THE RNA HELICASE DED1 INTERACTS WITH EIF4G1 TO CONTROL TRANSLATION INITIATION

Abstract

The DEAD-box RNA helicase Ded1 has an essential role in translation initiation in Saccharomyces cerevisiae. Mutations in the human homolog DDX3 have been implicated in several human diseases, particularly medulloblastoma, a pediatric brain cancer. Previous in vitro data and immunoprecipitation assays have shown that Ded1 can both bind to the initiation factor eIF4G1 and homo-oligomerize through its C-terminal domain. The interaction with eIF4G1 is essential to effectively synthesize proteins. Studies have also shown that ded1 mutants lacking a C-terminus (ded1-ΔCT) do not efficiently regulate translation, and a similar effect is also observed when only the last 14 amino acids of the C-terminus are deleted (ded1-Δ591-604). Here we characterized the in vivo effects of these interactions with the Ded1 C-terminus. In serial dilution growth assays, we observed growth defects in both ded1-ΔCT mutants and in strains lacking eIF4G1 (tif4631), along with a synthetic phenotype in ded1-ΔCT tif4631 double mutant strains. Similarly, ded1 double mutants lacking both eIF4G1 and the last 14 amino acids of the Ded1 C-terminus partially phenocopied the full ded1-ΔCT mutant. These growth defects suggest a decreased binding affinity by ded1 with eIF4G1 due to the lack of the C terminus that leads to diminished translation efficiency. Furthermore, we show that ded1 mutants show an increased resistance to rapamycin, a known target of the TOR pathway. This could suggest an important role for Ded1 C-terminus in regulating the TOR pathway in response to rapamycin stress. We also show that altering eIF4G1 levels in the presence of heat significantly affects cell viability. This might suggest a tight regulation of eIF4G1 levels during the cell’s response to heat stress.