Supplementary Protocols

Functional annotations

Probe sequence and their annotations for the custom microarray platform employed in the analysis (Blanchette et al. 2005) were downloaded from the NCBI GEO database through accession GPL7508. Due to the turnover of the FlyBase CG-identifiers employed in these annotations to assign probes to the transcripts that contain them, we have re-annotated the transcript and assignments to all the 42,034 probes as follows. First we downloaded from the UCSC Genome Browser all the FlyBase and RefSeq annotations on the Dm1 version of the Drosophila melanogaster genome which correspond, respectively, to 18,746 and 21,617 transcripts whose sequence was obtained from their genome annotation. Second, we built a non-redundant set of 25,965 FlyBase and Refseq transcript sequences and annotations. Third, we annotate probes to those transcripts for which we find a perfect match of the probe sequence. Through the transcript annotations in the genome we also annotated the genomic position of each probe within each matched transcript, the genomic position of the functional element associated to the probe (an intron for a junction probe and an exon for an exon probe) and the genomic position of the upstream and downstream exon in junction probes. Using this information we also annotated what probes map to multiple genomic locations. Fourth, using the Bioconductor organism-level annotation package for Drosophila melanogaster org.Dm.eg.db we fetch the Gene (EG) identifiers that correspond to each of the transcripts and annotate probes to the corresponding EG identifiers through the transcript(s) they are probing. Using this information we also annotated what probes map to multiple Entrez Gene identifiers. Fifth, using the pairs of probe and EG identifier and the

AnnotationDbi package from Bioconductor, we build a custom annotation package employed later throughout the analysis, storing all the retrieved information and other functional annotations that the Bioconductor infrastructure pulls out from other databases through the EG identifiers.

We also included into this custom annotation package probe annotations of alternative splicing events generated as follows. Using software from the UCSC Genome Browser (Kent et al. 2002) source code (http://genome.ucsc.edu/admin/cvs.html), in particular the tools

txBedToGraph and txgAnalyze, and the non-redundant set of 25,965 FlyBase and Refseq transcripts, we first generated a collection of 15,280 genome-wide alternative splicing (AS) event annotations. Next, we annotated junction probes to all those AS events whose genomic coordinates intersected those from the corresponding intron where, in the case of alternative transcription start site and alternative polyadenylation site, the intersection was considered also with respect to the upstream and downstream exons since such AS event annotations were overlapping the 5' and 3' end of exons, respectively.

Processing of the microarray data and non-specific filtering

Microarray data was processed using the limma package (Smyth 2004) from the Bioconductor software (Gentleman et al. 2004) and, more concretely, they were background-corrected using the "normexp" model with maximum likelihood estimation and an offset value of k=50 was added before taking

logarithms (i.e., M=log2[(R+k)/(G+k)]) in order to shift the intensities away from 0 and stabilize the variance of the log-ratios at low intensities (Silver et al. 2009). We normalized expression values within each array employing loess normalization and further normalized their scale between the arrays.

The initial set of 42,034 non-control probes was filtered to discard probes mapping to multiple genomic positions and probes that either were associated to more than one EG identifier or for which no EG identifier was found, leaving a set of 41,022 probes. This set was further filtered to discard junction probes for which their associated gene had no exon probes, and discard junction and exon probes belonging to with less than 2 exon probes, leaving a final set of 40,898 probes where 19,611 corresponded to splice (exon-exon) junction probes and 21,287 to exon probes.

Gene-level log2 expression ratios were obtained by, first, summarizing log2 expression values of exon probes with common EG identifier separately for male and female samples. This summarization was calculated by using the median polish algorithm, which is also employed in the summarization step of the RMA procedure for Affymetrix expression arrays (Irizarry et al. 2003) and aims to protect against outlier probe values. Second, the summarized log2 expression values from the female samples were subtracted from the ones of

the male samples in order to obtain a final set of 2,664 gene-level log2 expression ratios.

Finding differentially expressed genes

M Let xgk represent the log2 expression value in replicate k from a male sample hybridized to the Cy5 channel for gene g, obtained by summarizing the background-corrected and normalized expresssion values of the exon probes

F occurring within the gene, as previously described. Let xgk represent the analogous value from a female sample hybridized to the Cy3 channel. The

log2 ratio of expression between a male and a female fly for gene g in

replicate k is thus defined as

M F ygk = xgk − xgk .

Using the limma package (Smyth 2004) from Bioconductor and the following simple linear model

ygk = µg + εgk ,

where εgk are the residual error terms and µg is the expected log2 ratio of expresssion between male and female flies for gene g, we obtain estimates of µ and their standard errors for each gene by fitting the model to the log g 2

ratios ygk of the 3 replicates. Estimates and corresponding standard errors are

then employed to compute moderated t-statistics by the empirical Bayes shrinkage method (Smyth 2004) implemented in which borrows limma information across probes and increases the effective degrees of freedom, thus improving the rate of detection of differentially expressed. By setting cutoff values of minimum fold-change to 2 and maximum adjusted P-value to 1%, we call in this way 270 genes as being differentially expressed between male and female flies.

Finding differentially regulated splice junctions

We call a splice (exon-exon) junction differentially regulated if its corresponding junction probe changes significantly due to distinct post- transcriptional regulatory events of splicing between male and female flies. Following a similar approach to (Blanchette et al. 2005) we first estimate the

so-called log2 ratio of net-expression which corresponds to the log2 ratio of expression of a junction probe normalized by its corresponding gene-level

M log2 ratio of expression. More, concretely, let x jk represent the log2 expression value in replicate k from a male sample hybridized to the Cy5 channel for junction j. Let x F represent the analogous value from a female jk sample hybridized to the Cy3 channel. The log2 ratio of expression between a male and a female fly for junction j in replicate k is thus defined as

M F y jk = x jk − x jk .

Let the g(j) indicate the gene containing junction j and let yg( j )k be the log2 ratio of expression for gene g(j) and replicate k thus representing the same quantities as the previous y values but retrieving the corresponding gene- gk

level log2 ratio of expression for junction j. Then, the log2 ratio of net- expression for junction j and replicate k is calculated as

net M F M F y jk = y jk − yg( j )k = x jk − x jk − (xg( j )k − xg( j )k ) ,

which by re-organizing terms as follows

net M M F F y jk = x jk − xg( j )k − (x jk − xg( j )k ) ,

can be interpreted as the log2 ratio of expression between male and female flies for the expression value of a junction normalized by its corresponding gene-level expression. However, differently to (Blanchette et al. 2005) and analogously to the previously described gene expression analysis we fit again a simple linear model

net net y jk = µ j + εgk ,

net to the log2 ratios of net-expression y jk in order to estimate the expected log2

net ratio of net-expression µ j for junction j and its standard error. Again, using

the empirical Bayes method implemented in limma and setting cutoffs of fold- change to 2 and adjusted P-value to 1% we call 986 junctions as being

differentially regulated between male and female flies.

