Abstract
Ded1, an RNA‐dependent ATPase, promotes translation initiation of many mRNAs in yeast. Ded1 is extremely important for the translation of mRNAs predicted to have substantial secondary structure (Sen et al., 2015). Ded1 interacts directly with eIF4A and eIF4G and this complex has increased helicase activity compared to either Ded1 or eIF4A alone (Hilliker et al., 2011; Gao et al., 2016). Consistent with an important role in translation, DDX3, the human homolog of Ded1, has been shown to be misregulated in several cancers and is hijacked by HIV to allow translation of highly structured HIV mRNAs (Valiente‐Echeverria et al., 2015; Xiang et al., 2016). Because it is critical to translation and influences other translation initiation factors, we are interested in how Ded1 itself is regulated and how that regulation impacts translation.We use genetic selections to identify regulators of Ded1 function using a well characterized ded1 mutant. A cold sensitive ded1 mutant (ded1‐dam1) lacks two regions important for Ded1's ability to promote translation; one of these regions promotes the interaction between Ded1 and eIF4G. Yeast containing this mutant as the sole copy of ded1 show a growth defect, a dramatic drop in global translation, and an accumulation of P‐bodies at the restrictive temperature (Hilliker et al., 2011). We have isolated 38 putative mutations of the cold‐sensitive growth conferred by ded1‐dam1 assembly mutant. These 38 ded1‐dam1 suppressor mutants were isolated through one of four mutagenesis processes, including extended growth, EMS mutagenesis, UV mutagenesis and spontaneous mutations.Classical ways of identifying suppressor mutations, including library production or screening, are labor intensive. To accelerate the identification of suppressors, mutations will be identified using high throughput sequencing. Of the 38 putative suppressors of ded1‐dam1, 28 putative suppressors were sequenced using Ilumina HiSeq 2500 technology. Putative suppressors were chosen for sequencing based on results of mitochondrial mutation screens and the strength of suppressor mutation, as determined by frogging of serial dilutions. We are currently developing a pipeline for processing the data and identifying the relevant SNPs within the genomic data. By identifying suppressors of this well‐defined ded1 mutant, we will identify new regulators of Ded1, an RNA helicase essential for translation initiation.Support or Funding InformationThe Thomas F. and Kate Miller Jeffress Memorial Trust Awards Program in Interdisciplinary Research; HHMI Grant to University of Richmond; Dr. Martha Carpenter Fellowship, University of RichmondThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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