RNA Sequencing of HEK293T cells expressing 15xGly or 99xGly
Protein aggregates are a hallmark of neurodegenerative disease proposed to promote neurotoxicity through a diverse and growing set of mechanisms. Aggregation of polyglycine is specifically implicated in the pathogenesis of an emerging group of neurodegenerative GGC repeat expansion diseases. Here, we find that polyglycine aggregates incorporate endogenous proteins harboring glycine-rich sequences, including FAM98B, a conserved component of the vertebrate tRNA ligase complex. Through sequestration and accelerated proteosome-dependent turnover mediated by the FAM98B glycine-rich intrinsically disordered region (IDR), polyglycine depletes the ligase complex and disrupts tRNA processing. Overall design: HEK293T cells were infected with lentiviruses encoding doxycycline (DOX)-repressible fusion protein constructs encoding either 15xGly-eGFP-3xHA or 99xGly-eGFP-3xHA. After selection in blasticidin, single cell clones were grown and expanded. Clonal transduced cell lines were either maintained in growth media containing DOX (for suppression of transgene expression) or in DOX-lacking media (for induction of transgene expression) for 14 days before isolation of RNA for sequencing.