Direct ribosomal binding by a cellular inhibitor of translation

Abstract

During apoptosis and under conditions of cellular stress, several signaling pathways promote inhibition of cap-dependent translation while allowing continued translation of specific messenger RNAs encoding regulatory and stress-response proteins. We report here that the apoptotic regulator Reaper inhibits protein synthesis by binding directly to the 40S ribosomal subunit. This interaction does not affect either ribosomal association of initiation factors or formation of 43S or 48S complexes. Rather, it interferes with late initiation events upstream of 60S subunit joining, apparently modulating start-codon recognition during scanning. CrPV IRES-driven translation, involving direct ribosomal recruitment to the start site, is relatively insensitive to Reaper. Thus, Reaper is the first known cellular ribosomal binding factor with the potential to allow selective translation of mRNAs initiating at alternative start codons or from certain IRES elements. This function of Reaper may modulate gene expression programs to affect cell fate.Rapid changes in cellular gene expression are often brought about by regulation at the level of protein synthesis from existing mRNA transcripts. Such alterations are particularly important under conditions of cellular stress and apoptosis, and during certain stages of mitosis 1 . Cellular stresses such as viral infection or nutrient deprivation lead to an almost immediate shutdown of general translation accompanied by cleavage or covalent modification of one or more of the eukaryotic initiation factors (eIFs) 2-5 . However, this attenuation of translation is typically accompanied by a selective increase in the translation of specific regulatory proteins 6-9 whose sustained expression can affect cell fate 1,10 . How particular mRNAs are selectively translated during periods of global translation inhibition is not well understood.

Rapid selection against arbovirus-induced apoptosis during infection of a mosquito vector

“…To determine whether Reaper expression would also have an effect when the midgut barrier was bypassed, mosquitoes were infected by intrathoracic injection. Doses of 10, 100, or 1,000 plaque-forming units (PFU) were injected per mosquito, and samples were collected for TCID 50 assays at 1, 3, and 5 dpi. Mosquitoes injected with MRE/Rpr had significantly lower titers than control virus at 1 dpi with each of the injected doses (Fig.…”

“…TCID 50 Assay with Mosquito Samples. Individual mosquitoes were placed in 500 μL DMEM (Invitrogen) supplemented with 15 μg/mL penicillin/streptomycin (Invitrogen) and 1 ng/mL gentamycin (Cellgro).…”

“…Plates were scored for infection at 6 dpi by observing cytopathic effects. The number of infected wells per dilution was used to determine the TCID 50 per mosquito (56). Saliva Collection.…”

“…After salivation the FBS was added to 100 μL DMEM containing penicillin/streptomycin and gentamicin. Samples were vortexed, spun down, and then used for TCID 50 assays. Four independent biological replicates were performed per sample.…”

“…We can only speculate about what would have happened if the reaper insert was not able to be easily deleted from the MRE/Rpr genome, but we suspect that the effects on replication would have been even more drastic than what we observed. Reaper likely induces apoptosis by multiple mechanisms, including inhibition of IAP proteins, regulating mitochondrial fusion, and inhibiting cap-dependent protein translation (48)(49)(50). Because Reaper expression is up-regulated normally during Drosophila apoptosis (48), and many arbovirus mRNAs are capped, including SINV, inhibition of cap-dependent translation by Reaper homologs may play a role in decreasing arbovirus replication in apoptotic mosquito cells, regardless of how apoptosis is stimulated.…”