X ray crystallographic study of yeast dcp1 and dcp2 proteins insights into the mechanism and regulation of eukaryotic mRNA decapping

X-RAY CRYSTALLOGRAPHIC STUDY OF YEAST DCP1 AND DCP2 PROTEINS: INSIGHTS INTO THE MECHANISM AND REGULATION OF EUKARYOTIC mRNA DECAPPING SHE MEIPEI (B.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE Acknowledgements I would like to express my gratitude to all the people that make my graduate research possible and enjoyable. First of all, I would like to thank my supervisor Dr. Haiwei Song for giving me the opportunity to work on this interesting project and for his invaluable support and guidance, without which this thesis would not have been possible. I am greatly indebted to our collaborators Dr. Carolyn Decker and Professor Roy Parker at the University of Arizona for their contribution to this study (chapters 3.1.3 and 3.2.6). I would like to thank current and former SHW lab members for their constant help and comradeship, especially Cheng Zhihong, Kong Chunguang, Chen Nan, Zhou Zhihong, and Wu Mousheng. And I want to thank Sharon Ling for proofreading the manuscript. I would also like to thank previous supervisors in my first year of rotation, Dr. Qi Xie and Dr. Mohan Balasubramanian at the ex-Institute of Molecular Agrobiology, for their guidance and patience. I acknowledge the Institute of Molecular and Cell Biology for financial support and beamline scientists at SPring-8, ESRF and DESY for technical support. Finally, I am thankful to my father, my mother and my sister for their support and understanding throughout the years. I TABLE OF CONTENT Acknowledgments I Table of content II Summary V Abbreviations VII List of Figures X List of Tables XI Chapter 1. Introduction mRNA turnover 1.1 1.1.1 The life cycle of mRNA 1.1.2 Biological significance of mRNA decay 1.1.3 General mRNA decay pathways 1.1.4 Specialized mRNA decay pathways 1.1.5 cis-trans interaction affecting mRNA stability 12 1.1.6 mRNA degradation and diseases 13 5’-3’ decay pathway and processing bodies 1.2 14 1.2.1 Components of mRNA 5’decay machinery 14 1.2.2 mRNA cytoplasmic processing bodies 17 mRNA decapping enzymes 1.3 1.3.1 1.3.1.1 1.3.2 1.3.2.1 1.3.3 1.4 21 Characteristics of Dcp1 protein 21 The EVH1 domain 23 Characteristics of Dcp2 protein 25 The Nudix pyrophosphatase 27 Other decapping enzymes 29 Rationales of my study 33 II Chapter Material and Methods 2.1 Experimental materials 34 2.2 Molecular cloning 42 2.3 Protein analysis 46 2.4 Functional Analysis 47 2.5 Protein purification, crystallization and structure determination 53 Chapter Results Part I: Crystal structure of scDcp1p 63 3.1.1 Structural overview 63 3.1.2 scDcp1p belongs to the EVH1 family 63 3.1.3 Analysis on scDcp1p surface to identify regions for potential Dcp2p binding 67 3.1 3.2 Part II: Crystal structure of spDcp2n 75 3.2.1 Structural overview 75 3.2.2 Nudix domain as the catalytic center 77 3.2.3 The function of the N-terminal domain 81 3.2.4 The Dcp1 binding site 86 3.2.5 spDcp1p stimulates spDcp2p decapping activity 87 3.2.6 in vivo and in vitro study of S. cerevisiae Dcp2 protein 90 Part III: Structural basis for S. pombe Dcp1p and Dcp2p interaction 3.3 94 3.3.1 Structural overview of the S. pombe Dcp1p-Dcp2NT complex 94 3.3.2 The protein-protein interface in the Dcp1p-Dcp2NT complex 96 Chapter 4.1 Discussion Comparison of putative PRS binding site of scDcp1p with other EVH1 domains 98 III 4.2 The Dcp1-Dcp2 complex in lower and higher eukaryotes 99 4.3 Implication on the assembly and regulation of 5’ mRNA decay machinery 100 References 102 List and reprints of publications 113 IV Summary mRNA degradation is important in post-transcriptional gene regulation. There are two major mRNA decay pathways in eukaryotes, both initiated by the shortening of the poly(A) tail in the 3’ end of mRNA. After deadenylation, the transcript can be degraded in the 3’ pathway by the exosome complex. Alternatively, it can be degraded in a 5’ pathway, in which the 5’ guanosine cap is removed by the decapping enzyme and the transcript is hydrolyzed by the 5’→3’ exonuclease. The enzymes and factors involved in the 5’ decay pathway are co-localized into the cytoplasmic processing bodies, whereby nonsense-mediated mRNA decay and RNA interference also take place. As a rate-limiting step of 5’ decay pathway, the decapping reaction is carried out by the Dcp1-Dcp2 holoenzyme. Dcp2 is a Nudix pyrophosphatase and Dcp1 stimulates the activity of Dcp2. The crystal structures of yeast Dcp1 and Dcp2 proteins are presented in this study. The structure of the S.cerevisiae Dcp1 protein shows that it resembles the EVH1 domain, a protein-protein interaction module. Two highly conserved patches have been identified on the surface of Dcp1p: one corresponds to the ligand recognition site of the EVH1 family and the other is specific to Dcp1 proteins. Biochemical assays demonstrated that these two patches are not required for direct Dcp2p binding but it could be a putative binding site for other regulators. The N-terminal 300 residues of S.cerevisiae Dcp2p are necessary and sufficient for mRNA decapping. The crystal structure of the corresponding region of S. pombe Dcp2(1-266) shows that it consists of an N-terminal helical domain followed by a Nudix domain. The Nudix domain is the catalytic