Crystal structure of Dcp1p and its functional implications in mRNA decapping

Abstract

A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5′→3′ exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains. Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites. One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins. Moreover, a conserved hydrophobic patch is shown to be critical for decapping.The process of mRNA turnover is important in numerous aspects of eukaryotic mRNA physiology. These roles include the control of gene expression, antiviral defenses 1,2 and mRNA surveillance by recognizing and degrading the aberrant mRNAs 3 . Two major pathways of mRNA decay exist in eukaryotic cells 4 . In both yeast and mammals, mRNA degradation usually begins with the shortening of the poly(A) tail at the 3′ end of the mRNA. After deadenylation, the 5′ cap structure can be removed (decapping), thereby exposing the transcript to digestion by a 5′→3′ exonuclease Xrn1p 5-8 . Alternatively, after deadenylation in both yeast and mammals, mRNAs can be degraded in a 3′→5′ direction by the cytoplasmic exosome 9-12 . The resulting cap structure is hydrolyzed by the DcpS scavenger decapping enzyme, which is encoded by the DCS1 gene in yeast 13 .Decapping is a key step in the 5′→3′ decay pathway because it induces degradation of the mRNA, and thus it is subject to numerous control inputs. Decapping is also critical in an aspect of mRNA surveillance in which aberrant mRNAs containing nonsense codons are decapped without a requirement for deadenylation and are degraded in a 5′→3′ direction [14][15][16] RESULTS Overall structure of Dcp1pThe crystal structure of Dcp1p was solved by single wavelength anomalous dispersion (SAD) using selenomethionine (SeMet)-substituted crystals. The final model contains two copies of the Dcp1p, designated as molecules A and B, respectively. Several regions of the polypeptide are not visible in the electron density map and are assumed to be disordered, namely residues 1-21, 74-134 and 229-231 for molecule A, and residues 1-16 and 81-134 for molecule B in the crystallographic asymmetric unit. The current refined model has an R-factor of 22.7% and a R free value of 26.8% at a resolution of 2.3 Å with very good stereochemistry (see Table 1 and Methods). m 7 GDP could not be located in the electron density map or in washed crystals, suggesting that m 7 GDP probably does not bind in the crystal lattice (data not shown).No substantial differences are observed between the structures of the two Dcp1p molecules in the asymmetric uni...

DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

“…Yeast DCP2 interacts directly with DCP1 and this interaction is required for decapping in vivo and in vitro (3)(4)(5)(6)(7). In humans, the DCP2-DCP1 interaction requires additional proteins, which together assemble into multimeric decapping complexes that also include the enhancers of decapping 3 and 4 (EDC3 and ECD4), and the DEAD-box protein DDX6/RCK (8,9).…”

“…In humans, the DCP2-DCP1 interaction requires additional proteins, which together assemble into multimeric decapping complexes that also include the enhancers of decapping 3 and 4 (EDC3 and ECD4), and the DEAD-box protein DDX6/RCK (8,9). DCP2 is highly conserved and most information on DCP2 activation stems mainly from studies in S. cerevisiae and S. pombe (3)(4)(5)(6)(7). Fungi, however, lack EDC4 as well as many extensions and additional domains present in decapping activators of metazoan orthologs (8)(9)(10).…”

“…Fungi, however, lack EDC4 as well as many extensions and additional domains present in decapping activators of metazoan orthologs (8)(9)(10). For example, all eukaryotic DCP1 proteins contain an N-terminal EVH1 domain (3,5,6); however, DCP1 orthologs from metazoa and plants also have a proline-rich C-terminal extension (9, 10). The sequence of this extension is not conserved except for a 14-residue short motif (motif I, MI) conserved in metazoa with the exception of C. elegans ( Fig.…”

Functional insights from structures of coactivator-associated arginine methyltransferase 1 domains

“…Moreover, the surface buried at the interface of the CARM1 28-140 dimer hides the ligandbinding site found in all PH domains ( Figure 2B). To date, PH domain-like fold contains nine families in SCOP (Murzin et al, 1995) and includes proteins involved in a variety of biological processes such as signal transduction, cytoskeletal organization, nuclear transport and DNA repair (Blomberg et al, 1999;Ball et al, 2002;Lemmon et al, 2002;Gervais et al, 2004;She et al, 2004;Lemmon, 2007). This superfamily contains proteins that share a highly similar fold in spite of insignificant sequence similarity.…”

Isolation and characterization of a mutant defective in utilization of exogenous phosphatidylethanolamine in Saccharomyces cerevisiae

“…This mutation leads to a substitution of Gly to Arg at amino acid residue 141 within the highly conserved region (Fig. 2E) (She et al, 2004). This result suggests that M25 likely has an aberrant Dcp1p and this aberrant Dcp1p makes the yeast cells defective in utilizing exogenous PE when the synthesis of PE is suppressed.…”