Analysis of the hnRNP splicing microarray data

The raw hnRNP splicing microarray data from (Blanchette et al. 2009) was downloaded from GEO through accession GSE13940. Since these data was produced with the same microarray platform that used in this work, the same pre-processing and differential analysis steps were taken with the exception of the experimental design we employed which consisted of a common reference design with one control RNA source hybridized to the Cy3 channel while the different knockout experiments and replicates were hybridized to the Cy5 channel. We also employed a slightly smaller minimum fold-change cutoff value of 1.5 due to the narrower response to a knockout as opposed to the response observed for sex-specific differences while the maximum adjusted P-value cutoff was the same as in the sex-determination analysis (1%). This analysis yielded four lists of differentially regulated junctions, one for each hnRNP factor, which were considered for enrichment with respect to the junctions differentially regulated by sex-specific changes.

References

Blanchette M, Green RE, Brenner SE, Rio DC (2005) Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 19(11): 1306-1314. Blanchette M, Green RE, MacArthur S, Brooks AN, Brenner SE et al. (2009) Genome-wide analysis of alternative pre-mRNA splicing and RNA-binding specificities of the Drosophila hnRNP A/B family members. Mol Cell 33(4): 438-449. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome biology 5(10): R80. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ et al. (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics (Oxford, England) 4(2): 249-264. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH et al. (2002) The browser at UCSC. Genome research 12(6): 996-1006. Silver JD, Ritchie ME, Smyth GK (2009) Microarray background correction: maximum likelihood estimation for the normal-exponential convolution. Biostatistics (Oxford, England) 10(2): 352-363. Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology 3: Article3.

Hartmann_Suppl. Table1 results of qRT-PCR: AVG: average and STDEV: standarddeviation

No. of SXL- sites in 1000bp Fly Body Head Kc/S2 KC/Kc-sxl RNAi "tud" flies "tud" head window ubiquitous "event" AVG STDEV AVG STDEV AVG STDEV AVG STDEV AVERAGSTDEV AVG STDEV AVG STDEV category ccr4.exp 0.23 0.02 -0.31 0.03 -0.16 0.03 -0.18 0.02 -0.27 0.04 0.34 0.17 -0.74 0.22 ccr4.sjnc -7.87 0.14 -8.74 0.06 -4.83 0.12 -0.95 0.35 -0.84 0.14 -0.22 0.46 -0.81 0.32 1 jigr1.exp 3.61 0.06 3.50 0.06 -0.07 0.05 0.90 0.04 0.00 0.05 -1.02 0.09 -0.88 0.13 jigr1.sjnc 10.20 0.45 9.81 0.16 3.70 0.36 2.69 0.19 0.18 0.96 absent 2.55 0.27 6 exba.exp 1.29 0.14 1.65 0.27 0.12 0.04 0.01 0.07 0.06 0.17 0.15 0.31 -0.32 0.03 exba.sjnc 4.03 0.09 4.87 0.13 1.92 0.06 0.79 0.06 1.18 0.05 1.52 0.02 0.31 0.05 Hrb27c.exp 0.97 0.03 2.94 0.03 0.33 0.06 0.36 0.17 0.20 0.01 -0.27 0.18 -0.47 0.06 Hrb27c.sjnc1 0.19 0.25 1.87 0.20 -0.73 0.21 -1.92 0.39 -1.77 0.35 -0.64 0.32 -0.72 0.02 Hrb27c.sjnc2 -1.32 0.07 0.42 0.15 -1.63 0.10 -0.25 0.07 -2.01 0.09 -0.94 0.16 -0.96 0.07 3 rbp2.exp 2.25 0.07 0.14 0.03 0.23 0.13 1.61 0.03 0.18 0.06 -0.15 0.15 -0.28 0.13 rbp2.sjnc -0.06 0.09 -0.85 0.19 -0.84 0.07 0.26 0.14 0.58 0.33 -0.64 0.03 -0.89 0.02 sqd.exp 1.12 0.12 2.77 0.02 0.47 0.02 1.74 0.02 -0.15 0.10 0.99 0.02 0.07 0.54 sqd.sjnc -0.66 0.08 0.34 0.04 -0.43 0.12 0.57 0.04 -0.16 0.08 -0.11 0.39 0.33 0.12 1 Ubi-p63E.exp -1.33 0.00 -0.81 0.02 1.05 0.01 -3.03 0.01 1.59 0.07 0.56 0.06 -0.32 0.01 Ubi-p63.sjnc -11.71 0.69 -10.31 0.21 -1.12 0.74 -10.47 19.19 0.93 2.11 0.72 0.69 0.16 0.21 1 unc115.exp -2.39 0.04 -1.34 0.05 -1.05 0.07 -1.24 0.03 0.96 0.12 -0.53 0.03 -0.80 0.02 unc115.sjnc -7.59 0.81 -7.25 0.61 -4.25 0.52 -4.37 0.40 1.00 0.21 -0.43 0.21 -0.91 0.17 absent in cells LIMK1.exp -0.62 0.03 0.53 0.03 -0.41 0.07 0.07 0.05 -0.03 0.10 -0.43 0.13 -0.99 0.02 LIMK.sjnc -8.85 0.40 -9.17 0.02 -2.90 0.70 absent absent -0.60 0.33 -0.46 0.30 2 BicC.exp 4.36 0.04 3.44 0.06 4.28 0.08 absent absent absent absent BicC.sjnc 16.84 0.39 12.61 0.33 8.82 0.17 absent absent absent absent 3