domain, containing a Nudix motif characteristic of this family of pyrophosphatases. Mutagenesis study confirmed the V significance of two glutamic acid residues, Glu143 and Glu147, inside the Nudix motif. A third glutamic acid residue, Glu192, critical for decapping and outside of the Nudix motif was also identified based on structural analysis. The N-terminal domain is indispensable for mRNA turnover in vivo. In vitro, this region not only contributes to the decapping activity of the Nudix domain but also mediates the Dcp1-Dcp2 complex formation. A portion of the large conserved patch on the Dcp2 N-terminal domain was identified to be critical for Dcp1p binding by GST pull-down assay. The equivalent residues in S. cerevisiae Dcp2p critical for Dcp1p binding was demonstrated by yeast two-hybrid. Importantly, the association of Dcp1p to the Nterminal domain of Dcp2 is shown to be required for the stimulation of the Dcp2 protein activity. The crystal structure of S. pombe Dcp1p in complex with the N-terminal domain of Dcp2p confirmed the previous finding that the highly conserved residues on Dcp2 N-terminal domain are cricital for Dcp1p binding. In contrast to Dcp2p, Dcp1p binds to Dcp2p using mainly variant residues, suggesting that the direct interaction of Dcp1p with Dcp2p is not conserved across species, consistent with the notion that the binding of Dcp1 to Dcp2 in higher eukaryotes requires an additional factor. Based on these studies, the implication on mRNA decapping mechanism and regulation is discussed. VI Abbreviations 3AT 3-aminotriazole ADPRP ADP-ribose pyrophosphatase AMD ARE-mediated mRNA decay Ap4AP Ap4A pyrophosphatase ARE AU-rich elements ATP adenosine triphosphate CBC cap-binding complex CD circular dichroism CPSF cleavage and polyadenylation specificity factor CTD C-terminal domain DSE downstream sequence elements DTT dithiothreitol EDTA ethylene diamine tetra acetic acid EG ethylene glycol EJC exon junction complex EVH1 Enabled/VASP homology GST glutathione S-transferase GTP guanosine triphosphate HIT histidine triad IRES internal ribosome entry site Lsm Sm-like m7GDP 7-methylated guanosine diphosphate m7GMP 7-methylated guanosine monophosphate miRNA microRNA VII MPD 2-methyl-2, 4-pentanediol mRNA messenger RNA mRNP messenger ribonucleoprotein particle NCS non-crystallographic symmetry NGD no-go decay NMD nonsense-mediated mRNA decay NPC nuclear pore complex NSD non-stop decay NTD N-terminal domain PABP poly(A) binding protein PBC primary biliary cirrhosis P-body processing body PDB Protein Data Bank PH pleckstrin homology PMSF phenylmethanesulfonyl fluoride PRS proline-rich sequence PTC premature termination codon RISC RNA-induced silencing complex RNAi RNA interference RNAP II RNA polymerase II RNP ribonucleoprotein particle r.m.s.d root mean squared deviation rRNA ribosomal RNA SAD single wavelength anomalous dispersion scDcp1p S. cerevisiae Dcp1p VIII scDcp2p S.cerevisiae Dcp2p scDcp2ΔN S.cerevisiae Dcp2(102-970) scDcp2n S.cerevisiae Dcp2(1-300) SDS sodium dodecyl sulfate SeMet Seleno-L-methionine siRNA small interference RNA snoRNA small nucleolar RNA snRNA small nuclear RNA snRNP small nuclear ribonucleoprotein particle spDcp1p S. pombe Dcp1p spDcp2p S. pombe Dcp2p spDcp2n S. pombe Dcp2(1-266) TGF transforming growth factor TLC thin layer chromatography TMLA trimethyl lead acetate TNF-α tumor nercosis factor-α tRNA transfer RNA ts temperature sensitive TTP tristetraprolin UTR un-translated region WASP Wiskott-Aldrich syndrome protein IX 4. Discussion 4.1 Comparison of putative PRS binding site of scDcp1p with other EVH1 domains The structures of the yeast Dcp1 proteins revealed the core folding of the EVH1 domain with external helices. All previously characterized EVH1 domains utilize a highly conserved cluster of surface-exposed aromatic residues to recognize the PRS ligands with remarkably low affinity but high specificity (Ball et al., 2002). All EVH1 domains can be divided into three general classes based on their target PRS ligands, and the specificity is achieved by the variation of residues in the PRS binding site. Comparison of the putative PRS-binding site of Dcp1p with other EVH1 domains with known ligands helps to understand the characteristics of the site and to predict the sequence of a possible ligand. Mena, Homer and N-WASP are representatives of three different classes of the EVH1 domains and the comparison of residue composition and arrangement has revealed some similarities and marked differences as well (Figure 28). In scDcp1p, the potential PRS-binding site is lined with conserved aromatic residues including Trp49, Trp56, Leu190, Tyr47 and Trp204, of which only Trp56 is invariant in all four EVH1 proteins. Trp49 of Dcp1p is analogous to Tyr16 of the Mena protein; Tyr47 matches with Phe14 of Homer and Tyr46 of N-WASP; and Leu190 is the structural counterpart of phenylalanine of other EVH1 proteins. A unique feature of the Dcp1p pocket is Trp204, a highly conserved residue in all the Dcp1 proteins, but has no aromatic counterpart in other classes of EVH1 domains (Figure 11c). Thus the hydrophobic surface is much extended in Dcp1p, consisting of five conserved residues instead of three in other EVH1 domains. This observation suggests that 98 Dcp1p may represent a novel class of EVH1 domain and has specificity distinct from that of other EVH1 domains towards ligands. Dcp1 vs Mena (Class I) Dcp1 vs Homer (Class II) Dcp1 vs N-WASP (Class III) Figure 28. The potential ligand-binding site of scDcp1p. The comparison of PRSbinding site in Dcp1 protein (cyan) with that of other EVH1 domain-containing proteins representing three classes of proline-rich sequences is shown in Cα traces. The side chains of conserved residues are shown as sticks. 