Abstract
Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.
Highlights
Rapid modulation of gene expression is critical for cells to respond to environmental challenges and to initiate developmental programs
To differentiate whether loss of Dhh1 ATPase activity triggers formation of genuine processing bodies or whether these Dhh1DQAD-induced foci are anomalous granules, we monitored the localization of several PB components – namely Dcp1, Edc3, and Xrn1 – in both DHH1 and dhh1DQAD mutant cells
The stress granule marker Pab1 did not assemble into foci in dhh1DQAD cells (Figure 1—figure supplement 1A) demonstrating that Dhh1DQAD granules are composed of proteins found in bona fide PBs
Summary
Rapid modulation of gene expression is critical for cells to respond to environmental challenges and to initiate developmental programs. Eukaryotic cells have developed a variety of mechanisms to achieve tight regulation of gene expression This includes post-transcriptional control of messenger RNA (mRNA) levels by the regulation of translation or by varying the rates of mRNA degradation. Cytoplasmic mRNAs are marked by a 7-methylguanosine cap at the 5’ end and by a polyA tail at the 3’ end These modifications enable interaction with translation factors, including the cap-binding complex (eIF4F) and the polyA binding protein (Pab1) and protect the mRNA against degradation (Coller and Parker, 2004). Given their impact on both translation and mRNA decay, the status of the 5’ and 3’ ends of the mRNA, as well as the complement of proteins that bind the mRNA termini, are tightly controlled. While deadenylated mRNAs can be degraded from the 3’ end by the 10-subunit exosome complex (Chlebowski et al, 2013), mRNA decay in S. cerevisiae occurs predominantly via removal of the 5’ cap by the Dcp1-Dcp decapping enzyme, followed by degradation by the 5’-3’ exonuclease, Xrn (Garneau et al, 2007; Sun et al, 2013)
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