Clarifying Ded1 Function in Translational Control using an in vivo Fluorescence‐Based Reporter Assay

Abstract

Eukaryotic translation initiation is a critical node at which gene expression is regulated. Among the factors that are known to regulate this process, DEAD‐box RNA helicases are believed to promote translation by resolving secondary structure in the 5′ UTR of an mRNA. These helicases include the protein Ded1 in budding yeast, which is required to remove cap‐distal structure in the 5′ UTR of an mRNA, thus enabling scanning of the preinitiation complex. Yet the role of Ded1 in translation is complex; a translationally repressive role has also been demonstrated by Ded1 overexpression. Further confounding investigations of Ded1 function are the large number of protein‐protein and protein‐RNA contacts made by Ded1 to regulate activity. To study the diverse roles that Ded1 plays in translation, we have developed a system that reports on Ded1‐specific changes in translation in vivo. This system relies on a fluorescence‐based reporter that simultaneously promotes the transcription of two mRNA transcripts: one encoding RFP with a 5′ UTR that is translated independent of Ded1 activity, and the other encoding GFP with a 5′ UTR that requires Ded1‐activity for translation. We are using this system in combination with a library of random Ded1 mutants to broadly interrogate Ded1 activities for effects on translation initiation and repression. Support or Funding Information This work was supported by NIH grant R00GM119173 and start‐up funds from SUNY at Buffalo, College of Arts and Sciences.