Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

Abstract

Dhh1 and Pat1 in yeast are mRNA decapping activators/translational repressors thought to play key roles in the transition of mRNAs from translation to degradation. However, little is known about the physical and functional relationships between these proteins and the translation machinery. We describe a previously unknown type of diauxic shift-dependent modulation of the intracellular locations of Dhh1 and Pat1. Like the formation of P bodies, this phenomenon changes the spatial relationship between components involved in translation and mRNA degradation. We report significant spatial separation of Dhh1 and Pat1 from ribosomes in exponentially growing cells. Moreover, biochemical analyses reveal that these proteins are excluded from polysomal complexes in exponentially growing cells, indicating that they may not be associated with active states of the translation machinery. In contrast, under diauxic growth shift conditions, Dhh1 and Pat1 are found to co-localize with polysomal complexes. This work suggests that Dhh1 and Pat1 functions are modulated by a re-localization mechanism that involves eIF4A. Pull-down experiments reveal that the intracellular binding partners of Dhh1 and Pat1 change as cells undergo the diauxic growth shift. This reveals a new dimension to the relationship between translation activity and interactions between mRNA, the translation machinery and decapping activator proteins.

The DEAD-Box Protein Dhh1 Promotes Decapping by Slowing Ribosome Movement

“…Indeed, we and others have observed that Dhh1 binds ribosomes [38]. Moreover, Dhh1 represses translation in vitro of an mRNA harboring the Cricket Paralysis Virus IRES, which requires only 40S ribosomes to initiate translation [12].…”

“…We predicted that if endogenously expressed Dhh1 were performing the same function, Dhh1 should be found associated with polyribosomes. We and others have documented, however, that Dhh1 sediments with the soluble RNP in sucrose gradients [12],[38]. We reasoned that the association of Dhh1 with polyribosomes in cells with active decay machinery and minimal cues for translational repression (i.e.…”

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

“…An alternative source of altered mRNA stability can come from changes in the translation of mRNAs (Drummond et al, 2011; Huch and Nissan, 2014; Jacobson and Peltz, 1996). We therefore examined the polysome profiles of extracts from yeast exponentially growing in glucose to gain insight into their translation.…”

Not4‐dependent translational repression is important for cellular protein homeostasis in yeast

“…Yeast Dhh1 interacts physically with the Ccr4-Not complex (Hata et al, 1998;Coller et al, 2001;Maillet & Collart, 2002) and contributes to translational repression (Coller & Parker, 2005). Moreover, an interaction between Dhh1 and polysomes has been described (Drummond et al, 2011) and components of the Ccr4-Not complex including Not proteins and Dhh1 localize to cytoplasmic foci called P-bodies (Muhlrad & Parker, 2005;Parker & Sheth, 2007), where turnover of translationally repressed mRNA takes place. Indeed, we found that also Dhh1 contributes to the repression of K12 protein synthesis ( Fig 6B).…”