4.2 The Dcp1 and Dcp2 interaction in lower and higher eukaryotes In yeast, Dcp1p strongly associates with Dcp2p to form a holoenzyme with stimulated activity (Steiger et al., 2003). However, such direct interaction and activation effect were not observed with human or fly proteins in vitro (LykkeAndersen 2002; Cohen et al., 2005). This discrepancy is partly clarified based on the crystal structures of the S. pombe Dcp1p-Dcp2NT complex. As shown in Chapter 3.3.2, although the surface of Dcp2p involved in Dcp1p binding is highly conserved, the residues in Dcp1p that are involved in Dcp2p association have a higher degree of variation in higher eukaryotes than in yeast. Only three out of nine spDcp1p residues depicted in Figure 27 are identical in hDcp1a, versus seven invariant residues in scDcp1p. Accordingly, scDcp1p can form a heterogeneous complex with spDcp2n (residues 1-266) in vitro (data not shown). It is very likely that the protein-protein 99 interface seen in yeast is not preserved in other species or that the affinity is weak. Thus Dcp1 and Dcp2 proteins in higher eukaryotes might adopt a different mode of interaction. In line with this view, H. sapiens protein Hedls or A. thaliana VARICOSE, which lacks a homolog in yeast, has been shown to bridge the interactions between the Dcp1 and Dcp2 proteins in human and plant respectively (Fenger-GrØn et al., 2005; Xu et al., 2006; Simon et al., 2006). The divergence of the decapping proteins interaction in yeast and human, together with the observation that the human P-body contains additional components, reflects the evolution and developing complexity of the RNA degradation machinery from lower to higher eukaryotes. 4.3 Implication on the assembly and regulation of 5’ mRNA decay machinery The Dcp1p-Dcp2p complex is part of the 5’ mRNA decay machinery residing in the cytoplasmic P-bodies, including Dhh1p, Edc1p, Edc2p, Edc3p, Pat1p and Lsm17p, all of which are activators of decapping (Coller & Parker, 2004, Figure 29). In this study, several conserved surfaces on the Dcp1 and Dcp2 proteins are revealed, including patch and patch of Dcp1p and the conserved regions on the N-terminal domain and the Nudix domain of Dcp2p. Presumably, all or some of these surfaces are involved in binding other factors in the P-bodies. The 5’ decay factors participate at different steps of mRNA degeneration including translation repression, deadenylation and P-body formation (Coller & Parker, 2004). The interactions between these factors and the Dcp1p-Dcp2p complex suggest that mRNA degeneration is a coordinated and regulated process. And the activation effects on the Dcp1p-Dcp2p complex could be carried out through several mechanisms: by promoting the active conformation of the Dcp1p-Dcp2p holoenzyme 100 through protein-protein interaction; by reducing the inhibitory structures of mRNA; or by facilitating the access of the 5’ termini of mRNAs to the catalytic center. And the structural and functional analysis on the Dcp1 and Dcp2 proteins in this study provide a molecular basis for further investigation in the field of mRNA decay study. m7Gppp AAAAA m7Gppp Translation termination factors AAAAA deadenylases Dhh1p, Pat1p ribosome Dhh1p, Pat1p Dcp1p Dcp1p Dcp2p Lsm1-7p OH Dcp1p Dcp2p Pat1p Dhh1p Dcp2p OH Pat1p p pp Dhh1p 7G m Edc3p Dcp2p Lsm1-7p Pat1p Dhh1p Edc3p Edc3p m7Gppp Lsm1-7p OH Xrn1p m7Gpp mRNA processing body Figure 29. The model for mRNA decapping mechanism. In the cytoplasm, the closed-loop mRNA serves as the template for protein synthesis. When translation is terminated, the poly(A) tail of mRNA is degraded by the deadenylase complex. The translation repressors, Pat1 and Dhh1 proteins, link the translation termination with the decapping step and mediate mRNA into the cytoplasmic processing body (grey area). The P-body factors, including Dcp1p, Edc3p, Lsm1-7p and Dhh1pPat1p, regulate the decapping activity of Dcp2p through its conformational change to anchor the mRNA to the Box B region (blue crescent) and orient its 5’ cap to the active site (red star) for catalysis. When the cap is hydrolyzed, the m7GDP product is released and the mRNA body is quickly degraded by the 5’ exonuclase 101 References 1. Abdelghany, H.M., Bailey, S., Blackburn, G.M., Rafferty, J.B., McLennan, A.G. (2003). Analysis of the catalytic and binding residues of the diadenosine tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans by sitedirected mutagenesis. J Biol Chem. 278(7):4435-9. 2. Aguilera, A. (2005). Cotranscriptional mRNP assembly: from the DNA to the nuclear pore. Curr Opin Cell Biol 17, 242-250. 3. Albrecht, M. and Lengauer, T. (2004). Novel Sm-like proteins with long Cterminal tails and associated methyltransferases. FEBS Lett 569, 18-26. 4. Allmang, C., Petfalski, E., Podtelejnikov, A., Mann, M., Tollervey, D., and Mitchell, P. (1999). The yeast exosome and human PM-Scl are related complexes of 3' --> 5' exonucleases. Genes Dev 13, 2148-2158. 