Supervisors info:
Παναγιώτα Καφάσλα, Ερευνήτρια Γ, ΕΚΕΒΕ "Αλέξανδρος Φλέμινγκ"
Γεώργιος Παναγιώτου, Ερευνητής Α, ΕΚΕΒΕ "Αλέξανδρος Φλέμινγκ"
Αθανάσιος Κοτσίνας, Επίκουρος Καθηγητής, Ιατρική, ΕΚΠΑ
Summary:
Alternative splicing (AS) is an important post-transcriptional regulatory process of gene expression, which has recently emerged as a “hallmark of cancer”. Regulation of AS is coordinated by ribonucleoprotein complexes (RNPs) in response to changes in environmental or physiological conditions communicated through signaling pathways. However the mechanisms through which signaling pathways communicate with the splicing machinery and drive the interactions which orchestrate the switch of the AS profile to promote tumour growth, invasion and metastasis remain largely unknown.
This master’s thesis project aimed to investigate a novel finding of this lab: the interaction of the scaffold protein Iqgap1 with one of the splicing regulators, hnRNPM, in the nucleus of cancer cell-lines. This finding has interesting implications, since the role of scaffold proteins in post-transcriptional regulation in the nucleus has not been explored. Based on information highlighting the importance of a switch in AS profile for gastric cancer patient survival, we applied co-immunoprecipitation and proteomics approaches to a normal gastric epithelial (HFE-145) and a gastric cancer cell line (NUGC-4) and identified 138 nuclear proteins interacting with IQGAP1, including 44 components of splicing machinery. Validation experiments confirmed the protein-protein interaction of IQGAP1 with hnRNPM, as well as RNA-dependent interactions with SRSF1, hnRNPA1, hnRNPA2B1, hnRNPC1/C2, DDX17 and other splicing regulators. Our results indicate that IQGAP1 participates in the formation of at least two different RNPs with potential roles in splicing and/or RNA metabolism. Phosphoproteomics analysis of IQGAP1 interacting partners also showed IQGAP1 interaction with phosphorylated spliceosome components, including PRPF6 and PRPF31.
In order to study the role of IQGAP1 depletion on gastric cancer cell phenotype and splicing patterns, CRISPR-Cas9 IQGAP1 knock-out clones of the cell lines of interest were subjected to phenotypical assays. The depletion of IQGAP1 appeared to have no effect on cell cycle progression in asynchronous culture, while, in contrast to HFE-145, the clonogenic capacity of NUGC-4 cells appeared to be increased after IQGAP1 depletion. At the same time, the results of a preliminary assay of hnRNPM-dependent splicing using the DUP51M splicing construct indicate that IQGAP1 depletion affects hnRNPM-dependent splicing partners.
Our current hypothesis is that IQGAP1 plays a significant role in the assembly of RNP complexes participating in pre-mRNA splicing and post-transcriptional regulation in gastric epithelial and gastric cancer cells, orchestrating patterns of AS which may be linked to phenotypical changes of gastric cancer cell lines. Interactions of IQGAP1 with post-transcriptional regulators emerge, therefore, as potential drivers of AS in gastric cancer- a result which, after systematic study, could contribute potential candidates for the development of novel therapeutics against a rare and deadly disease.
Keywords:
Biochemistry, Molecular biology, RNA splicing, Cancer
