Pat1 contributes to the RNA binding activity of the Lsm1-7-Pat1 complex

Abstract

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5'-to-3' exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7-Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7-Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7-Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7-Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7-Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7-Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.

Keywords: Lsm1-7–Pat1 complex; Pat1; Sm-like proteins; decapping; mRNA decay.

FIGURE 1.

Lsm1-7 complex assembles in a pat1Δ mutant. Lsm1-7–Pat1 complex purified from wild-type cells (left) and the Lsm1-7 complex purified from pat1Δ cells (right) were separated by SDS-PAGE and visualized by silver staining.

FIGURE 2.

Lsm1-7 complex is severely impaired in its ability to bind RNA and does not exhibit a binding preference for oligoadenylated RNA. BSA or increasing concentrations of Lsm1-7–Pat1 complex purified from wild-type cells or the Lsm1-7 complex purified from pat1Δ cells (indicated on top in A and B) were subjected to gel shift assays using uniformly radiolabeled PGK1 and PGK1-A₅ RNAs (A) or MFA2 and MFA2-A₅ RNAs (B). Plots of the percentage of RNA bound vs. the concentration of the complex used are shown on the right of the phosphorimages of the gels. Bands of bound and unbound RNA are labeled.

FIGURE 3.

The Pat1 fragments are severely impaired in their ability to bind RNA and do not exhibit a binding preference for oligoadenylated RNA. BSA or increasing concentrations of Pat1M+C or Pat1C expressed and purified from E. coli (indicated on top) were subjected to gel shift assays using uniformly radiolabeled MFA2 and MFA2-A₅ RNAs. Plots of the percentage of RNA bound (quantitated using phosphorimager) vs. the concentration of the protein used are shown directly below the phosphorimages of the corresponding gels. Bands corresponding to the bound RNA are marked with asterisks on the left of the phosphorimages. At least two gel retarded bands are observed with Pat1C. The lower of the two bands may be the result of disassembly during the gel run of the RNP complex(s) representing the upper band.

FIGURE 4.

Pat1 directly contacts RNA. (A) Silver-stained SDS-PAGE of Pat1C and Pat1M+C purified from bacteria. (B,C) Lsm1-7–Pat1 complex purified from wild-type yeast (2 pmol), Lsm1-7 complex purified from pat1Δ yeast (3 and 5 pmol), Pat1C (45 pmol), or Pat1M+C (24 pmol) expressed and purified from E. coli or BSA was subjected to UV crosslinking in the presence of uniformly radiolabeled PGK1 RNA for varying lengths of time (indicated at the bottom). After ribonuclease treatment, the crosslinked proteins were visualized by denaturing PAGE and autoradiography.

FIGURE 5.

Association of Lsm1-7 complex with Pat1 fragments results in restoration of RNA binding activity. (A) BSA or purified Lsm1-7 complex was incubated with purified Pat1C or Pat1M+C or just buffer (as indicated on top) and then bound to anti-Flag antibody matrix. After washing followed by incubation of the matrix with uniformly radiolabeled PGK1 RNA and washing again, the RNA retained on the matrix was extracted and visualized by denaturing PAGE and autoradiography (top). (Bottom) A bar diagram of the amount of RNA retained on the matrix after each reaction (quantitated using phosphorimager) normalized to the value obtained with Lsm1-7 complex pre-incubated with just buffer. (B) BSA or increasing concentrations of Lsm1-7–Pat1M+C complex purified from pat1Δ cells expressing Pat1M+C fragment were subjected to gel shift assays using uniformly radiolabeled PGK1 and PGK1-A₅ RNAs. A plot of the percentage of RNA bound (quantitated using phosphorimager) vs. the concentration of the complex used and phosphorimages of the gels are shown on the lower right and upper panels, respectively. Lower left panel shows the silver-stained SDS-PAGE of the purified Lsm1-7–Pat1M+C complex.

FIGURE 6.

Pat1C does not copurify with the Lsm1-7 complex from yeast. (A) Lsm1-7 complex was purified from pat1Δ cells that express Pat1C or Pat1M+C, and the purified material was separated by SDS-PAGE and visualized by silver staining. (B) BSA or increasing concentrations of the complex purified from pat1Δ cells expressing Pat1C were subjected to gel shift assays using uniformly labeled PGK1 or PGK1-A₅ RNAs. Plots of the percentage of RNA bound (quantitated using phosphorimager) vs. the concentration of the protein used are shown directly below the phosphorimages of the gels. (C) The Lsm1-7 complex was purified from a pat1Δ strain expressing Flag-Pat1C and the input lysate, eluates of the first (anti-Flag antibody matrix pull-down), and second (Ni-NTA matrix pull-down) steps of purification and cell lysate from a control strain in which neither Lsm1 nor Pat1C is Flag tagged were subjected to Western analysis using anti-Flag antibodies.

FIGURE 7.

Inability of Pat1C to interact with the Lsm1-7 complex is not due to phosphorylation. (A) Lsm1-7 complex was pulled down using Ni-NTA matrix from the lysates of pat1Δ cells that express Pat1C, Flag-Pat1C, or Flag-Pat1C-AA (indicated on top) from a CEN plasmid followed by Western analysis using anti-Flag antibodies of the lysate and pull-down samples. Two independent transformants expressing Flag-Pat1C-AA were used for the experiment. (B) pat1Δ cells expressing Pat1C were grown in glucose containing medium to log phase and then transferred to fresh medium that contains or lacks glucose for 30 min before they were pelleted and lysed. The Lsm1-7 complex was pulled down from the lysates using Ni-NTA matrix followed by Western analysis using anti-Flag antibodies of the lysate and pull-down samples. (C) Lsm1-7 complex purified from pat1Δ yeast was incubated with Pat1C or Pat1C-EE purified from E. coli or BSA and then bound to the anti-Flag antibody matrix. After washing to remove unbound proteins, the bound proteins were eluted, separated by SDS-PAGE, and visualized by silver staining (left panel). (Right) Silver-stained SDS-PAGE of purified Pat1C and Pat1C-EE.