5. Amrani, N., Ganesan, R., Kervestin, S., Mangus, D.A., Ghosh, S., and Jacobson, A. (2004). A faux 3'-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay. Nature 432, 112-118. 6. Anantharaman, V. and Aravind, L. (2004). Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability. BMC Genomics 5, 45. 7. Anderson P.A. (2005). Place for RNAi. Dev Cell. (3):311-312. 8. Anderson, J.S. and Parker, R.P. (1998). The 3' to 5' degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3' to 5' exonucleases of the exosome complex. EMBO J 17, 1497-1506. 9. Araki, Y., Takahashi, S., Kobayashi, T., Kajiho, H., Hoshino, S., and Katada, T. (2001). Ski7p G protein interacts with the exosome and the Ski complex for 3'-to-5' mRNA decay in yeast. EMBO J 20, 4684-4693. 10. Badis, G., Saveanu, C., Fromont-Racine, M., and Jacquier, A. (2004). Targeted mRNA degradation by deadenylation-independent decapping. Mol Cell 15, 5-15. 11. Bai, R.Y., Koester, C., Ouyang, T., Hahn, S.A., Hammerschmidt, M., Peschel, C., and Duyster, J. (2002). SMIF, a Smad4-interacting protein that functions as a co-activator in TGFbeta signalling. Nat Cell Biol 4, 181-190. 12. Bailey, S., Sedelnikova, S.E., Blackburn, G.M., Abdelghany, H.M., Baker, P.J., McLennan, A.G., and Rafferty, J.B. (2002). The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms. Structure 10, 589-600. 102 13. Ball, L.J., Jarchau, T., Oschkinat, H., and Walter, U. (2002). EVH1 domains: structure, function and interactions. FEBS Lett 513, 45-52. 14. Ball, L.J., Kuhne, R., Hoffmann, B., Hafner, A., Schmieder, P., VolkmerEngert, R., Hof, M., Wahl, M., Schneider-Mergener, J., Walter, U., Oschkinat, H., and Jarchau, T. (2000). Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity. EMBO J 19, 4903-4914. 15. Beelman, C.A., Stevens, A., Caponigro, G., LaGrandeur, T.E., Hatfield, L., Fortner, D.M., and Parker, R. (1996). An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382, 642-646. 16. Beneken, J., Tu, J.C., Xiao, B., Nuriya, M., Yuan, J.P., Worley, P.F., and Leahy, D.J. (2000). Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition. Neuron 26, 143-154. 17. Bentley D.L. (2005). Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr Opin Cell Biol. 17(3):251-256. 18. Bessman, M.J., Frick, D.N., and O'handley, S.F. (1996). The MutT proteins or "Nudix" hydrolases, a family of versatile, widely distributed, "housecleaning" enzymes. J Biol Chem 271, 25059-25062. 19. Bonnerot, C., Boeck, R., and Lapeyre, B. (2000). The two proteins Pat1p (Mrt1p) and Spb8p interact in vivo, are required for mRNA decay, and are functionally linked to Pab1p. Mol Cell Biol 20, 5939-5946. 20. Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M., and Seraphin, B. (2000). A Sm-like protein complex that participates in mRNA degradation. EMBO J 19, 1661-1671. 21. Brengues, M., Teixeira, D., and Parker, R. (2005). Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310, 486-489. 22. Brennan, C.M. and Steitz, J.A. (2001). HuR and mRNA stability. Cell Mol Life Sci 58, 266-277. 23. Brünger, A.T. et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. 24. Cagney, G., Uetz, P., and Fields, S. (2000). High-throughput screening for protein-protein interactions using two-hybrid assay. Methods Enzymol 328, 314. 25. Callebaut, I. (2002). An EVH1/WH1 domain as a key actor in TGFbeta signalling. FEBS Lett 519, 178-180. 103 26. Caponigro, G. and Parker, R. (1995). Multiple functions for the poly(A)binding protein in mRNA decapping and deadenylation in yeast. Genes Dev 9, 2421-2432. 27. Caponigro, G., Muhlrad, D., and Parker, R. (1993). A small segment of the MAT alpha transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons. Mol Cell Biol 13, 5141-5148. 28. Chen, C.Y. and Shyu, A.B. (1995). AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci 20, 465-470. 29. Chen, N., Walsh, M.A., Liu, Y., Parker, R., and Song, H. (2005). Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping. J Mol Biol 347, 707718. 30. Cheng, H., Dufu, K., Lee, C.S., Hsu, J.L., Dias, A., Reed R. (2006). Human mRNA export machinery recruited to the 5' end of mRNA.Cell 127(7):13891400. 31. Cheng, Z., Coller, J., Parker, R., and Song, H. (2005). Crystal structure and functional analysis of DEAD-box protein Dhh1p. RNA 11, 1258-1270. 32. Chernokalskaya, E., Dubell, A.N., Cunningham, K.S., Hanson, M.N., Dompenciel, R.E., and Schoenberg, D.R. (1998). A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family. RNA 4, 1537-1548. 33. Clement, S.L. and Lykke-Andersen, J. (2006). No mercy for messages that mess with the ribosome. Nat Struct Mol Biol 13, 299-301. 34. Cohen, L.S., Mikhli, C., Jiao, X., Kiledjian, M., Kunkel, G., and Davis, R.E. (2005). Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent. Mol Cell Biol 25, 8779-8791. 35. Collaborative Computational Project, Number 4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 36. Coller, J. and Parker, R. (2004). Eukaryotic mRNA decapping. Annu Rev Biochem 73, 861-890. 37. Coller, J. and Parker, R. (2005). General translational repression by activators of mRNA decapping. Cell 122, 875-886. 38. Coller, J.M., Tucker, M., Sheth, U., Valencia-Sanchez, M.A., and Parker, R. (2001). The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. RNA 7, 17171727. 