Analysis of oncogenic Kras-induced changes in the RNA-bound proteome of PDAC cells

In order to reveal the impact of oncogenic KRAS signaling on the RNA-bound proteome of Pancreatic Ductal Adenocarcinoma (PDAC), we carried out quantitative whole Transcriptome RNA Interactome Capture (RIC) analysis in tumour cells from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). For RIC, we employed Orthogonal Organic Phase Separation (OOPS)(Queiroz et al., 2019). In this method, UV-C crosslinking coupled with phenol-chloroform based phase separation is used to isolate RNA-crosslinked proteins, which concentrate in the interface fraction, whilst free proteins concentrate in the organic fraction. SILAC was employed for quantitative analysis of Dox induced (24hrs) changes in the RNA-bound proteome. To assess the impact of ERK signalling, an independent experiment was carried out in which Trametinib (10nM) was added to the cells at the same time as Dox (both with or without Dox cells). A total of 6 biological replicates without Trametinib, and 4 biological replicates with Trametinib, were conducted, with switching the Heavy and Light SILAC labels for +Dox and -Dox conditions. Both the Interface as well as the organic phase fractions were analysed by mass spectrometry. In a separate experiment, the total proteome changes in response to dox (24hrs) were quantified from two reciprocally SILAC labelled mixes of heavy and light iKras PDAC whole cell lysates (See the READ ME.XLS for details of the experimental conditions).