Tissue-restricted Fly Body Head Kc/S2 KC/Kc-sxl RNAi "tud" flies "tud" head category "event" AVG STDEV AVG STDEV AVG STDEV AVG STDEV AVERAGSTDEV AVG STDEV AVG STDEV Body-specific Bj1.exp 2.03 0.16 4.14 0.04 -0.57 0.17 1.05 0.24 ND -0.06 0.12 Bj1.sjnc1 -1.81 0.05 -0.43 0.19 -0.51 0.17 1.51 0.07 ND -0.20 0.81 CaMKI.exp -0.76 0.20 -0.28 0.06 -0.01 0.04 1.42 0.29 -0.03 0.17 -1.76 0.01 CaMKI.sjnc 0.63 0.18 0.81 0.09 0.65 0.75 3.02 0.04 1.17 0.01 -2.20 0.30 capu. exp -0.49 0.51 absent absent 0.06 bad Tm curve capu.constjnc -1.77 0.04 -2.78 0.07 -0.80 0.11 absent absent 0.00 1.27 0.01 2 capu.sjnc1 2.33 0.27 2.61 0.14 0.04 0.06 absent absent 2.69 ND CG14098.exp -7.25 0.15 -6.30 0.09 -1.06 0.06 -1.84 0.25 -0.52 0.74 0.34 0.08 CG14098.sjnc -12.11 0.12 -8.89 0.10 -1.35 0.63 absent absent 0.92 0.52 1 CKII.b.exp 1.05 0.09 3.05 0.06 0.62 0.55 1.43 0.01 0.07 0.07 -0.42 0.02 CKII.b.sjnc -0.64 0.15 -0.93 0.12 1.10 0.20 1.21 0.10 0.88 0.13 -0.55 0.02 3 cnn.exp 0.80 0.05 1.07 0.15 -0.98 0.14 1.85 0.13 ND 0.51 0.25 cnn.jnc -7.62 0.42 -1.93 0.12 -0.58 0.18 1.51 0.08 ND 0.23 0.50 DopR2.exp 0.21 0.26 1.07 0.05 -0.63 0.13 -0.51 0.06 0.33 0.24 absent DopR2.sjnc 0.90 0.17 0.85 0.07 -0.69 0.08 absent absent absent eIF3-S9.exp 0.51 0.22 2.38 0.08 -0.43 0.77 0.02 0.02 -0.04 0.01 0.20 0.03 eIF3-S9.sjnc -10.54 0.88 -7.00 0.17 absent 0.96 absent absent absent 1 ftz-f1.exp 2.02 0.15 4.23 0.06 -1.00 0.06 -1.55 0.07 2.75 0.03 absent ftz-f1.sjnc 13.26 22.77 0.84 1.03 -0.91 0.48 absent absent absent 1 Grb28b.exp -0.80 0.29 3.17 0.04 -1.00 0.19 2.77 0.17 -0.52 0.93 -1.47 0.06 Grb28b.sjnc 0.53 0.15 15.00 0.47 -1.20 0.80 absent absent -1.41 0.30 imp.exp -3.50 0.12 -4.26 0.04 -0.25 0.18 -2.76 0.05 0.76 0.02 1.14 0.08 imp.sjnc -11.63 0.27 -10.97 0.27 absent absent absent 0.18 absent 2 ps.exp -2.02 0.05 -2.28 0.09 0.45 0.07 -1.57 0.15 ND 0.18 0.45 ps.sjnc -0.06 0.13 0.59 0.14 0.66 0.04 absent ND 0.40 0.41 rm62.exp 1.84 0.01 2.28 0.08 -0.63 0.05 -1.09 0.06 0.65 0.02 -0.85 0.02 rm62.sjnc 0.38 0.44 -0.28 0.21 -0.71 0.19 -0.34 0.13 0.56 0.22 -0.64 0.23 smg.exp 5.96 0.05 5.80 0.02 0.26 0.10 -0.60 0.42 -0.36 0.06 0.11 0.12 smg.sjnc 3.13 0.70 2.75 0.58 0.07 0.18 -1.06 0.35 -0.42 0.54 absent Ald.exp -0.83 0.30 -1.40 0.08 -0.23 0.10 2.34 0.01 0.84 0.08 0.35 0.04 Ald.sjnc -9.29 0.93 -6.78 0.12 -0.07 0.14 2.19 0.02 0.60 0.11 0.92 0.40 B52.exp 2.19 0.31 2.94 0.03 -0.01 0.18 0.25 0.08 0.66 0.04 -1.68 0.04 B52.sjnc -0.07 0.08 1.30 0.08 -0.25 0.06 1.02 0.04 0.54 0.14 -1.62 0.03

Head-specific bcd.exp 3.44 0.17 2.68 0.46 -0.23 0.11 1.43 0.29 0.52 0.11 -0.27 0.13 -0.27 0.13 bcd.sjnc 8.52 0.29 3.27 0.13 3.25 0.15 1.67 1.06 0.36 0.38 -0.37 0.08 -0.37 0.08 Supplementary Table 1: Results of female/male ratios determined by quantitative RT-PCR for the 28 splicing changes validated from the microarray results in flies, bodies, heads, cell lines, and “tudor”-mutant flies and heads. Ratios were calculated from Cp values according to Pfaffl [85] and presented as average in log2 (AVG) and standard deviation

(STDEV) of triplicate experiments for each the general expression of the gene (exp) and the splice-junction (sjnc). The genes were sorted in the following categories: 1. ubiquitous group (changes observed in fly, body, head and cell lines, but also including genes that were absent in the cell lines) 2. tissue-specific group: changes observed in the fly and body only, or fly and head only. Expression is called absent if the CP values were equal or higher than 32, often with high variation between triplicates. The last column shows the number of potential SXL-binding sites identified in a 1000bp window surrounding the sjnc. ND: not done