104 39. Conti, E. and Izaurralde, E. (2005). Nonsense-mediated mRNA decay: molecular insights and mechanistic variations across species. Curr Opin Cell Biol 17, 316-325. 40. Cougot, N., Babajko, S., and Seraphin, B. (2004a). Cytoplasmic foci are sites of mRNA decay in human cells. J Cell Biol 165, 31-40. 41. Cougot, N., van Dijk, E., Babajko, S., and Seraphin, B. (2004b). 'Captabolism'. Trends Biochem Sci 29, 436-444. 42. Daugeron, M.C., Mauxion, F., and Seraphin, B. (2001). The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation. Nucleic Acids Res 29, 2448-2455. 43. de la Fortelle, E. & Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction method. Methods Enzymol. 276, 472– 494. 44. Decker, C.J. and Parker, R. (1993). A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev 7, 1632-1643. 45. Ding, L., Spencer, A., Morita, K., and Han, M. (2005). The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol Cell 19, 437-447. 46. Dodson, R.E. and Shapiro, D.J. (2002). Regulation of pathways of mRNA destabilization and stabilization. Prog Nucleic Acid Res Mol Biol 72, 129-164. 47. Doma, M.K. and Parker, R. (2006). Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation. Nature 440, 561-564. 48. Dunckley, T. and Parker, R. (1999). The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J 18, 5411-5422. 49. Dunckley, T., Tucker, M., and Parker, R. (2001). Two related proteins, Edc1p and Edc2p, stimulate mRNA decapping in Saccharomyces cerevisiae. Genetics 157, 27-37. 50. Emsley, P. and Cowtan, K (2004). Coot: Model-Building Tools for Molecular Graphics. Acta D 60, 2126-2132 51. Eystathioy, T., Jakymiw, A., Chan, E.K., Seraphin, B., Cougot, N., and Fritzler, M.J. (2003). The GW182 protein co-localizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies. RNA 9, 1171-1173. 52. Fedorov, A.A., Fedorov, E., Gertler, F., and Almo, S.C. (1999). Structure of EVH1, a novel proline-rich ligand-binding module involved in cytoskeletal dynamics and neural function. Nat Struct Biol 6, 661-665. 105 53. Fenger-GrØn, M., Fillman, C., Norrild, B., and Lykke-Andersen, J. (2005). Multiple Processing Body Factors and the ARE Binding Protein TTP Activate mRNA Decapping. Mol Cell 20, 905-915. 54. Fischer, N. and Weis, K. (2002). The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1. EMBO J 21, 2788-2797. 55. Frischmeyer, P.A. and Dietz, H.C. (1999). Nonsense-mediated mRNA decay in health and disease. Hum Mol Genet 8, 1893-1900. 56. Frischmeyer, P.A., van Hoof, A., O'Donnell, K., Guerrerio, A.L., Parker, R., and Dietz, H.C. (2002). An mRNA surveillance mechanism that eliminates transcripts lacking termination codons. Science 295, 2258-2261. 57. Fukuhara, N., Ebert, J., Unterholzner, L., Lindner, D., Izaurralde, E., and Conti, E. (2005). SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway. Mol Cell 17, 537-547. 58. Gabelli, S.B., Bianchet, M.A., Bessman, M.J., and Amzel, L.M. (2001). The structure of ADP-ribose pyrophosphatase reveals the structural basis for the versatility of the Nudix family. Nat Struct Biol 8, 467-472. 59. Gallouzi, I.E., Parker, F., Chebli, K., Maurier, F., Labourier, E., Barlat, I., Capony, J.P., Tocque, B., and Tazi, J. (1998). A novel phosphorylationdependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability. Mol Cell Biol 18, 3956-3965. 60. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A. (2005). Protein Identification and Analysis Tools on the ExPASy Server; (In) pp. 571-607 61. Gatfield, D. and Izaurralde, E. (2004). Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, 575578. 62. Ghosh, T., Peterson, B., Tomasevic, N., and Peculis, B.A. (2004). Xenopus U8 snoRNA binding protein is a conserved nuclear decapping enzyme. Mol Cell 13, 817-828. 63. Gu, M., Fabrega, C., Liu, S.W., Liu, H., Kiledjian, M., and Lima, C.D. (2004). Insights into the structure, mechanism, and regulation of scavenger mRNA decapping activity. Mol Cell 14, 67-80. 64. He, W. and Parker, R. (2001). The yeast cytoplasmic LsmI/Pat1p complex protects mRNA 3' termini from partial degradation. Genetics 158, 1445-1455. 65. Hollams, E.M., Giles, K.M., Thomson, A.M., and Leedman, P.J. (2002). MRNA stability and the control of gene expression: implications for human disease. Neurochem Res 27, 957-980. 66. Holm L., Sander C. (1993). Protein structure comparison by alignment of distance matrices. J Mol Biol. 233(1):123-138. 106 67. Hsu, C.L. and Stevens, A. (1993). Yeast cells lacking 5'-->3' exoribonuclease contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure. Mol Cell Biol 13, 4826-4835. 68. Ingelfinger, D., Arndt-Jovin, D.J., Luhrmann, R., and Achsel, T. (2002). The human LSm1-7 proteins co-localize with the mRNA-degrading enzymes Dcp1/2 and Xrnl in distinct cytoplasmic foci. RNA 8, 1489-1501. 69. Jakymiw, A., Lian, S., Eystathioy, T., Li, S., Satoh, M., Hamel, J.C., Fritzler, M.J., and Chan, E.K. (2005). Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol 7, 1267-1274. 70. Jiao X., Wang Z., and Kiledjian M. (2006). Identification of an mRNAdecapping regulator implicated in X-linked mental retardation. Mol Cell, 24(5):713-22. 71. Jing,Q., Huang,S., Guth,S., Zarubin,T., Motoyama,A., Chen,J., Di Padova,F., Lin,S.C., Gram,H., and Han,J. (2005). Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120, 623-634. 72. