Pat1 contains distinct functional domains that promote P-body assembly and activation of decapping

Abstract

The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.

FIG. 1.

Deletion constructs of Pat1. (A) Regions of homology from a fungal alignment. Pat1 fungal homologs from S. cerevisiae, S. paradoxus, S. mikatae, S. bayanus, S. castellii, and S. kudriavzevii were downloaded from the Saccharomyces Genome Database and imported into Vector NTI (Invitrogen, CA). We then aligned the sequences using the AlignX module of Vector NTI and determined the regions of similarity from within the alignment module. CTD, C-terminal domain; NTD, N-terminal domain. (B) Format of the deletion constructs used to determine functional domains of Pat1 showing placement of the blunt-cutting restriction endonucleases. (C) Observed phenotypes of the respective deletion constructs. (D) Western blot showing expression levels of the Pat1 deletion constructs. wt, wild type.

FIG. 2.

Residues 254 to 422 of Pat1 are required for growth at 35°C in a pat1Δ strain. Shown is growth of pat1Δ yeast cells at 35°C transformed with plasmids containing empty vector (pat1Δ), wild-type Pat1 (wt), or the deletion constructs. Yeast strains were grown on minimal medium plates supplemented with 2% dextrose and placed at 35°C for 3 days.

FIG. 3.

Residues 254 to 422 of Pat1 are required for the restoration of decapping in a pat1Δ strain. (A) Shown is the steady-state decay analysis of the unstable MFA2 reporter mRNA in a pat1Δ strain. Each lane represents the strain containing empty vector (pat1Δ), wild-type PAT1 on a CEN vector (wt on CEN), or deletion constructs. SCR1 RNA is shown below each panel as a loading control. The diagram indicates the relative positions of the full-length mRNA and the poly(G) decay intermediate. M indicates the molecular size marker. (B) Graph depicting the relative percentages of the poly(G) decay fragment versus full-length mRNA for pat1Δ, the wild type on the CEN plasmid, and each respective deletion construct-containing pat1Δ strain. Error bars indicate ranges of values obtained. Asterisks indicate groupings that showed statistically significant differences from each other assessed by a Mann-Whitney nonparametric test and with a P value of less than 0.02.

FIG. 4.

Residues 422 to 763 of Pat1 are required for Lsm1-GFP to accumulate in P bodies. Using Lsm1-GFP as a marker for P bodies, we observed P bodies in either a wild-type (wt), pat1Δ, or deletion construct-containing pat1Δ strain.

FIG. 5.

Residues 422 to 763 of Pat1 are required for Pat1-GFP to accumulate in P bodies. pat1Δ strains were transformed with plasmids containing deletion constructs of Pat1 tagged with a C-terminal GFP. P bodies were observed only in constructs that contained residues 422 to 763.

FIG. 6.

Residues 422 to 697 of Pat1 are required for the overexpression growth defect, P-body increase, and translational repression caused by Pat1. (A) Cartoon illustrating the construction of the chromosomally integrated galactose overexpression cassettes of Pat1 deletions. The KanMX6 GAL upstream activating sequence GST cassette was integrated upstream of full-length PAT1 as well as each respective PAT1 deletion, allowing the galactose-driven overexpression of each respective construct. CTD, C-terminal domain; NTD, N-terminal domain. (B) Growth of yeast cells of each respective construct when plated on galactose medium. YEP, yeast extract-peptone. (C) P-body accumulation in cells following overexpression of the respective constructs. Times after the addition of galactose are indicated. P bodies were visualized using Dcp2-GFP. (D) Histogram showing the percentage of P-body-containing cells with two or more large, visible P bodies per cell both pre- and postinduction with galactose for each respective PAT1 deletion. (E) Histogram showing incorporation of [³⁵S]methionine in cells overexpressing Pat1 deletions 10 min after the addition of label. The values for incorporation following sucrose induction were set as 100% for each deletion strain, and the values for galactose induction are represented as a percentage of the sucrose values.

FIG. 7.

Residues 422 to 763 of Pat1 are required for Dcp2-GFP to accumulate in P bodies at mid-log phase. (A) Using Dcp2-GFP as a marker for P bodies, we observed P bodies in a mid-log-phase growing pat1Δ strain containing the indicated deletion constructs on CEN plasmids. (B) Histogram indicating the percentage of cells for each construct that contained visible P bodies and the proportion of those P bodies that were larger than typical P bodies. wt, wild type.

FIG. 8.

Lsm1, Dcp1, and Edc3 interact with different domains of Pat1, and Pat1 interacts with the Lsm domain of Edc3. (A) Using a two-hybrid analysis, we determined that Lsm1, Dcp1, and Edc3 all interact with full-length Pat1. By repeating the two-hybrid analysis with the deletion constructs of Pat1, we showed the specific regions of Pat1 with which the proteins interact. +++ indicates a strong interaction with Pat1, while - - - indicates no interaction with Pat1. a.a., amino acids; NTD, N-terminal domain; CTD, C-terminal domain. (B) Using a two-hybrid analysis, we determined the interaction of Pat1 with domains of Edc3. Pat1 specifically interacts with the Lsm domain of Edc3. +++ indicates a strong interaction with Pat1, while - - - indicates no interaction with Pat1. FDF, FDF amino acid motif.

FIG. 9.

Pat1 binds to RNA homopolymers. Binding of in vitro-translated [³⁵S]methionine-labeled Pat1 and Pat1 domains to poly(U)-Sepharose was done in the presence of yeast tRNA at 0.5 μg/ml as a nonspecific competitor. Binding reactions were carried out in the presence of specific competitors at 10- and 100-fold excesses. No binding to the protein G-Sepharose occurred. Lanes marked input show 10% of the volume of the in vitro-translated products used for the binding reaction of full-length Pat1, 10% of the volume for residues 10 to 254, 5% of the volume for residues 254 to 422, and 5% of the volume for residues 422 to 763.

FIG. 10.

Model for Pat1 function. LSM complex, Lsm1-7 protein complex.