Hartmann_Suppl.Table2

A) enrichment (biological process) of genes containing differentially regulated junctions Term OddsRatio Count Size GeneSyms negative regulation of oskar mRNA translation Inf 4 4 BicC, Hrb27C, apt, aret regulation of nuclear mRNA splicing, via spliceosome Inf 3 3 Hrb27C, Pep, Sxl establishment or maintenance of polarity of larval imaginal disc epithelium Inf 3 3 Moe, dlg1, l(2)gl female somatic sex determination Inf 3 3 Sxl, dsx, tra2 primary sex determination 19.17 5 6 Stat92E, Sxl, gro, otu, ovo G1/S transition of mitotic cell cycle 15.30 4 5 CycE, E2f, cdc2c, dap syncytial blastoderm mitotic cell cycle 15.30 4 5 CycA, CycB, grp, toc positive regulation of translation 11.53 6 8 Dp1, Hrb27C, orb, osk, pAbp, pum DNA damage checkpoint 11.44 3 4 grp, lok, pr-set7 nuclear-transcribed mRNA poly(A) tail shortening 11.44 3 4 Pop2, smg, twin DNA unwinding during replication 11.44 3 4 Dsp1, Top1, dre4 mRNA splice site selection 11.44 3 4 B52, SC35, pUf68 mRNA export from nucleus 11.44 3 4 Hel25E, nxf2, sqd pole plasm localization 11.44 3 4 Moe, csul, osk negative regulation of antimicrobial humoral response 11.44 3 4 Dsp1, Jra, Stat92E response to inorganic substance 11.44 3 4 CG7843, Cam, ZnT35C female germ-line sex determination 11.44 3 4 Sxl, otu, ovo negative regulation of protein modification process 11.44 3 4 Ptp61F, dap, pr-set7 DNA geometric change 11.44 3 4 Dsp1, Top1, dre4 negative regulation of immune response 11.44 3 4 Dsp1, Jra, Stat92E condensation 10.29 8 11 Bj1, Dp1, Su(var)2-10, Su(var)2-HP2, Top1, eIF-4E, pr-set7, skpA JAK-STAT cascade 9.58 5 7 CycD, CycE, Stat92E, Su(var)2-10, cdc2c negative regulation of translation 7.76 6 9 Sxl, bru-3, exba, pum, smg, sqd spindle assembly 7.68 6 9 Eb1, Grip84, chb, grp, mud, pbl negative regulation of cell cycle 7.64 4 6 CycA, Pten, dap, pum positive regulation of protein metabolic process 6.73 7 11 Dp1, Hrb27C, Nedd4, orb, osk, pAbp, pum germ-line cyst formation 6.38 5 8 aret, bol, hts, orb, otu posttranscriptional regulation of gene expression 5.55 25 43 AGO1, BicC, Dp1, Hrb27C, Hsc70-3, Hsc70-4, Pten, Ptp61F, Rm62, Sxl, Upf2, apt, aret, bol, brat, bru-3, exba, gw, loqs, orb, osk, pAbp, pum, smg, sqd negative regulation of protein metabolic process 5.46 14 24 BicC, Hrb27C, Ptp61F, Sxl, apt, aret, bru-3, chb, dap, exba, pr-set7, pum, smg, sqd neuromuscular synaptic transmission 5.09 4 7 Ca-P60A, Pka-R1, apt, tkv interphase 4.60 6 11 CycA, CycB, CycE, E2f, cdc2c, dap cytoplasm organization 4.60 13 24 Hrb27C, Moe, Nacalpha, Tm1, capu, csul, exu, orb, osk, spir, sqd, syt, yl mitotic sister chromatid segregation 4.48 7 13 Bj1, CycA, CycB, Su(var)2-HP2, chb, eIF-4E, toc pole plasm RNA localization 4.29 10 19 Hrb27C, Nacalpha, Tm1, capu, exu, orb, osk, spir, sqd, syt embryonic pattern specification 3.93 7 14 Gug, Tl, osa, retn, spz, stck, wun oocyte anterior/posterior axis specification 3.88 13 26 Hrb27C, Moe, Nacalpha, Tm1, capu, csul, exu, gus, orb, osk, spir, sqd, syt intracellular mRNA localization 3.87 11 22 Hrb27C, Nacalpha, Tm1, capu, csul, exu, orb, osk, spir, sqd, syt pole plasm oskar mRNA localization 3.85 9 18 Hrb27C, Nacalpha, Tm1, capu, exu, orb, spir, sqd, syt ribonucleoprotein complex biogenesis 3.85 8 16 AGO1, B52, CG5033, SC35, Ubi-p63E, me31B, pUf68, tra2 negative regulation of transcription from RNA polymerase II promoter 3.84 7 14 Aef1, chn, dsx, gro, ovo, retn, tim axon choice point recognition 3.82 5 10 Nedd4, ab, exba, lola, plexA positive regulation of transport 3.82 5 10 Cam, Kap-alpha3, Nedd4, Pld, olf186-F regulation of alternative nuclear mRNA splicing, via spliceosome 3.58 12 25 B52, CG6995, CG7879, Doa, Hel25E, Rm62, SC35, aret, dom, pUf68, sqd, tra2 negative regulation of metabolic process 3.53 8 17 Hsc70-3, Hsc70-4, Rm62, Su(var)2-HP2, Upf2, dom, gw, loqs regulation of cell cycle process 3.42 8 17 CycA, CycE, aret, bol, chb, dap, otu, skpA mRNA catabolic process 3.36 7 15 Pop2, Upf2, gw, pum, smg, sqd, twin posttranscriptional gene silencing by RNA 3.36 7 15 AGO1, Hsc70-3, Hsc70-4, Rm62, Upf2, gw, loqs oocyte differentiation 3.27 20 44 Hrb27C, Moe, Nacalpha, Pka-R1, Sxl, Tm1, capu, chb, csul, dap, exu, growl, gus, hts, orb, osk, spir, spz, sqd, syt oocyte construction 3.01 17 39 Hrb27C, Moe, Nacalpha, Pka-R1, Tm1, capu, chb, csul, exu, growl, gus, orb, osk, spir, spz, sqd, syt centrosome organization 2.98 7 16 Cam, Grip84, chb, cnn, grp, hts, skpA RNA splicing 2.93 18 42 B52, CG13900, CG6995, CG7879, CG7971, Doa, Hel25E, Hrb27C, Pep, Rm62, SC35, Sxl, aret, dom, dsx, pUf68, sqd, tra2 regulation of cellular protein metabolic process 2.91 25 59 BicC, CycE, Dp1, Hrb27C, Nedd4, Pka-R1, Ptp61F, Sxl, apt, aret, bol, brat, bru-3, chb, dap, exba, grp, orb, osk, pAbp, pr-set7, pum, smg, sqd, ssh asymmetric cell division 2.90 12 28 CycE, G-oalpha47A, Stat92E, aret, brat, chb, dap, hts, neur, pUf68, pum, tkv RNA splicing, via transesterification reactions with bulged adenosine as nucleophile 2.88 17 40 B52, CG13900, CG6995, CG7879, Doa, Hel25E, Hrb27C, Pep, Rm62, SC35, Sxl, aret, dom, dsx, pUf68, sqd, tra2 M phase of meiotic cell cycle 2.66 9 22 Grip84, Sxl, bol, c(3)G, cnn, eIF-4E, grp, mod(mdg4), mud mRNA processing 2.61 20 50 AGO1, B52, CG13900, CG6995, CG7879, Doa, Hel25E, Hrb27C, Pep, Rm62, SC35, Sxl, aret, dom, dsx, orb, pUf68, sm, sqd, tra2 chromosome segregation 2.58 14 35 Bj1, CG11265, CycA, CycB, Dp1, Eb1, Su(var)2-HP2, Top1, chb, cnn, eIF-4E, grp, mod(mdg4), toc nucleocytoplasmic transport 2.57 10 25 Bj1, Cam, Hel25E, Kap-alpha3, Nup154, nxf2, olf186-F, ran, sqd, tim regulation of cellular localization 2.57 10 25 Bj1, Cam, Hrb27C, Kap-alpha3, Nacalpha, Pld, exu, olf186-F, syt, tim stem cell division 2.56 8 20 CycA, Gbeta13F, Ggamma1, Stat92E, aret, pum, tkv, trol developmental maturation 2.50 18 46 Hrb27C, Moe, Nacalpha, Pka-R1, Tm1, capu, chb, csul, exu, growl, gus, orb, osk, rk, spir, spz, sqd, syt regulation of cell cycle 2.49 12 31 Bj1, E2f, Grip84, Pitslre, brat, cdc2c, dlg1, grp, l(2)gl, lok, pUf68, pr-set7 germ cell development 2.43 25 66 Act5C, Hrb27C, Moe, Nacalpha, Ntl, Pka-R1, Stat92E, Sxl, Tm1, capu, chb, csul, dj, exu, growl, gus, lok, osk, pUf68, pum, spir, spz, sqd, syt, tkv Wnt receptor signaling pathway 2.40 10 26 Axn, CkIIalpha, G-oalpha47A, Stat92E, dlp, fry, gish, gro, osa, skd negative regulation of biosynthetic process 2.39 19 50 AGO1,, Aef1, BicC, , CG31716, y , y ,Hrb27C, g , Sxl, apt, , aret, pbru-3, , chn, , dsx, g exba, , gro, ovo, , pum, , retn, p , smg, p , sqd, p , tim , , j, , g , g , g , , , p p, p , p , y , multicellular organismal development 2.36 31 88 tim, toc, tra2, wun M phase of mitotic cell cycle 2.35 14 37 APC7, Bj1, CycA, CycB, CycE, CycJ, Grip84, Su(var)2-HP2, aret, chb, csul, eIF-4E, pum, toc positive regulation of developmental process 2.35 11 29 CG7263, CycE, E2f, Eip75B, Nedd4, alph, cac, l(2)gl, lok, mod(mdg4), tkv nuclear division 2.32 15 40 APC7, Bj1, CycA, CycB, CycE, CycJ, Grip84, Su(var)2-HP2, aret, chb, cnn, csul, eIF-4E, pum, toc regulation of cellular component biogenesis 2.20 12 33 Hrb27C, Nacalpha, Nedd4, Pten, Tm1, cac, exu, pum, spz, ssh, syt, tkv

