In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or posttranslational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock.IQGAP1 controls hnRNPM's sumoylation, subnuclear localization and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation. knockout generation, splicing assays, microscopy techniques and quantitation, immunostaining) are described in the Supplementary Material and Methods section. Cell culturesThe human STAD cell lines AGS, KATOIII, MKN45 and NUGC4 were used. When indicated, cells were treated with the sumoylation inhibitor III 2-D08 (Millipore, Cat# 505156) at 100 μM for 12 h. Mass spectrometry and Proteomics analysisAnti-IQGAP1 immunoprecipitation samples were processed in collaboration with the Core Proteomics Facility at EMBL Heidelberg. See Supplementary Materials and Methods. RNA-seq analysisAS was analysed by using VAST-TOOLS v2.2.2 (29) and expressed as changes in percentspliced-in values (∆PSI). To generate RNA maps, we used the rna_maps function (30), using sliding windows of 15 nucleotides as described in Supplementary Materials and Methods. Quantification and Statistical AnalysisData were analysed using GraphPad Prism 7 software (GraphPad Software). Student's t test (comparisons between two groups), one-way ANOVA were used as indicated in the legends. p <0.05 was considered statistically significant. ACCESSION NUMBERSThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 69 partner repository with the dataset identifier PXD017842.RNA-seq data have been deposited in GEO: GSE146283.

150 words): 17 Alternative Splicing (AS) is extensively regulated during the cell cycle and is also 18 involved in the progression of distinct cell cycle phases. Stressing agents, such as heat 19 shock, halts AS affecting mainly post-transcriptionally spliced genes. Stress-dependent 20 regulation of AS relies possibly on the subnuclear location of its determinants, such us 21 Serine-Arginine rich (SR) and heterogeneous nuclear ribonucleoproteins, hnRNPs. 22 42role so far reported for hnRNPM is in spliceosome assembly and splicing itself 10,11 , 43 including regulation of AS of other as well as its own pre-mRNAs [12][13][14] . The association 44 of hnRNPM with the spliceosome is abolished under heat-induced stress 11,15 , known to 45 affect largely post-transcriptional splicing events 16 , suggesting hnRNPM's response in 46 stress-related signalling pathways. 47 HnRNPM-regulated AS events have been linked to disease development and 48 progression. Specifically, hnRNPM-mediated AS is deterministic for the metastatic 49 potential of breast cancer cells, due to cell-type specific interactions with other splicing 50 factors [17][18][19] . Furthermore, in Ewing sarcoma cells, hnRNPM abundance and subnuclear 51 localization change in response to a chemotherapeutic inhibitor of the PI3K/mTOR 52 pathway, resulting in measurable AS changes 20 . Very recently, hnRNPM was shown to 53 respond to pattern recognition receptor signalling, by modulating the AS outcome of an 54 RNA-regulon of innate immune transcripts in macrophages 21 . 55Despite such growing evidence on the cross-talk between hnRNPM-dependent AS and 56 cellular signalling, how distinct signals are transduced to hnRNPM and the splicing 57 machinery remains unclear. Here, we present conclusive evidence of how this could be 58 achieved. We describe a novel interaction between hnRNPM and the scaffold protein 59 IQGAP1 (IQ Motif Containing GTPase Activating Protein 1) in the nucleus of gastric 60 cancer cells. Cytoplasmic IQGAP1 acts as a signal integrator in a number of signalling 61 pathways 22 , but there is no defined role for the nuclear pool of IQGAP1. With IQGAP1 62 mRNA being overexpressed in many malignant cell types, the protein seems to regulate 63 cancer growth and metastatic potential [23][24][25] . Moreover, aged mice lacking IQGAP1 64 develop gastric hyperplasia suggesting an important in vivo role for IQGAP1 in 65 maintaining the gastric epithelium 26 . In the present study we show that this novel, 66 nuclear interaction between hnRNPM and IQGAP1 links heat-induced signals to 67 hnRNPM-regulated AS in gastric cancer, a cancer type that has been associated with a 68 significantly high incidence of AS changes 6,7 . Additionally, we show that depletion of 69 both interacting proteins results in alternative splicing changes that disfavour tumour 70 growth, which makes them and their interaction interesting cancer drug targets. 71 72 4 Results 73 hnRNPM and IQGAP1 expression levels are significantly altered in gastric 74 cancer. 75 Analysis of t...

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