The scaffold protein IQGAP1 links heat-induced stress signals to alternative splicing regulation in gastric cancer cells

Abstract

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.

IQGAP1 and RNA Splicing in the Context of Head and Neck via Phosphoproteomics

“…One specific example of the interactions between IQGAP1 and splicing proteins is a study by Birladeanu et al that links hnRNPs, another large family of splicing factors, to IQGAP1 in the context of gastric cancer . Their study showed that IQGAP1 regulates alternative splicing through hnRNPs.…”

“…One specific example of the interactions between IQGAP1 and splicing proteins is a study by Birladeanu et al that links hnRNPs, another large family of splicing factors, to IQGAP1 in the context of gastric cancer. 94 Their study showed that IQGAP1 regulates alternative splicing through hnRNPs. In a gastric cancer cell line, they found that IQGAP1 can interact with spliceosome component proteins, including hnRNPs and SR proteins.…”

“…Among the IQGAP1-dependent alternative splicing events identified in Birladeanu et al, the Fanconi Anemia complementation group I (FANCI) was one of the most frequently occurring events. 94 The FANCI protein is part of the Fanconi Anemia (FA) complex for DNA repair. FA deficiency has been indicated to be associated with HNSCC for a long time.…”

Communication between the nucleus and the mitochondria via NDUFS4 alternative splicing in gastric cancer cells

“…We compared NUGC4 and NUGC4-IQGAP1 KO cells in a wound healing assay and found more than 50% reduction in the migration rate of the KO cells compared to the parental ones (Figure 4F, S4F). Of note, IQGAP1 depletion did not impair migration of MKN45 cells (Birladeanu et al 2021), in which NDUFS4 AS and expression were also unaffected (Figure S2D-F). Overall, our data point towards a direct effect of IQGAP1 on the mitochondrial respiratory chain, at least partially through the regulation of AS of NDUFS4, the deregulation of which can be linked to other mitochondrial defects (mitochondrial fragmentation/aggregation, reduction of mitochondrial ROS production) observed in the KO cells.…”

“…Given that nuclear IQGAP1 is differently distributed in the nuclei of gastric cancer cells of different origin 11 , we studied further the consecutive different AS regulation by IQGAP1. We present herein conclusive evidence on the participation of IQGAP1 in the oxidative metabolism of gastric cancer cells via its involvement in the regulation of AS of components of the mitochondrial electron transport chain.…”