AGO1, Actn, Atpalpha, B52, CG13349, CycE, DAAM, Df31, Dp1, E2f, Fps85D, G-oalpha47A, His3.3B, Hrb27C, Jra, Mhc, Moe, Nacalpha, Pten, SC35, Su(var)2-HP2, cellular component assembly 2.17 46 133 Tm1, TppII, betaTub56D, capu, csul, dec-1, dlg1, exu, gus, l(2)gl, me31B, nrv2, nuf, orb, osk, pUf68, qua, spir, spz, sqd, ssh, syt, tkv, tra2, unc-13 positive regulation of cellular process 2.17 18 51 CG7263, CycD, CycE, Eip75B, Pld, alph, aret, bol, cac, dlp, eIF-4E, l(2)gl, lok, mod(mdg4), neur, skd, skpA, tkv determination of anterior/posterior axis, embryo 2.16 14 39 Hrb27C, Moe, Nacalpha, Tm1, capu, csul, exu, gus, orb, osk, pum, spir, sqd, syt cellular macromolecular complex assembly 2.16 14 39 AGO1, B52, CG13349, Df31, His3.3B, Mhc, SC35, betaTub56D, l(2)gl, me31B, pUf68, tkv, tra2, unc-13 negative regulation of cellular metabolic process 2.14 23 65 AGO1, Aef1, BicC, CG31716, Hrb27C, Ptp61F, Sxl, apt, aret, bru-3, chb, chn, dap, dsx, exba, gro, ovo, pr-set7, pum, retn, smg, sqd, tim salivary gland cell autophagic cell death 2.11 11 31 Akap200, CG14995, CG4091, Doa, Eip75B, NAT1, eIF-4E, eRF1, emp, ftz-f1, l(2)gl positive regulation of biosynthetic process 2.06 16 46 Dp1,, E2f, ,Hrb27C, y , Tl, ,chn, ,dsx, p lola, , orb, p osa, , osk, , otu, ,ovo, p pAbp, , pum, retn, , pspz , , , , g , , , , y, ( g ), , , , p p, p , q , oogenesis 2.00 34 104 ras, retn, spri, tkv, toc, twin, yl

B) Gene Ontology enrichment (biological process) of differentially expressed genes Term OddsRatio Count Size GeneSyms DNA damage checkpoint 27.37 3 4 grp, lok, pr-set7 regulation of DNA replication 27.37 3 4 CycE, mus209, otu negative regulation of oskar mRNA translation 27.37 3 4 BicC, Hrb27C, aret regulation of nuclear mRNA splicing, via spliceosome 18.66 2 3 Hrb27C, Pep DNA topological change 18.15 2 3 Top1, dom cell cycle arrest 18.15 2 3 dap, grp regulation of mitotic cell cycle, embryonic 18.15 2 3 CycA, grp negative regulation of cell cycle process 18.15 2 3 CycA, dap histone lysine methylation 18.15 2 3 Su(var)3-9, pr-set7 sensory organ precursor cell division 18.15 2 3 CycE, dap lateral inhibition 18.15 2 3 CkIIalpha, Dl cell cycle checkpoint 13.68 3 5 grp, lok, pr-set7 G1/S transition of mitotic cell cycle 13.68 3 5 CycE, cdc2c, dap syncytial blastoderm mitotic cell cycle 13.68 3 5 CycA, CycB, grp protein targeting to mitochondrion 9.11 3 6 Hsp60, Hsp60C, Tom40 regulation of exit from mitosis 9.11 3 6 CycA, CycE, aret female sex determination 9.11 3 6 dsx, otu, ovo negative regulation of cell cycle 9.11 3 6 CycA, dap, pum interphase 7.66 5 11 CycA, CycB, CycE, cdc2c, dap negative regulation of translation 7.44 4 9 exba, pum, smg, sqd G2/M transition of mitotic cell cycle 6.83 3 7 CycA, CycB, cdc2c protein amino acid methylation 6.83 3 7 Su(var)3-9, bin3, pr-set7 negative regulation of protein metabolic process 5.61 9 24 BicC, Hrb27C, aret, dap, exba, pr-set7, pum, smg, sqd RNA catabolic process 5.54 6 16 CG11265, CG6967, Upf2, pum, smg, sqd positive regulation of translation 5.46 3 8 Hrb27C, osk, pum germ-line cyst formation 5.46 3 8 aret, bol, otu long-term memory 5.22 4 11 exba, osk, pum, yu chromosome condensation 5.22 4 11 Bj1, Top1, eIF-4E, pr-set7 nuclear-transcribed mRNA catabolic process 5.10 5 14 CG6967, Upf2, pum, smg, sqd mitotic sister chromatid segregation 4.06 4 13 Bj1, CycA, CycB, eIF-4E DNA-dependent DNA replication 4.06 4 13 Top1, dre4, mus209, otu methylation 4.06 4 13 CG14478, Su(var)3-9, bin3, pr-set7 regulation of cell cycle process 3.69 4 14 CycE, aret, bol, otu negative regulation of transcription from RNA polymerase II promoter 3.65 4 14 Aef1, dsx, ovo, retn translational initiation 3.65 4 14 Rbp2, Su(var)3-9, eIF-4E, eIF5 posttranscriptional regulation of gene expression 3.65 12 43 BicC, Hrb27C, Rm62, Upf2, aret, bol, brat, exba, osk, pum, smg, sqd regulation of alternative nuclear mRNA splicing, via spliceosome 3.59 7 25 B52, Hel25E, Rm62, Rox8, aret, dom, sqd RNA splicing, via transesterification reactions with bulged adenosine as nucleophile 3.56 11 40 B52, CG13900, Hel25E, Hrb27C, Pep, Rm62, Rox8, aret, dom, dsx, sqd RNA splicing 3.32 11 42 B52, CG13900, Hel25E, Hrb27C, Pep, Rm62, Rox8, aret, dom, dsx, sqd mRNA processing 3.32 13 50 B52, CG13900, Dek, Hel25E, Hrb27C, Pep, Rm62, Rox8, aret, dom, dsx, sm, sqd pole plasm mRNA localization 3.27 5 19 Hrb27C, capu, mael, osk, sqd cytoplasm organization 3.06 6 24 Hrb27C, capu, mael, osk, sqd, yl negative regulation of biosynthetic process 2.96 12 50 Aef1, BicC, Hrb27C, Su(var)3-9, aret, dsx, exba, ovo, pum, retn, smg, sqd regulation of cellular protein metabolic process 2.94 14 59 BicC, CycE, Hrb27C, aret, bol, brat, dap, exba, grp, osk, pr-set7, pum, smg, sqd nucleocytoplasmic transport 2.90 6 25 Bj1, Hel25E, Kap-alpha3, Nup154, dco, sqd oocyte anterior/posterior axis specification 2.75 6 26 Dl, Hrb27C, capu, mael, osk, sqd negative regulation of cellular metabolic process 2.58 14 65 Aef1, BicC, Hrb27C, Su(var)3-9, aret, dap, dsx, exba, ovo, pr-set7, pum, retn, smg, sqd M phase of mitotic cell cycle 2.54 8 37 Bj1, CycA, CycB, CycE, CycJ, aret, eIF-4E, pum chromatin organization 2.38 9 44 BEAF-32, Df31, HmgD, Su(var)3-9, Ubi-p63E, dom, dre4, eIF-4E, pr-set7 nuclear division 2.30 8 40 Bj1, CycA, CycB, CycE, CycJ, aret, eIF-4E, pum oogenesis 2.29 28 148 BEAF-32, BicC, Cbl, CycE, Dl, Hrb27C, Hsp60C, Jra, Su(var)3-9, Top1, aret, brat, capu, dap, dec-1, dom, exba, growl, mael, mus209, osk, otu, ovo, pum, ras, retn, sqd, yl cell division 2.19 12 63 CycA, CycB, CycE, Dl, Sep1, Tl, aret, bol, brat, dap, dco, pum cellular macromolecule catabolic process 2.18 9 47 CG11265, CG31169, CG6967, CYLD, Ubi-p63E, Upf2, pum, smg, sqd

