iCLIP analysis of full-length and deletion mutants of myc-LARP6

Intrinsically disordered regions (IDRs) are prevalent in RNA-binding proteins (RBPs), yet their roles in RNA interactions remain poorly defined. We examined the structured and disordered RNA-binding activities of LARP6, an RBP with a diverse RNA-binding repertoire. U87 glioblastoma cells stably expressing myc-tagged full-length or various deletion mutants of LARP6 under a doxycycline switch were induced to express myc-LARP6 variants at near endogenous levels, before individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) was performed to assess each variants' RNA-binding targets on the transcriptome. A total RNA-seq experiment was also performed on the cells in parallel to distinguish between changes in RNA bidning and those that are affected via expression level variations. Overall design: iCLIP of myc-LARP6 from full-length (FL), N-terminal Region deleted (deltaNTR), La-module deleted (delta LaMod), N-terminal domain deleted (deltaNTD), and C-terminal region deleted (deltaCTR1) U87 cells, along with untransfected control cells was performed as indicated in (https://doi.org/10.1101/2024.09.20.614075). To normalise iCLIP differences based on total RNA variations, an rRNA depletion whole transcriptome sequencing of each stably expressing U87 cell-line following LARP6 induction was also performed in parallel, using short single read (SE50) and long paired end read (PE150) sequencing. These were used as input control.