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119. 73. Kapp, L.D. and Lorsch, J.R. (2004). The molecular mechanics of eukaryotic translation. Annu Rev Biochem 73, 657-704. 74. Khanna R. and Kiledjian M. (2004). Poly(A)-binding-protein-mediated regulation of hDcp2 decapping in vitro. EMBO J. 23(9):1968-76. 75. Kofuji, S., Sakuno, T., Takahashi, S., Araki, Y., Doi, Y., Hoshino, S., and Katada, T. (2006). The decapping enzyme Dcp1 participates in translation termination through its interaction with the release factor eRF3 in budding yeast. Biochem Biophys Res Commun 344, 547-553. 76. Korner, C.G., Wormington, M., Muckenthaler, M., Schneider, S., Dehlin, E., and Wahle, E. (1998). The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes. EMBO J 17, 5427-5437. 77. Kshirsagar, M. and Parker, R. (2004). Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae. Genetics 166, 729-739. 78. Kufel, J., Allmang, C., Petfalski, E., Beggs, J., and Tollervey, D. (2003). Lsm Proteins are required for normal processing and stability of ribosomal RNAs. J Biol Chem 278, 2147-2156. 79. Kufel, J., Allmang, C., Verdone, L., Beggs, J.D., and Tollervey, D. (2002). Lsm proteins are required for normal processing of pre-tRNAs and their efficient association with La-homologous protein Lhp1p. Mol Cell Biol 22, 5248-5256. 107 80. Ladomery, M., Wade, E., and Sommerville, J. (1997). Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes. Nucleic Acids Res 25, 965-973. 81. LaGrandeur, T.E. and Parker, R. (1998). Isolation and characterization of Dcp1p, the yeast mRNA decapping enzyme. EMBO J 17, 1487-1496. 82. Lall, S., Piano, F., and Davis, R.E. (2005). Caenorhabditis elegans decapping proteins: localization and functional analysis of Dcp1, Dcp2, and DcpS during embryogenesis. Mol Biol Cell 16, 5880-5890. 83. Lejeune, F. and Maquat, L.E. (2005). Mechanistic links between nonsensemediated mRNA decay and pre-mRNA splicing in mammalian cells. Curr Opin Cell Biol 17, 309-315. 84. Lejeune, F., Li, X., and Maquat, L.E. (2003). Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol Cell 12, 675-687. 85. Lin, M.D., Fan, S.J., Hsu, W.S., and Chou, T.B. (2006). Drosophila decapping protein 1, dDcp1, is a component of the oskar mRNP complex and directs its posterior localization in the oocyte. Dev Cell 10, 601-613. 86. Liu, H., Rodgers, N.D., Jiao, X., and Kiledjian, M. (2002). The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases. EMBO J 21, 4699-4708. 87. Liu, J., Rivas, F.V., Wohlschlegel, J., Yates, J.R., III, Parker, R., and Hannon, G.J. (2005). A role for the P-body component GW182 in microRNA function. Nat Cell Biol 7, 1261-1266. 88. Liu, J., Valencia-Sanchez, M.A., Hannon, G.J., and Parker, R. (2005). MicroRNA-dependent localization of targeted mRNAs to mammalian Pbodies. Nat Cell Biol 7, 719-723. 89. Liu, S.W., Jiao, X., Liu, H., Gu, M., Lima, C.D., and Kiledjian, M. (2004). Functional analysis of mRNA scavenger decapping enzymes. RNA 10, 14121422. 90. Lykke-Andersen, J. (2002). Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay. Mol Cell Biol 22, 8114-8121. 91. Lykke-Andersen, J. and Wagner, E. (2005). Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev 19, 351-361. 92. Mildvan, A.S., Xia, Z., Azurmendi, H.F., Saraswat, V., Legler, P.M., Massiah, M.A., Gabelli, S.B., Bianchet, M.A., Kang, L.W., and Amzel, L.M. (2005). Structures and mechanisms of Nudix hydrolases. Arch Biochem Biophys 433, 129-143. 108 93. Mitchell, P. and Tollervey, D. (2000). mRNA stability in eukaryotes. Curr Opin Genet Dev 10, 193-198. 94. Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M., and Tollervey, D. (1997). The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases. Cell 91, 457-466. 95. Muhlrad, D. and Parker, R. (1999). Aberrant mRNAs with extended 3' UTRs are substrates for rapid degradation by mRNA surveillance. RNA 5, 12991307. 96. Muhlrad, D., Decker, C.J., and Parker, R. (1994). Deadenylation of the unstable mRNA encoded by the yeast MFA2 gene leads to decapping followed by 5'-->3' digestion of the transcript. Genes Dev 8, 855-866. 97. Mukherjee, D., Gao, M., O'Connor, J.P., Raijmakers, R., Pruijn, G., Lutz, C.S., and Wilusz, J. (2002). The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements. EMBO J 21, 165-174. 98. Naitow, H., Tang, J., Canady, M., Wickner, R.B., and Johnson, J.E. (2002). LA virus at 3.4 Å resolution reveals particle architecture and mRNA decapping mechanism. Nat Struct Biol 9, 725-728. 99. Nakamura, A., Amikura, R., Hanyu, K., and Kobayashi, S. (2001). Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development 128, 3233-3242. 100. Navarro, R.E., Shim, E.Y., Kohara, Y., Singson, A., and Blackwell, T.K. (2001). cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans. Development 128, 3221-3232. 101. Neidhardt, F.C., Bloch, P.L., and Smith, D.F. (1974). Culture medium for enterobacteria. J Bacteriol 119, 736-747. 102. Piccirillo, C., Khanna, R., and Kiledjian, M. (2003). Functional characterization of the mammalian mRNA decapping enzyme hDcp2. RNA 9, 1138-1147. 103. Pillai, R.S., Bhattacharyya, S.N., Artus, C.G., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E., and Filipowicz, W. (2005). Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573-1576. 