Elimination of the 5' cap of eukaryotic mRNAs, known as decapping, is considered to be a crucial, irreversible and highly regulated step required for the rapid degradation of mRNA by Xrn1, the major cytoplasmic 5'-3' exonuclease. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor. However, this complex has a low intrinsic enzymatic activity and requires several accessory proteins such as the Lsm1-7 complex, Pat1, Edc1-Edc2 and/or Edc3 to be fully active. Here we present the crystal structure of the active form of the yeast Kluyveromyces lactis Dcp1-Dcp2 enzyme bound to its product (mGDP) and its potent activator Edc3. This structure of the Dcp1-Dcp2 complex bound to a cap analog further explains previously published data on substrate binding and provides hints as to the mechanism of Edc3-mediated Dcp2 activation.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82–R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3′-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317–352–Hit1p70–164 complex reveals a novel mode of protein–protein association explaining the strong stability of the Rsa1p–Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p–Rsa1p–Hit1p heterotrimer.

The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species.