seCLIP of C. elegans EIF-3.G in the cholinergic motor neurons
Protein translation is tightly regulated at the initiation phase, which entails exceedingly complex interactions among multiple EIF initiation factors to bring the ribosome to the mRNA and scan the 5' leader sequence for the start codon. EIF-3 contains multiple subunits with essential and emerging regulatory activities throughout the translation initiation pathway.  However, it remains poorly understood how individual components of the EIF3 complex contribute to the selective regulation of translation in multicellular organisms. Here, through studies of a missense mutation in C. elegans EIF-3.G, an RNA-binding subunit of EIF3, we have dissected mechanisms of how regulation of protein translation initiation modulates neuronal excitation.  EIF-3.G(C130Y) alters a conserved Zinc Finger and behaves as a gain-of-function.  We show that while EIF-3.G(C130Y) permits essential aspects of translation initiation, this variant functionally involves its RNA binding activity in regulating protein synthesis. We mapped EIF-3.G binding sites in the C. elegans cholinergic motor neuron transcriptome and identified a significant representation of mRNAs containing long and GC-rich 5' UTRs and in mRNAs exhibiting activity-dependent transcript level changes. Our results suggest that modulation of the scanning process of translation initiation by EIF3 contributes homeostatic regulation to synaptic activity changes. Overall design: Two replicate experiments were performed in strains containing 3xFLAG-tagged EIF-3.G(+), EIF-3.G(C130Y), or EIF-3.G(?RRM). A single replicate seCLIP experiment was performed with a negative control without a transgene in a wild type C. elegans background (N2). EIF-3.G(+) and EIF-3.G(?RRM) transgenes were in the background of acr-2(n2420) and EIF-3.G(C130Y) transgene was in the background of eif-3.G(C130Y); acr-2(n2420).