Numerous interactions act redundantly to assemble a tunable size of P bodies in Saccharomyces cerevisiae

Abstract

Eukaryotic cells contain multiple RNA-protein assemblies referred to as RNP granules, which are thought to form through multiple protein-protein interactions analogous to a liquid-liquid phase separation. One class of RNP granules consists of P bodies, which consist of nontranslating mRNAs and the general translation repression and mRNA degradation machinery. P bodies have been suggested to form predominantly through interactions of Edc3 and a prion-like domain on Lsm4. In this work, we provide evidence that P-body assembly can be driven by multiple different protein-protein and/or protein-RNA interactions, including interactions involving Dhh1, Psp2, and Pby1. Moreover, the relative importance of specific interactions can vary with different growth conditions. Based on these observations, we develop a summative model wherein the P-body assembly phenotype of a given mutant can be predicted from the number of currently known protein-protein interactions between P-body components.

Keywords: P bodies; RNP granules; mRNA decay.

Fig. 1.

edc3Δ lsm4ΔC yeast assemble P bodies in stationary phase. (A) Representative images of Dcp2-GFP– or Dhh1-GFP–containing wild-type and edc3Δ lsm4ΔC yeast grown in SComplete media containing 2% dextrose (dex). Images were taken with cells in midlog phase under glucose-depleted conditions and in stationary phase. (B) Quantification of P bodies in cells from the images obtained in A, in which images were similarly thresholded for fluorescence intensity and the percentage of cells with at least one Dcp2-GFP or Dhh1-GFP granule was determined. Error bars indicate the SD of mean values. Statistical significance for changes in cells with at least one P body was determined using a Student’s t test (**P ≤ 0.01). (C) Dcp2-GFP–containing wild-type and edc3Δ lsm4ΔC strains used in A and B were cotransformed with plasmid expressing Lsm1-RFP and grown to stationary phase to test colocalization with Dcp2-GFP granules. (Magnification: A and C, 100×.)

Fig. 2.

Overexpression of Dhh1 in edc3Δ lsm4ΔC yeast led to partial recovery of P-body assembly under glucose-depleted conditions. (A) edc3Δ lsm4ΔC yeast were transformed with either GFP-only or GFP-tagged variants of Dcp2, Dhh1, Pat1, Dcp1, and Lsm1, and tested for P-body assembly in glucose starvation under midlog growth. Figures contain representative images of cells observed via fluorescence microscopy. For comparison, edc3Δ lsm4ΔC with a genomically GFP-tagged Dcp2 was similarly treated and imaged. (B) Quantification of cells with GFP-positive puncta from A. (C) Western blot indicating two- to threefold overexpression of Dhh1-GFP off a CEN vector. Wild-type and edc3Δ lsm4ΔC yeast containing genomic Dhh1-GFP were transformed with either vector (−) or an additional copy of Dhh1-GFP (+), and expression was tested under midlog growth. (D) Representative images and quantification of overexpression of untagged Dhh1 on a CEN vector in edc3Δ lsm4ΔC Dcp2-GFP or edc3Δ lsm4ΔC Dhh1-GFP. (E) Colocalization of overexpressed Dhh1-mCherry with Dcp2-GFP in edc3Δ lsm4ΔC Dcp2-GFP yeast under midlog glucose-depleted conditions. (F) Dhh1-GFP–overexpressing edc3Δ lsm4ΔC yeast were glucose-starved in the presence of cycloheximide (100 µg/mL) and tested for its effect on P-body formation. (G) Wild-type and edc3Δ lsm4ΔC yeast were cotransformed with an untagged Dhh1 and Pab1-GFP, and tested for stress granule formation (Pab1-GFP foci) after 30 min of glucose starvation. The number of yeast cells with at least one Pab1-GFP stress granule was quantified. Error bars indicate the SD of the mean. Statistical significance was determined using a Student’s t test (**P ≤ 0.01, ***P ≤ 0.001). (Magnification: A and D–G, 100×.)

Fig. 3.

