META-ANALYSIS OF MRP4/ABCC4 TO DETERMINE HOT SPOTS FOR CRISPR-Cas9 EDITING DESIGN TO STUDY THE REGULATION OF ITS NUCLEAR LOCALIZATION.

DE SOUSA, MAXIMILIANO; LAGOS, RODRIGO; SAMANTA GANCEDO; GÓMEZ, NATALIA

Congreso; LVX Reunion Anual de la Sociedad Argentina de Investigacion Clinica; 2020

SAIC

Recent findings show that high MRP4 levels are critical for pancreatic ductal adenocarcinoma(PDAC) cell proliferation and associated with a more aggressive phenotype. We havedemonstrated the presence of MRP4 in the nucleus in pancreatic human PDACs tissues. Also, wehave observed this MRP4 novel localization in both hepatic and pancreatic cancer cell lines, usingtechniques such as western blot prior to subcellular fractionation and confocal microscopy. Theaim of the present work is to make a computational analysis to study possible sequences to betargeted with CRISPR-Cas 9 to study the mechanism underlying this novel localization. Our analysisof the complete protein sequence, consisting in 1325 amino acids, using cNLS Mapper SOFTWAREfound a nuclear localization sequence (NLS) located from I33 to D42 (IGHKRRLEED) with a score of7 that corresponds to proteins partially located in the nucleus. Bibliographic recopilation describespost-translational modifications, as phosphorylations, near NLS regions that could regulate thepossibility of interaction between NLS and the proteins involved in the transporting. The analysisof the sequence described that the Y45i s a phosphorylation site linked evolutively with aconserved site of ubiquitination located in K1278, closely to the previously described PDZ domain(ETAL1325). It is known that this domain is decisive for its location in the plasma membrane due toits interaction with adapter proteins such as NHERF1, NHERF3 or MPP1. In conclusion, we proposeto explore the effect of three different CRISPR cas9 constructions on the transport of MRP4 to thenucleus. The first one will be focused on producing changes in the NLS sequence without changingthe reading frame and the other two to modify punctually Y45 near NLS and the K1278 near PDZdomain.