Yeast DEAD-box helicase Ded1 stimulates translation initiation, particularly of mRNAs with structured 5'UTRs. Interactions of the Ded1 N-terminal domain (NTD) with eIF4A, and Ded1-CTD with eIF4G, subunits of eIF4F, enhance Ded1 unwinding activity and stimulation of preinitiation complex (PIC) assembly in vitro. However, the importance of these interactions, and of Ded1-eIF4E association, in vivo were poorly understood. We identified separate amino-acid clusters in the Ded1-NTD required for binding to eIF4A or eIF4E in vitro. Disrupting each cluster selectively impairs native Ded1 association with eIF4A or eIF4E, and reduces cell growth, polysome assembly, and translation of reporter mRNAs with structured 5'UTRs. It also impairs Ded1 stimulation of PIC assembly on a structured mRNA in vitro. Ablating Ded1 interactions with eIF4A/eIF4E unveiled a requirement for the Ded1-CTD for robust initiation. Thus, Ded1 function in vivo is stimulated by independent interactions of its NTD with eIF4E and eIF4A, and its CTD with eIF4G.
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 9 and 9-supplement 1.
- Suna Gulay
- Neha Gupta
- Jon R Lorsch
- Alan G Hinnebusch
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Nahum Sonenberg, McGill University, Canada
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis, and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted, including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.
A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.