Psp2 and Pby1 overexpression recovers P-body assembly in edc3Δ lsm4ΔC yeast. (A) edc3Δ lsm4ΔC Dhh1-GFP yeast transformed with CEN and 2-μ (2u) vectors containing Psp2, Pby1, Psp1, and Pgd1 were tested for P-body assembly under midlog glucose-depleted conditions. (Magnification: 100×.) (B) The number of cells with at least one Dhh1-GFP–positive P body was quantified. (C) The number of cells with at least one Dcp2-GFP–positive P body in edc3Δ lsm4ΔC Dcp2-GFP yeast transformed with Psp2 and Pby1 on CEN and 2-μ vectors was quantified. Error bars indicate the SD of the mean. Statistical significance was determined using a Student’s t test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; n.s., not significant). For C, the significance was determined by comparing with P bodies in a CEN vector-only control.

Fig. 4.

Multivalent interactions of Dhh1 are essential for recovery of P-body assembly. (A) Table showing the mutations made in Dhh1. (B) GFP-tagged wild-type and mutant Dhh1 proteins were expressed in edc3Δ lsm4ΔC and its corresponding isogenic wild-type yeast, and the effect of the mutation on P-body recovery was tested. (C) Quantification of P-body assembly in wild-type yeast using Dhh1-GFP (wild type and mutants) to test recruitment of Dhh1-GFP variants to P bodies. (D) The number of cells with at least one Dhh1-GFP–positive (for tagged wild-type and mutant Dhh1 proteins transformed into edc3Δ lsm4ΔC yeast; black bars) or Dcp2-GFP–positive P body (for untagged wild-type and mutant proteins transformed into edc3Δ lsm4ΔC Dcp2-GFP yeast; patterned bars) was quantified. N.T., not tested. (E) Western blot showing expression of GFP-tagged Dhh1 mutants in edc3Δ lsm4ΔC yeast. (F) Dhh1 overexpression in edc3Δ pat1Δ yeast does not rescue P-body assembly. Wild type, edc3Δ lsm4ΔC, and edc3Δ pat1Δ yeast were cotransformed with plasmids encoding Dcp2-GFP and untagged Dhh1 (or vector-only), and P-body assembly was tested under glucose-depleted conditions. Error bars indicate the SD of the mean. Statistical significance was determined using a Student’s t test (**P ≤ 0.01, ***P ≤ 0.001; n.s., not significant). (Magnification: B and F, 100×.)

Fig. 5.

Dhh1 contributes to residual P-body assembly in vivo. (A and B) The contribution of Dhh1-mediated interactions on residual P-body assembly in (A) midlog phase and in (B) stationary phase (G0) was tested. (C) Quantification of P-body assembly by visualizing Dcp2-GFP–positive foci in wild-type, edc3Δ lsm4ΔC, edc3Δ lsm4ΔC dhh1Δ, and dhh1Δ only (stationary phase only). Error bars indicate the SD of the mean. Statistical significance was determined using a Student’s t test (**P ≤ 0.01, ***P ≤ 0.001). (Magnification: A and B, 100×.)

Fig. 6.

Submicroscopic P body-related mRNP assemblies exist in visible P body-deficient yeast. (A) Traces derived from nanoparticle tracking analysis of Dcp2-GFP, Dhh1-GFP, and GFP-only particles obtained from wild-type (Wt) yeast cells. Total cell lysates derived from yeast grown under glucose-replete (Top; + glucose) and glucose-depleted (Bottom; − glucose) conditions were analyzed using NTA. The dashed line indicates the peak height (395.5 nm) of the largest Dcp2-GFP granule observed in wild type. (B) Cycloheximide (Cyh; 100 μg/mL) treatment reduces P-body assembly as assessed by visualizing Dcp2-GFP particles from wild-type yeast. (C) Deletion of xrn1Δ increases mean P-body size compared with wild type. These experiments were conducted in an alternate yeast genetic background strain. (D) Distinct reduction in the mean size of Dcp2-GFP particles obtained from glucose-starved edc3Δ lsm4ΔC or edc3Δ lsm4ΔC dhh1Δ yeast. The particles observed in edc3Δ lsm4ΔC Dcp2-GFP and edc3Δ lsm4ΔC dhh1Δ Dcp2-GFP yeast exhibit enrichment of Dcp2-GFP over GFP in the corresponding control experiment (green trace; − glucose). (E) Overexpression of wild-type Dhh1 (blue) but not the RNA-binding mutant, RAKA (green), partially restores P-body assembly in edc3Δ lsm4ΔC Dcp2-GFP yeast. (F) Cycloheximide inhibits assembly of Dcp2-GFP particles in edc3Δ lsm4ΔC but not edc3Δ lsm4ΔC dhh1Δ yeast. The shaded area around the trend line in all traces represents the SEM derived from replicates.