C) Gene Ontology enrichment (biological process) of differentially expressed genes that change at transcriptional level only. Term OddsRatio Count Size GeneSyms nuclear migration 8.92 2 9 Cp190, mael mitotic spindle organization 2.81 5 62 CG7033, RpL3, RpS4, RpS9, mus209 translation 2.39 7 102 Aats-ala, Aats-ile, Ef1gamma, RpL3, RpS4, RpS9, Su(var)3-9 microtubule-based process 2.26 8 124 CG1193, CG7033, Cp190, RpL3, RpS4, RpS9, mael, mus209

D) Gene Ontology enrichment (biological process) of genes that change at the post-transcriptional level of splicing only Term OddsRatio Count Size GeneSyms establishment or maintenance of polarity of larval imaginal disc epithelium Inf 3 3 Moe, dlg1, l(2)gl response to inorganic substance 18.53 3 4 CG7843, Cam, ZnT35C negative regulation of cell adhesion 9.26 3 5 Pten, Timp, dlg1 cystoblast division 9.26 3 5 chb, hts, pUf68 somatic sex determination 9.26 3 5 Sxl, gro, tra2 positive regulation of synaptogenesis 9.26 3 5 Nedd4, cac, tkv neuromuscular synaptic transmission 8.26 4 7 Ca-P60A, Pka-R1, apt, tkv spindle assembly 7.77 5 9 Eb1, Grip84, chb, mud, pbl regulation of Notch signaling pathway 6.19 4 8 Nedd4, l(2)gl, lqf, neur neuromuscular process 6.19 4 8 Atpalpha, Doa, lola, neur spliceosome assembly 6.17 3 6 SC35, pUf68, tra2 primary sex determination 6.17 3 6 Stat92E, Sxl, gro establishment of spindle localization 6.17 3 6 Eb1, chb, dlg1 flight behavior 5.17 5 11 Actn, Eb1, Gpdh, Sema-2a, tutl regulation of Wnt receptor signaling pathway 4.95 4 9 Axn, Stat92E, gro, skd establishment or maintenance of epithelial cell apical/basal polarity 4.95 4 9 Moe, dlg1, l(2)gl, trol septate junction assembly 4.43 5 12 Atpalpha, G-oalpha47A, dlg1, l(2)gl, nrv2 asymmetric protein localization 4.14 6 15 Gbeta13F, Ggamma1, Moe, csul, dlg1, l(2)gl axon choice point recognition 4.12 4 10 Nedd4, ab, lola, plexA sleep 4.12 4 10 5-HT1A, Hsc70-3, Rdl, tim regulation of photoreceptor cell differentiation 4.12 4 10 Mbs, alph, neur, sina positive regulation of transport 4.12 4 10 Cam, Nedd4, Pld, olf186-F centrosome organization 3.73 6 16 Cam, Grip84, chb, cnn, hts, skpA taxis 3.39 6 17 Sema-2a, Timp, shep, tim, wun, yuri oocyte anterior/posterior axis specification 3.31 9 26 Moe, Nacalpha, Tm1, csul, exu, gus, orb, spir, syt cytoplasm organization 3.12 8 24 Moe, Nacalpha, Tm1, csul, exu, orb, spir, syt cell junction assembly 3.10 6 18 Atpalpha, G-oalpha47A, Pten, dlg1, l(2)gl, nrv2 imaginal disc-derived wing hair organization 3.10 6 18 G-oalpha47A, LIMK1, Mbs, fry, pk, qua pole plasm oskar mRNA localization 3.10 6 18 Nacalpha, Tm1, exu, orb, spir, syt posttranscriptional gene silencing by RNA 3.09 5 15 AGO1, Hsc70-3, Hsc70-4, gw, loqs actin filament organization 2.95 14 44 Actn, DAAM, Fps85D, Gbeta13F, Ggamma1, Pak3, Pka-R1, Pten, gro, hts, qua, ran, spir, ssh oocyte differentiation 2.95 14 44 Moe, Nacalpha, Pka-R1, Sxl, Tm1, chb, csul, exu, gus, hts, orb, spir, spz, syt intracellular mRNA localization 2.90 7 22 Nacalpha, Tm1, csul, exu, orb, spir, syt pole plasm RNA localization 2.86 6 19 Nacalpha, Tm1, exu, orb, spir, syt hair cell differentiation 2.86 6 19 G-oalpha47A, LIMK1, Mbs, fry, pk, qua positive regulation of developmental process 2.81 9 29 CG7263, E2f, Eip75B, Nedd4, alph, cac, l(2)gl, mod(mdg4), tkv oocyte construction 2.80 12 39 Moe, Nacalpha, Pka-R1, Tm1, chb, csul, exu, gus, orb, spir, spz, syt posttranscriptional regulation of gene expression 2.73 13 43 AGO1, Dp1, Hsc70-3, Hsc70-4, Pten, Ptp61F, Sxl, apt, bru-3, gw, loqs, orb, pAbp regulation of cellular component biogenesis 2.72 10 33 Nacalpha, Nedd4, Pten, Tm1, cac, exu, spz, ssh, syt, tkv actin filament-based process 2.51 20 71 Actn, DAAM, Fps85D, G-oalpha47A, Gbeta13F, Ggamma1, LIMK1, Mhc, Moe, Pak3, Pka-R1, Pten, gro, hts, nuf, pbl, qua, ran, spir, ssh developmental maturation 2.48 13 46 Moe, Nacalpha, Pka-R1, Tm1, chb, csul, exu, gus, orb, rk, spir, spz, syt negative regulation of signal transduction 2.46 11 39 Axn, Cam, Gug, Nedd4, Pten, Stat92E, Su(var)2-10, alph, gro, l(2)gl, stck regulation of localization 2.46 11 39 Ca-P60A,, Cam, , Nacalpha, , Nedd4, , p Pld, , Pten, , p Ptp61F, , exu, olf186-F, , , syt, , tim p , , ( ) , , , , g , , , p , q , p , p , , y , , cellular component assembly 2.16 28 115 tra2, unc-13 regulation of neurogenesis 2.15 9 35 LIMK1, Mbs, alph, fry, l(2)gl, neur, pbl, sina, ssh cellular macromolecular complex assembly 2.15 10 39 AGO1, CG13349,, ,His3.3B, , Mhc,p , SC35, , l(2)gl, pUf68, , p, tkv, tra2, , unc-13p p, p , ( ) , , , , , p, , g, , q, q, , , , , anatomical structure development 2.05 29 131 olf186-F, skd, stai, tutl macromolecular complex subunit organization 2.05 15 61 AGO1, CG13349, Cam, His3.3B, Mhc, SC35, chb, eRF1, l(2)gl, pUf68, spz, ssh, tkv, tra2, unc-13 Supplementary Table 2: Full list of GO-enrichment terms containing statistically significant categories (pvalue <0.005) for

