EIF4A3 associated splicing and nonsense mediated decay defined by a systems analysis with novel small molecule inhibitors

Abstract

Chemical biology approaches to the global functions of splicing reactions are gaining momentum, with an increasing repertoire of small molecule probes becoming available. Here we map the association of eIF4A3 with transcript expression, NMD and alternative splicing using a set of selective novel small molecule allosteric helicase inhibitors whose synthesis and chemical properties we have recently described. We show through analysis of dose monotonic transcriptional responses to increasing inhibition that both full length and NMD prone transcripts link eIF4A3 to normal functioning of cell division including chromosome segregation and cell cycle checkpoints, pointing to a conserved role of splicing and transcript quality processing in cell cycle functions. Cell cycle analysis and microscopy of inhibitor treated cells demonstrates chromosome mis-segregation and spindle defects, associated with a G2/M arrest, validating this observation. Through analysis of conserved alternative splicing patterns exhibiting monotonic responses, we find that eIF4A3 dependent alternative splicing involves exons that are longer and introns that are shorter than transcripts not modulated by eIF4A3. Moreover we observe conservation of over/under representation of RBP binding motif density over introns and exons implicated eIF4A3 modulated skipped exon and retained introns. The distribution of motif densities over 5 and branch intron sites and 5 exons is consistent with function of the exon-junction complex. Taken together we have defined a fraction of the transcrip-tome dependent on eIF4A3 functions and revealed a link between eIF4A3 and cell cycle regulation. The systems approach described here suggests additional avenues for therapeutic exploitation of eIF4A3 functions in cancer and related diseases.