mRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave

Abstract

The relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress. It appears that the relocalization of mRNA to Pbodies after stress is biphasic some mRNAs are present early, whereas others are recruited much later concomitant with recruitment of translation initiation factors, such as eIF4E. We also find that Bfr1p is a latephaselocalizing Pbody protein that is important for the delayed entry of certain mRNAS to Pbodies. Therefore, for the mRNAs tested, relocalization to Pbodies varies both in terms of the kinetics and factor requirements. This work highlights a potential new regulatory juncture in gene expression that would facilitate the overall rationalization of protein content required for adaptation to stress.

Keywords: Glucose regulation; Pbodies; Stress granules; Yeast; mRNA localization.

Fig. 1.

Earlyphase mRNAs are present in Pbodies early after glucose depletion. Fluorescence microscopy images of yeast cells at two different time points after glucose depletion. The RPS16A, RPS23B and PGK1 mRNAs are followed using the mTAG system MS2GFP, mRNA decay components are followed using CFPtagged Dcp2p and components of the closed loop complex are followed using RFPtagged eIF4E across the same cells. The colored inset overlay images after 50minutes of glucose depletion depict examples where the mRNAs colocalize with Pbodies yellow triangles but not with EGPbodies white diamonds. Graphs to the right represent the percentage of Pbodies or EGPbodies that harbor each mRNA after 10minutes white bars and 50minutes gray bars of glucose depletion. Scale bars 5 m.

Fig. 2.

Latephase mRNAs enter Pbodies after an extended period of glucose starvation. As Fig.1, following SPG4, SUE1, VNX1, TDP1 and RRP43 mRNA relative to mRNA decay and closed loop complex components. The colored inset overlay images after 50minutes of glucose depletion depict examples where the mRNAs colocalize with Pbodies yellow triangles but not with EGPbodies white diamonds. Graphs to the right show that the percentage of Pbodies harboring each mRNA increases from 10minutes white bars to 50minutes gray bars of glucose depletion, whereas minimal colocalization with EGPbodies was observed. Scale bars 5 m.

Fig. 3.

Time course of localization of a latephase mRNA to Pbodies relative to eIF4E. Fluorescence microscopy images of yeast cells over a time course after glucose depletion. The TDP1 mRNA is followed using the mTAG system via MS2GFP middle row, mRNA decay components are followed using CFPtagged Dcp2p top row and components of the closed loop complex are followed using RFPtagged eIF4E bottom row. A white triangle highlights the time point where eIF4E and the mRNA are first observed colocalizing with the Pbody marker. Scale bar 5 m.

Fig. 4.

Latephase mRNA localization relies upon Pbody formation. Fluorescence images of yeast cells after 50minutes of glucose depletion. lsm4C edc3 mutant strains were generated that carry the MS2tagged mRNAs labeled on the left. This mutant is deficient in Pbody formation, as shown by the lack of localization for Dcp2pCFP. The RPS16A mRNA provides an example where earlyphase mRNAs still aggregate, whereas localization is not observed for two latephase mRNAs, VNX1 and TDP1. Scale bars 5 m.

Fig. 5.

Bfr1p interacts with Dcp2p and Xrn1p via RNA and enters Pbodies. A Western blots IB from TAP TAPIP on strains bearing TAPtagged Bfr1p, as well as Myctagged Xrn1p or Dcp2p. B The reciprocal affinity purification of either Xrn1pTAP or Dcp2pTAP in strains containing Bfr1pMyc. In both A and B, TAPtagged proteins were detected with a protein A peroxidase conjugate PAP, Myctagged proteins were detected with an antibody against Myc, the presence of Tef1p was detected using an antibody against Tef1p. In both sets of experiments, an untagged wildtype strain was used as a negative control on the same gel. C Localization of Bfr1p in exponentially growing cells glucose or after 10 or 50minutes of glucose starvation glucose. Scale bar 5 m.

Fig. 6.

Latephase mRNA localization to Pbodies requires Bfr1p. Images of bfr1 mutant strains containing Dcp2pCFP and MS2tagged RPS16A, VNX1 or TDP1. The mRNA and Dcp2p localization is shown after A no starvation, B 10minutes of glucose starvation, C 50 minutes of glucose starvation. Scale bars 5 m.

Fig. 7.

A model depicting the two phases of mRNA relocalization to Pbodies. Phase I. Following cellular stress, such as glucose starvation, earlyphase mRNAs relocalize to Pbodies with the mRNA decay machinery. Here, the mRNAs are either degraded or held in a translationally repressed state. Phase II. More prolonged glucose starvation leads to a release of latephase mRNAs that have been associated with the translation initiation machinery in a repressed state. These mRNAs are relocalized to Pbodies in a Bfr1pdependent manner.