-offs for up- or down-regulation were set at 2 standard deviations from unchanged. (C) Size of the sets of alternative and constitutive junctions that are strongly and consistently up- and down-regulated following knockdown of each splicing factor. dASF/SF2 affects the largest number of splicing events; PSI affects the smallest number. (D) Number of splicing events strongly and consistently affected by RNAi knockdown of each of two splicing factors. In parentheses are the expected sizes of each set assuming that each splicing factor affects in-dependent sets of splicing events. For each combination except B52 and PSI, the number of affected events is larger than expected by chance. (E) Antagonistically affected splice junctions. The numbers of splice events that were increased following knockdown of one splicing factor and decreased following knockdown of another are shown. Also shown is the number expected under the random model in which each splicing factor's affects are independent of the affects of the other splicing factors.

B52/SRp55-binding motifs near B52/SRp55 uniquely affected splice junctions. (A) Sequence logo of the previously identified B52/SRp55-binding motif (Shi et al. 1997). Lines connecting residues indicate predicted base-pairing interactions. Stars underneath residues indicate B52/SRp55 footprint contacts (Shi et al. 1997). (B) Pairs of motifs similar to the previously identified B52/SRp55-binding motif (Shi et al. 1997) are overrepresented around 5′ splice sites that are down-regulated when B52/SRp55 is knocked-down relative to the 5′ splice sites affected in the other experiments. No significant difference is seen in 5′ splice site regions around up-regulated junctions or in either up- or down-regulated 3′ splice site regions. Error bars are determined analytically using the binomial distribution and correspond to 1 standard deviation: sqrt[np(1 - p)]/n, where n is the number of sequences searched and p is the observed probability of having sites of given score.

Experimental design and clustering results. (A) Thirty-six-mer probes were selected for all alternatively spliced junctions from the GadFly 3.2 Drosophila genome annotation (“a” probes). For each gene, two exonic probes were selected from regions common to all isoforms to gauge total gene expression (“e” probes). Up to two constant constitutive junction probes were also selected (“c” probes). (B) Immunoblot analysis confirmed effective RNAi knockdown of the hnRNP proteins PSI and hrp48 and of the SR proteins B52/SRp55 and dASF/SF2. (C) Hierarchical clustering using average log expression ratios from all splice junction probes was performed to assess the global affects of biological replicates of each splicing factor RNAi knockdown and to compare between splicing factors. This analysis indicates that the dASF/SF2, B52/SRp55, and hrp48 experiments produce a characteristic splicing response. The PSI results, however, were more variable (see text). The global splicing response to dASF/SF2 or B52/SRp55 knockdown includes more similarities than either does to hrp48 or PSI knockdown.

RT-PCR validation of selected targets. Shifts in alternatively spliced isoforms predicted from the microarray analysis were monitored by RT-PCR for three different targets using oligonucleotides flanking the affected alternative splice sites. (Bottom panel) Together with the RT-PCR gel analysis, the alternative spliced junction expression computed from the microarray data are shown. The densitometry of the gels are shown on the left expressed as a log₂ ratio of the two measured isoforms. (A) CG6084 is a predicted target of the SR protein B52/SRp55. B52/SRp55 knockdown promoted skipping of the alternative cassette exon. (B) CG6143 (PEP) is a predicted B52/SRp55 target whose cassette exon is included upon knockdown of B52/SRp55. (C) CG4912 is a predicted target of the hnRNP protein PSI. The knockdown of PSI promotes skipping of the alternative cassette exon.