An aberrant cytoplasmic intron retention programme is a blueprint for ALS-related RBP mislocalization

We recently described aberrant cytoplasmic SFPQ intron-retaining transcripts (IRTs) and concurrent SFPQ protein mislocalization as a new hallmark of amyotrophic lateral sclerosis (ALS). However the generalizability and potential roles of cytoplasmic IRTs in health and disease remain unclear. Here, using time-resolved deep-sequencing of nuclear and cytoplasmic fractions of hiPSCs undergoing motor neurogenesis, we reveal that ALS-causing VCP gene mutations lead to compartment-specific aberrant accumulation of IRTs. Specifically, we identify >100 IRTs with increased cytoplasmic (but not nuclear) abundance in ALS samples. Furthermore, these aberrant cytoplasmic IRTs possess sequence-specific attributes and differential predicted binding affinity to RNA binding proteins (RBPs). Remarkably, TDP-43, SFPQ and FUS - RBPs known for nuclear-to-cytoplasmic mislocalization in ALS - avidly and specifically bind to this aberrant cytoplasmic pool of IRTs, as opposed to any individual IRT. Our data are therefore consistent with a novel role for cytoplasmic IRTs in regulating compartment-specific protein abundance. This study provides new molecular insight into potential pathomechanisms underlying ALS and highlights aberrant cytoplasmic IRTs as potential therapeutic targets. Overall design: Bulk RNA-seq from fractionated cells at different timepoints throughout a 35-day differentiation protocol that converted iPSC cells to highly enriched motor neurons.