The C-terminal domain from S. cerevisiae Pat1 displays two conserved regions involved in decapping factor recruitment

Abstract

Eukaryotic mRNA decay is a highly regulated process allowing cells to rapidly modulate protein production in response to internal and environmental cues. Mature translatable eukaryotic mRNAs are protected from fast and uncontrolled degradation in the cytoplasm by two cis-acting stability determinants: a methylguanosine (m(7)G) cap and a poly(A) tail at their 5' and 3' extremities, respectively. The hydrolysis of the m(7)G cap structure, known as decapping, is performed by the complex composed of the Dcp2 catalytic subunit and its partner Dcp1. The Dcp1-Dcp2 decapping complex has a low intrinsic activity and requires accessory factors to be fully active. Among these factors, Pat1 is considered to be a central scaffolding protein involved in Dcp2 activation but also in inhibition of translation initiation. Here, we present the structural and functional study of the C-terminal domain from S. cerevisiae Pat1 protein. We have identified two conserved and functionally important regions located at both extremities of the domain. The first region is involved in binding to Lsm1-7 complex. The second patch is specific for fungal proteins and is responsible for Pat1 interaction with Edc3. These observations support the plasticity of the protein interaction network involved in mRNA decay and show that evolution has extended the C-terminal alpha-helical domain from fungal Pat1 proteins to generate a new binding platform for protein partners.

Figure 1. Structure of S. cerevisiae Pat1 C domain.

A and B. Left panels: ribbon representation of ScPat1C crystal structure. The ARM repeats are depicted using different colors. Middle panel: Mapping of the sequence conservation at the surface of the ScPat1C domain. Coloring is from grey (low conservation) to cyan (highly conserved). The conservation score was calculated using the CONSURF server and using an alignment made from the sequences of 30 Pat1 fungal orthologues. Right panel: Mapping of the electrostatic potential at the surface of the ScPat1C domain. Positively (10 k_BT/e^-) and negatively (−10 k_BT/e^-) charged regions are colored in blue and red, respectively. The electrostatic potential was calculated using PBEQ Solver server . Orientation in B differs from A by a 180° rotation along the horizontal axis. C. Ribbon representation of the superimposition between human Pat1C domain (green; [36]) and ScPat1C domain. The core conserved from yeast to human is colored in yellow. The fungi specific C-terminal extension from ScPat1C is colored in red. D. Surface representation of ScPat1 domain. Same color code as panel C.

Figure 2. Sequence alignment of Pat1 orthologues.

Alignment was performed using ClustalW . Strictly conserved residues are in white on a black background. Partially conserved amino acids are boxed. Secondary structure elements assigned from the ScPat1C structure are indicated above the alignment. Black stars below the sequences indicate residues mutated in this study. This figure was generated using the ESPript server .

Figure 3. The conserved residues at the N-terminal residues are involved in interaction with Lsm1-7.

A. ScPat1C structure with the residues mutated in this study shown as ball and sticks. Color scheme identical to Fig.1A. B. Growth analysis of PAT1 mutants. Serial dilutions of the different strains transformed with vector, a plasmid encoding wild type ScPat1, or the indicated mutant thereof, were deposited on plates and incubated at the indicated temperatures. C. Pull-down experiment of untagged Lsm1-7 complex with His₆ ScPat1C wild type or mutants. Input (top) and eluted (bottom) samples were analyzed by 22% SDS-PAGE and Coomassie Blue staining. The asterisk denotes a contaminant protein that co-purifies with the Lsm1-7 complex but which is not retained on the HisPur™ Ni-NTA Magnetic Beads used for these pull-down experiments. Molecular weights (kDa) of the markers are indicated on the left of the gels.

Figure 4. The yeast specific residues located at the C-terminus of ScPat1C are functionally important.

A. ScPat1C structure with the residues mutated in this study shown as ball and sticks. Color scheme identical to Fig.1A. The region absent in the ScPat1ΔC68 construct is colored in grey. B. Growth analysis of PAT1 mutants. Serial dilutions of the different strains transformed with vector, a plasmid encoding wild type ScPat1, or the indicated mutant thereof, were deposited on plates and incubated at the indicated temperatures. C. Monitoring ScPat1 interaction with Edc3, Rps28 or Scd6 using the two-hybrid assay. The wild-type or edc3Δ strain was transformed with the indicated pairs of vectors and interaction between the factors encoded by these two plasmids was scored by assaying β-galactosidase activity.