Analysis of LARP6 impact on RNA localisation to cell protrusions of Glioblastoma cells

LARP6 is a conserved RNA-binding domain that regulates the localisation of Ribosomal Protein mRNAs to the protrusions of mesenchymal-like cells. Our results had revealed that in addition to its folded La-module RNA binding domain, LARP6 makes direct contacts with its RNA targets via its disordered N-terminal region, which precedes the La-module. To assess the contribution of this region to the regulation of RNA localisation by LARP6, we carried out a spatial transcriptomics analysis of U-87 MG Glioblastoma cells, treated with scrambled control vs LARP6 3'UTR targeting siRNAs, with or without rescuing the LARP6 expression using a doxycycline-inducible full-legth or delta N-terminal mutant of LARP6. The cells were subjected to siRNA knockdown for 72hrs, before being seeded on 3um transwell filters and allowed to protrude through the pores for 90 mins. The protrusions and cell-bodies on opposite sides of the filter were isolateed, with total RNA extracted and subjected to RNA-sequencing using Lexogen QUANTSEQ 3' mRNA sequencing library preparation. Three independent biological replicates of each experiment were performed. Overall design: A total of three independent experiments were performed, each with the following conditions: 1- U87-MG control cells + scrambled (scr) siRNA; 2- U87-MG control cells + LARP6 3'UTR targeting (si3UTR) siRNA; 3- U87-MG cells + Full-length (FL) myc-LARP6 + scr siRNA; 4- U87-MG cells + FL myc-LARP6 + si3UTR; 5- U87-MG cells + delta-NTR myc-LARP6 + scr siRNA; 6- U87-MG cells + delata-NTR myc-LARP6 + si3UTR; Each sample was then fractionated into protrusion and cell-body fractions using transwell filters, before RNA extraction and sequencing using Lexogen's Quantseq 3' mRNA sequencing. For more details, please see the associated preprint: https://doi.org/10.1101/2024.09.20.614075.