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 (m7G) cap and a poly(A) tail at their 5′ and 3′ extremities, respectively. The hydrolysis of the m7G 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.

A unique surface on Pat1 C-terminal domain directly interacts with Dcp2 decapping enzyme and Xrn1 5′–3′ mRNA exonuclease in yeast

“…The Pat1C Domain Binds Dcp2 That Bridges Interaction with Edc3. We have previously shown using a yeast two-hybrid assay that the fungal-specific and conserved region located at the C-terminal part of Pat1C was important for interaction with Edc3 (21). Analysis of a deletion mutant demonstrated that the same region was required as well for growth at 37°C (21) revealing its functional importance.…”

“…This region displays a strongly conserved surface responsible for the direct interaction with Lsm2 and Lsm3 proteins from the Lsm1-7 complex, which binds to the 3′ end of oligoadenylated mRNAs (9,10,(18)(19)(20)(21). Pat1C is also important for Dcp2 and Xrn1 binding in yeast and human (8,17,22) as well as for yeast Edc3 (21) and human EDC4 recruitment (12). While the Dcp2, Xrn1, and EDC4 binding sites on Pat1C remain to be identified, the region responsible for yeast Edc3 binding has been mapped using the two-hybrid assay to a conserved region, which is only present at the C-terminal extremity of fungal Pat1 proteins (21).…”

“…Pat1C is also important for Dcp2 and Xrn1 binding in yeast and human (8,17,22) as well as for yeast Edc3 (21) and human EDC4 recruitment (12). While the Dcp2, Xrn1, and EDC4 binding sites on Pat1C remain to be identified, the region responsible for yeast Edc3 binding has been mapped using the two-hybrid assay to a conserved region, which is only present at the C-terminal extremity of fungal Pat1 proteins (21). However, whether this Pat1-Edc3 interaction is direct or bridged by other factors remains unclear.…”

RPS28B mRNA acts as a scaffold promoting cis-translational interaction of proteins driving P-body assembly

“…In principle, rps28aΔ and rps28bΔ strains could affect P-body assembly due to decreased levels of Rps28 protein. Indeed, Rps28 interacts with Edc3, Pat1, Lsm2/4/8 and Pby1 PB proteins as assessed by yeast two hybrid assays [36,37]. However, Rps28b protein reportedly only accounts for ~8% of the Rps28 protein population [20,38,39], and yet rps28bΔ strains exhibit a striking PB assembly defect, greater than that seen with rps28aΔ strains.…”

Pat1 activates late steps in mRNA decay by multiple mechanisms

“…The disordered N-terminal region of Pat1 contains a conserved motif that interacts with the DEAD-box ATPase Dhh1, but is generally dispensable for cell growth and normal mRNA turnover (35, 36). The α-α superhelical C-terminal domain (PatC) interacts with Lsm1-7, which is required for bulk 5’-3’ mRNA degradation in vivo (27, 28, 37–39). Removal of the unstructured middle domain severely attenuates decapping and 5’-3’ degradation, though the mechanism remains poorly understood (36, 40).…”