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

Drummond, Sheona P.; Hildyard, John; Firczuk, Helena; Reamtong, Onrapak; Li, Ning; Kannambath, Shichina; Claydon, Amy J.; Beynon, Robert J.; Eyers, Claire E.; McCarthy, John E. G.

One model for the mode of action of Dhh1 and Pat1 in the control of mRNA degradation is that they promote re-localization of mRNAs by inhibiting translation, either on individual mRNA species or generically. This would seem to be consistent with the proposal that the rates of translation and mRNA degradation are inversely related to each other, so that suppression of translation will automatically lead to accelerated decay (3). We decided to explore the idea that the mechanism of action of these proteins is mediated by changes in translation. A key premise of this model is that translational repression leads to mRNA destabilization. If this is true, constraints imposed on the activity of the translation machinery by limiting translation factor activity should result in accelerated mRNA decay. We utilized rpb1-1 strains in which the expression of CDC33 (encoding eIF4E) had been placed under the control of the tet07 operator, thus allowing modulation of gene synthesis via the addition of doxycycline. Using doxycycline to reduce the intracellular abundance of eIF4E to 75 and to 50%, respectively, of the wild-type level, which in turn reduces protein synthesis to 83 and 72% of the wild-type rate, we determined whether there are measurable changes in the half-lives of S. cerevisiae mRNAs which manifest distinct stabilities, i.e. those of ACT1 and MF2 (Supplementary Figure S9) . The results suggest that the global rates of translation and decay, at least over the range explored here, are not universally coupled to each other. For comparison, we performed parallel experiments using a tet07::DHH1 rpb1-1 strain (Supplementary Figure S9) , confirming that repression of intracellular Dhh1 does lead to mRNA stabilization (3). We also examined whether limiting translation in vivo can induce formation of P bodies, since Dhh1 and Pat1 are both known to accumulate in these bodies in response to the diauxic growth shift. This was investigated using strains that contain tet07-regulated translation factor genes. Starting with an initiation factor, we found that suppression of the level of eIF4A in the cell led to reduced rates of translation but did not, in itself, induce the formation of P bodies during exponential growth ( Figure 6 ). Interestingly, restricting the availability of eIF4A also limited the observed degree of association of Dhh1 with actively translating polysomal complexes. Moreover, inhibition of in vivo translation via suppression of an elongation factor (in a tet07::TEF3 strain) also did not trigger formation of P bodies in the exponential phase (Supplementary Figure S10) . In summary, slowing translation in vivo by imposing inhibition at either the initiation step or the elongation step had no effect on the relationship between cellular growth phase and P body formation.