104. Prehoda, K.E., Lee, D.J. and Lim, W.A. (1999). Structure of the enabled/VASP homology domain-peptide complex: a key component in the spatial control of actin assembly. Cell 97, 471-480. 105. Proudfoot, N.J., Furger, A., and Dye, M.J. (2002). Integrating mRNA processing with transcription. Cell 108, 501-512. 109 106. Ruiz-Echevarria, M.J., Gonzalez, C.I., and Peltz, S.W. (1998). Identifying the right stop: determining how the surveillance complex recognizes and degrades an aberrant mRNA. EMBO J 17, 575-589. 107. Sachs, A.B. (1993). Messenger RNA degradation in eukaryotes. Cell 74, 413421. 108. Sakuno, T., Araki, Y., Ohya, Y., Kofuji, S., Takahashi, S., Hoshino, S., and Katada, T. (2004). Decapping reaction of mRNA requires Dcp1 in fission yeast: its characterization in different species from yeast to human. J Biochem (Tokyo) 136, 805-812. 109. Scarsdale, J.N., Peculis, B.A., and Wright, H.T. (2006). Crystal structures of U8 snoRNA decapping nudix hydrolase, X29, and its metal and cap complexes. Structure 14, 331-343. 110. Schwartz, D., Decker, C.J., and Parker, R. (2003). The enhancer of decapping proteins, Edc1p and Edc2p, bind RNA and stimulate the activity of the decapping enzyme. RNA 9, 239-251. 111. Sen, G.L. and Blau, H.M. (2005). Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol 7, 633636. 112. Sheldrick, G.M. (1998). In direct methods for solving macromolecular structures. (ed. Fortier, S.) Kluwer Academic, Dordrecht, The Netherlands. 401-411. 113. Sheth, U. and Parker, R. (2003). Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805-808. 114. Simon, E., Camier,S., and Seraphin,B. (2006). New insights into the control of mRNA decapping. Trends Biochem Sci. 31: 241-243. 115. Solinger, J.A., Pascolini, D., and Heyer, W.D. (1999). Active-site mutations in the Xrn1p exoribonuclease of Saccharomyces cerevisiae reveal a specific role in meiosis. Mol Cell Biol 19, 5930-5942. 116. Steiger, M., Carr-Schmid, A., Schwartz, D.C., Kiledjian, M., and Parker, R. (2003). Analysis of recombinant yeast decapping enzyme. RNA 9, 231-238. 117. Tang, J., Naitow, H., Gardner, N.A., Kolesar, A., Tang, L., Wickner, R.B., and Johnson, J .E. (2005). The structural basis of recognition and removal of cellular mRNA 7-methyl G 'caps' by a viral capsid protein: a unique viral response to host defense. J Mol Recognit. 18, 158-168. 118. Teixeira, D., Sheth, U., Valencia-Sanchez, M.A., Brengues, M., and Parker, R. (2005). Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11, 371-382. 119. Terwilliger, T.C. (2002). Automated structure solution, density modification and model building. Acta Crystallogr. D Biol. Crystallogr. 58, 1937–1940. 110 120. Tharun, S. and Parker, R. (1999). Analysis of mutations in the yeast mRNA decapping enzyme. Genetics 151, 1273-1285. 121. Tharun, S., He, W., Mayes, A.E., Lennertz, P., Beggs, J.D., and Parker, R. (2000). Yeast Sm-like proteins function in mRNA decapping and decay. Nature 404, 515-518. 122. Tharun, S., Muhlrad, D., Chowdhury, A., and Parker, R. (2005). Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection. Genetics 170, 33-46. 123. Unterholzner, L., and Izaurralde, E. (2004). SMG7 acts as a molecular link between mRNA surveillance and mRNA decay. Mol. Cell 16(4):587-96. 124. van Dijk, E., Cougot, N., Meyer, S., Babajko, S., Wahle, E., and Seraphin, B. (2002). Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. EMBO J 21, 6915-6924. 125. van Dijk, E., Le Hir, H., and Seraphin, B. (2003). DcpS can act in the 5'-3' mRNA decay pathway in addition to the 3'-5' pathway. Proc Natl Acad Sci U S A 100, 12081-12086. 126. van Hoof, A. and Parker, R. (2002). Messenger RNA degradation: beginning at the end. Curr Biol 12, R285-R287. 127. Vasudevan, S., Peltz, S.W., and Wilusz, C.J. (2002). Non-stop decay--a new mRNA surveillance pathway. Bioessays 24, 785-788. 128. Vetter, I.R., Nowak, C., Nishimoto, T., Kuhlmann and J., Wittinghofer, A (1999). Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport. Nature 398, 39-46. 129. Vilela, C., Velasco, C., Ptushkina, M., and McCarthy, J.E. (2000). The eukaryotic mRNA decapping protein Dcp1 interacts physically and functionally with the eIF4F translation initiation complex. EMBO J 19, 43724382. 130. Volkman, B.F., Prehoda, K.E., Scott, J.A., Peterson, F.C., and Lim, W.A. (2002). Structure of the N-WASP EVH1 domain-WIP complex: insight into the molecular basis of Wiskott - Aldrich syndrome. Cell 111, 565-576. 131. Wang Y., Liu C.L., Storey J.D., Tibshirani R.J., Herschlag D. and Brown P.O. (2002a) Precision and functional specificity in mRNA decay.Proc Natl Acad Sci U S A. 99(9):5860-5865. 132. Wang, Z. and Kiledjian, M. (2001). Functional link between the mammalian exosome and mRNA decapping. Cell 107, 751-762. 133. Wang, Z., Jiao, X., Carr-Schmid, A., and Kiledjian, M. (2002b). The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc Natl Acad Sci U S A 99, 12663-12668. 111 134. Weeks, C.M. and Miller, R. (1999). The design and implementation of SnB v2.0, J. Appl. Cryst.32, 120-124. 135. Xu J., Yang J.Y., Niu Q.W. and Chua NH. (2006). Arabidopsis DCP2, DCP1, and VARICOSE Form a Decapping Complex Required for Postembryonic Development. Plant Cell. 18(12):3386-3398. 136. Yang, Z., Jakymiw, A., Wood, M.R., Eystathioy, T., Rubin, R.L., Fritzler, M.J., and Chan, E.K. (2004). GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation. J Cell Sci 117, 55675578. 