A) genes containing differentially regulated sjncs (DRsjnc) B) genes regulated at the transcriptional level (DE genes) C)

genes regulated at the transcriptional level only D) genes regulated at the level of splicing only. Columns correspond to:

1: GO Term

2: odds ratio

3: No. of genes belonging to this category

4. No of genes belonging to this category in the background dataset

5. Genes with differentially regulated sjnc belonging to this category

Hartmann_Suppl. Table 3

gene synonym probe DE gene AS event type of AS event NMD DR sjnc: log2FCnetExp log2_FC_ log2FC_ge Entrez_ID ID affects: log2FCne AdjP-value gene ne_adjust tExp ed_p_valu e aret 2668 YES UTR alt promoter, intron retention 4.56 0.00025 -1.07 0.00016 34648 aret 2682 YES UTR bleeding exon 2.84 0.00045 -1.07 0.00016 34648 aret 2669 YES CDS alt promoter 2.07 0.00049 -1.07 0.00016 34648 aret 2670 YES CDS alt promoter -1.99 0.00074 -1.07 0.00016 34648 aret 2683 YES UTR const. jnc 1.25 0.00065 -1.07 0.00016 34648 aret 2680 YES CDS cassette exon 1.19 0.00236 -1.07 0.00016 34648 aret 2678 YES CDS cassette exon 1.04 0.00328 -1.07 0.00016 34648 aret 2675 YES UTR cassette exon 1.04 0.00893 -1.07 0.00016 34648 B52 12514 YES CDS cassette exon, alt5'ss, intron retention YES 1.31 0.00103 -1.02 0.00031 41670 B52 12513 YES CDS cassette exon, alt5'ss, intron retention 1.20 0.00153 -1.02 0.00031 41670 B52 12516 YES CDS cassette exon, intron retention YES 1.03 0.00094 -1.02 0.00031 41670 BicC 3116 YES UTR alt promoter -2.28 0.00074 -2.99 0.00001 34946 BicC 3119 YES CDS alt promoter 1.67 0.00434 -2.99 0.00001 34946 BicC 3123 YES CDS const. jnc 1.25 0.00135 -2.99 0.00001 34946 bol 8838 YES CDS const. jnc 2.35 0.00029 1.23 0.00003 39049 bol 8846 YES UTR alt promoter, cassette exon YES 1.51 0.00023 1.23 0.00003 39049 bol 8845 YES UTR cassette exon YES -1.09 0.00072 1.23 0.00003 39049 bol 8839 YES CDS const. jnc -1.05 0.00177 1.23 0.00003 39049 bru-3 9590 NO CDS const. jnc -1.04 0.00125 0.59 0.00045 39527 caz 6234 YES CDS alt5'ss, alt3'ss 1.38 0.00176 -1.29 0.00021 32587 CG13900 7212 YES UTR const. jnc 1.44 0.00054 -1.7 0.00011 38093 CG17838 13681 YES CDS cassette exon 2.59 0.00229 1.7 0.00002 42460 CG17838 13675 YES CDS const. jnc YES -1.95 0.00023 1.7 0.00002 42460 CG17838 13663 YES CDS alt promoter, cassette exon 1.85 0.00063 1.7 0.00002 42460 CG17838 13666 YES CDS alt promoter -1.85 0.00017 1.7 0.00002 42460 CG17838 13670 YES CDS cassette exon YES 1.81 0.00040 1.7 0.00002 42460 CG17838 13665 YES CDS alt promoter -1.78 0.00028 1.7 0.00002 42460 CG17838 13673 YES CDS cassette exon -1.41 0.00034 1.7 0.00002 42460 CG17838 13678 YES CDS cassette exon YES 1.27 0.00138 1.7 0.00002 42460 CG17838 13679 YES CDS cassette exon -1.13 0.00036 1.7 0.00002 42460 CG17838 13672 YES CDS cassette exon YES 1.11 0.00134 1.7 0.00002 42460 CG17838 13671 YES CDS cassette exon -1.03 0.00062 1.7 0.00002 42460 CG31716 2408 NO UTR alt promoter, cassette exon, bleeding exon 1.18 0.00188 -0.56 0.00057 34416 CG6995 14527 NO CDS const. jnc YES -1.37 0.00905 -0.51 0.00302 42958 CG7082 844 NO UTR alt promoter, bleeding exon 2.10 0.00029 0.32 0.00885 33401 CG7879 7421 NO UTR cassette exon 1.32 0.00128 -0.51 0.00134 38184 CG7879 7420 NO UTR cassette exon 1.13 0.00055 -0.51 0.00134 38184 CG7971 7469 NO UTR alt promoter YES 1.53 0.00066 -0.51 0.00121 38206 CG7971 7460 NO CDS cassette exon, alt poly A 1.07 0.00076 -0.51 0.00121 38206 CG9809 11012 YES UTR const. jnc YES -1.48 0.00343 -2.12 0.00001 40562 CG9809 11011 YES CDS const. jnc YES -1.16 0.00314 -2.12 0.00001 40562 CG9809 11006 YES CDS bleeding exon, intron retention 1.12 0.00101 -2.12 0.00001 40562 cpo 13220 NO UTR alt promoter, cassette exon -1.09 0.00053 0.84 0.00047 45840 Doa 15081 NO CDS alt promoter, alt5'ss, alt3'ss, bleeding exon 1.16 0.00050 -0.4 0.01553 43415 Dp1 18609 NO UTR cassette exon 1.54 0.00032 -0.94 0.00027 37116 Dp1 18611 NO UTR cassette exon 1.30 0.00071 -0.94 0.00027 37116 Dp1 18605 NO UTR alt promoter, alt5'ss, alt3'ss 1.18 0.00035 -0.94 0.00027 37116 eIF3-S9 18465 NO CDS bleeding exon, intron retention 1.54 0.00017 -0.98 0.00038 36981 Hel25E 1395 YES CDS const. jnc -1.07 0.00095 -1.25 0.00007 33781 Hel25E 1400 YES UTR alt promoter, alt3'ss, bleeding exon -1.05 0.00107 -1.25 0.00007 33781 