137. Yu, J.H., Yang, W.H., Gulick, T., Bloch, K.D., and Bloch, D.B. (2005). Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body. RNA 11, 1795-1802. 138. Zarrinpar, A., Bhattacharyya, R.P., and Lim, W.A. (2003). The structure and function of proline recognition domains. Sci STKE, RE8. 587–596. 112 List of publications She M., Decker C.J., Chen N., Tumati S., Parker R., and Song H. (2006). Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe.Nat Struct Mol Biol. 13(1):63-70. She M., Decker C.J., Sundramurthy K., Liu Y., Chen N., Parker R., and Song H. (2004). Crystal structure of Dcp1p and its functional implications in mRNA decapping. Nat Struct Mol Biol. 11(3):249-56. 113 [...]... 23 spDcp1p stimulates the decapping activity of spDcp2n 88 X Figure 24 In vivo decapping assay of the wild-type and mutants spDcp2n 92 Figure 25 Analysis of scDcp1p-scDcp2n interaction and decapping activity 93 Figure 26 Crystal structure of the S pombe Dcp1p-Dcp2NT complex 95 Figure 27 The protein-protein interface in the Dcp1p-Dcp2NT complex 97 Figure 28 The Potential ligand-binding site of scDcp1p... Edc3p and other auxiliary RNA-binding proteins Figure is modified from Coller and Parker, 2004 Dcp1p/Dcp2p) Decapping enzyme complex are composed of two subunits: the regulatory unit Dcp1p and the catalytic unit Dcp2p The features of these two proteins will be addressed in Chapter 1.3 Dhh1p is a DEAD-box RNA helicase of 58 kDa that actively represses translation and stimulates the assembly of the decapping. .. After rounds of translation, mRNA is finally destabilized and degraded mRNA decay is generally initiated by the removal of the poly(A) tail, thereafter the mRNA body is subjected to exonucleolytic digestion from either the 5’ end or 3’ end The mechanism and regulation of mRNA decay will be addressed in the following chapter 1.1.2 Biological significance of mRNA decay As the final step of mRNA metabolism,... Activity of mutants in the hydrophobic surface patch of scDcp1p 74 Figure 17 Crystal structure of spDcp2n 76 Figure 18 Comparison of spDcp2n with other Nudix enzymes 77 Figure 19 The Nudix motif of spDcp2n is the catalytic center 80 Figure 20 Functional analysis of two individual domains of spDcp2n 82 Figure 21 Sequence alignment and surface view of spDcp2n 83 Figure 22 The spDcp1p binding region in the. .. degraded from the 3’ end The 3’ pathway requires the exosome, which is a complex containing multiple 3' to 5' exoribonucleases and RNA binding proteins (Allmang et al., 1999; Mitchell et al., 1997) Together with the Ski complex (including Ski2p, Ski3p, Ski8p and the adapter Ski7p), exosome degrades mRNA to release the cap and the remaining mRNA of only a few nucleotides in length (Anderson and Parker,... Purification of recombinant spDcp2n 55 Figure 10 Purification of the S pombe Dcp1p-Dcp2NT complex 56 Figure 11 Crystal structure of scDcp1p 64 Figure 12 Comparison of scDcp1p with the homologous EVH1/PH domains 66 Figure 13 Surface representation of scDcp1p 68 Figure 14 Activity of mutants in the conserved patch 1 of scDcp1p 70 Figure 15 Activity of mutants in the conserved patch 2 of scDcp1p 72 Figure... mediating the posterior localization of osk mRNA to the oocyte (Lin et al., 2006) 231 S.Cerevisiae Dcp1p S Pombe Dcp1p D.Melanogaster Dcp1 H.sapiens Dcp1a H.sapiens Dcp1b 127 372 582 617 Figure5 Schematic diagram of Dcp1 proteins Domain organization of S.cerevisiae Dcp1p, S pombe Dcp1p, D melanogaster Dcp1, H sapiens Dcp1a and Dcp1b respectively The conserved N-terminal regions among all Dcp1 proteins. .. rendering the mRNA more stability than the wild-type one In α-thalassemia, certain variant causes a translation read-through and the displacement of the stabilizing factor bound to the 3’ARE This is associated with a decrease in the mRNA level and hence the development of the disease 1.2 mRNA 5’ decay machinery and processing bodies The machinery of the 5’ decay pathway is composed of multiple proteins. .. converging input stimuli to control the quantity and quality of mRNA Firstly, the half-lives of eukaryotic mRNAs may vary up to three orders of magnitude and they are correlated with the physical functions of encoded proteins (Sachs, 1993; Wang et al., 2002a) For example, the transcripts of structural proteins tend to have longer halflives, while the transcripts of proteins in signaling pathways usually... (Sheth and Parker, 2003) In contrast, there is no specific locus for the 3’ decay pathway, and the exosome and Ski proteins are evenly distributed in the cytosol In higher eukaryotes, extra components that have no yeast counterparts are identified in P-bodies One of them is Ge-1, or Hedls Ge-1 stimulates hDcp2 decapping activity and is suggested to mediate the hDcp1a and hDcp2 protein complex formation . X-RAY CRYSTALLOGRAPHIC STUDY OF YEAST DCP1 AND DCP2 PROTEINS: INSIGHTS INTO THE MECHANISM AND REGULATION OF EUKARYOTIC mRNA DECAPPING SHE MEIPEI (B.Sc.) A THESIS. by the Dcp1- Dcp2 holoenzyme. Dcp2 is a Nudix pyrophosphatase and Dcp1 stimulates the activity of Dcp2. The crystal structures of yeast Dcp1 and Dcp2 proteins are presented in this study. The. from either the 5’ end or 3’ end. The mechanism and regulation of mRNA decay will be addressed in the following chapter. 1.1.2 Biological significance of mRNA decay As the final step of mRNA