Hrb27C 1661 YES UTR alt promoter 1.71 0.00021 -1.54 0.00005 33968 Hrb27C 1660 YES UTR alt promoter 1.64 0.00062 -1.54 0.00005 33968 Imp 5361 NO UTR alt promoter, cassette exon 2.54 0.00010 -0.3 0.01804 32009 Imp 5355 NO CDS alt promoter, cassette exon, alt5'ss 1.08 0.00046 -0.3 0.01804 32009 La 3590 YES CDS alt promoter, bleeding exon 1.85 0.00014 -1.2 0.00037 35305 La 3588 YES UTR bleeding exon, intron retention 1.34 0.00030 -1.2 0.00037 35305 mask 14344 NO CDS const. jnc -1.33 0.00066 -0.36 0.00537 50070 me31B 2336 YES CDS alt promoter 2.86 0.00152 -3.05 0.00003 34364 me31B 2337 YES UTR alt promoter 2.61 0.00035 -3.05 0.00003 34364 me31B 2339 YES CDS const. jnc 1.78 0.00244 -3.05 0.00003 34364 nito 16217 YES UTR bleeding exon 1.36 0.00061 -1.24 0.00007 35756 nito 16219 YES UTR const. jnc 1.33 0.00029 -1.24 0.00007 35756 orb 14106 NO CDS alt promoter 2.45 0.00010 -0.54 0.00236 42752 orb 14102 NO CDS alt promoter 2.36 0.00017 -0.54 0.00236 42752 orb 14107 NO CDS alt promoter -1.91 0.00029 -0.54 0.00236 42752 orb 14108 NO CDS const. jnc -1.80 0.00225 -0.54 0.00236 42752 pAbp 18567 NO UTR alt promoter, alt5'ss, alt3'ss, intron retention, cassette exon, bleeding exon 2.22 0.00043 -0.54 0.00190 37070 pAbp 18568 NO UTR alt promoter, alt5'ss, bleeding exon, intron retention 1.30 0.00661 -0.54 0.00190 37070 pAbp 18575 NO UTR const. jnc -1.01 0.00066 -0.54 0.00190 37070 pit 13888 NO CDS const. jnc -1.18 0.00122 -0.56 0.00067 42595 Pof 19806 YES UTR alt promoter, bleeding exon, intron retention -2.19 0.00032 2.16 0.00012 37947 Pof 19809 YES CDS const. jnc 1.24 0.00280 2.16 0.00012 37947 Pof 19808 YES CDS const. jnc -1.02 0.00259 2.16 0.00012 37947 pUf68 7351 NO CDS cassette exon, alt5'ss -1.12 0.00045 0.09 0.35496 38173 Rbp2 6334 YES CDS alt5'ss, alt3'ss 1.31 0.00029 -1.14 0.00031 49809 rin 12500 YES UTR alt promoter, alt5'ss, intron retention, cassette exon, bleeding exon 1.53 0.00017 -1.51 0.00004 47998 Rm62 11406 YES CDS cassette exon 1.69 0.00046 -1.16 0.00036 40739 Rm62 11407 YES CDS cassette exon 1.35 0.00037 -1.16 0.00036 40739 Rm62 11411 YES UTR alt promoter, cassette exon 1.27 0.00044 -1.16 0.00036 40739 Rm62 11410 YES UTR alt promoter, cassette exon 1.09 0.00056 -1.16 0.00036 40739 SC35 2703 NO CDS const. jnc -1.73 0.00096 0.07 0.54439 53444 SC35 2704 NO CDS alt promoter -1.27 0.00195 0.07 0.54439 53444 shep 8169 NO CDS alt3'ss 1.03 0.00091 -0.33 0.00592 38605 sm 18830 YES CDS alt promoter, cassette exon 1.99 0.00035 1.54 0.00009 37254 sm 18834 YES CDS cassette exon -1.76 0.00207 1.54 0.00009 37254 sm 18832 YES CDS alt promoter, cassette exon -1.51 0.00071 1.54 0.00009 37254 sm 18842 YES UTR alt promoter -1.44 0.00206 1.54 0.00009 37254 sm 18825 YES CDS const. jnc 1.32 0.00096 1.54 0.00009 37254 sm 18831 YES CDS alt promoter, cassette exon -1.31 0.00103 1.54 0.00009 37254 sm 18827 YES CDS const. jnc 1.26 0.00043 1.54 0.00009 37254 sm 18828 YES CDS const. jnc 1.02 0.00175 1.54 0.00009 37254 sqd 12495 YES UTR cassette exon 1.76 0.00030 -1.66 0.00014 41666 sqd 12497 YES UTR cassette exon 1.62 0.00055 -1.66 0.00014 41666 sqd 12489 YES CDS cassette exon 1.47 0.00025 -1.66 0.00014 41666 SRPK 17872 NO CDS alt promoter 1.57 0.00020 -0.67 0.00027 36706 Sxl 4955 NO UTR cassette exon, alt3'ss YES 1.56 0.00017 -0.79 0.00049 3772180 Sxl 4962 NO UTR alt promoter, alt5'ss, alt3'ss 1.27 0.00092 -0.79 0.00049 3772180 tra2 17723 NO UTR alt promoter, alt3'ss, bleeding exon YES -1.02 0.00092 -0.25 0.07679 36619 Supplementary Table 3: Sex-specifically regulated splice-junctions in RNA binding . Columns correspond to:

1. Gene synonym (as in Flybase)

2. Probe ID: Probe on the microarray

3. DE gene: transcriptionally regulated genes

4. AS event effects: changes in sjnc predicted to affect the coding or UTR regions of the transcripts

5. AS event: the sjnc is involved in the indicated type of AS event.

6. NMD: the sjnc corresponds to a transcript that contains a premature stop codon and could potentially be a target of

NMD

7: Net expression ratio (male/female) from the microarray for the sjnc (in log2;)

8: p-value corresponding to the data in column 7.

9: fold-change (male/female in log2) of change in general transcript level (transcriptional change)

10: p-value corresponding to the data in column 9.