Reconstitution, Activities, and Structure of the Eukaryotic RNA Exosome

Summary The RNA exosome is a multisubunit 3′ to 5′ exoribonuclease complex that participates in degradation and processing of cellular RNA. To determine the activities and structure of the eukaryotic exosome, we report the reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. Comparative biochemical analysis between purified subunits and reconstituted exosomes using AU-rich, polyadenylated (poly[A]), generic, and structured RNA substrates reveals processive phosphorolytic activities for human Rrp41/Rrp45 and the 9-subunit human exosome, processive hydrolytic activities for yeast Rrp44 and the yeast 10-subunit exosome, distributive hydrolytic activities for Rrp6, and processive and distributive hydrolytic activities for the yeast 11-subunit exosome. To elucidate the architecture of a eukaryotic exosome, its conserved surfaces, and the structural basis for RNA decay, we report the X-ray structure determination for the 286 kDa nine-subunit human exosome at 3.35 A.

[1]  Friedrich Lottspeich,et al.  An exosome‐like complex in Sulfolobus solfataricus , 2003, EMBO reports.

[2]  Itay Mayrose,et al.  ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures , 2005, Nucleic Acids Res..

[3]  Peer Bork,et al.  A complex prediction: three‐dimensional model of the yeast exosome , 2002, EMBO reports.

[4]  J. Wilusz,et al.  Bringing the role of mRNA decay in the control of gene expression into focus. , 2004, Trends in genetics : TIG.

[5]  W A Hendrickson,et al.  Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three‐dimensional structure. , 1990, The EMBO journal.

[6]  M. Moore.  From Birth to Death: The Complex Lives of Eukaryotic mRNAs , 2005, Science.

[7]  B. Séraphin,et al.  Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase , 2005, Cell.

[8]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[9]  Bertrand Séraphin,et al.  Subunit architecture of multimeric complexes isolated directly from cells , 2006, EMBO reports.

[10]  R. Parker,et al.  Yeast Exosome Mutants Accumulate 3′-Extended Polyadenylated Forms of U4 Small Nuclear RNA and Small Nucleolar RNAs , 2000, Molecular and Cellular Biology.

[11]  M. Mann,et al.  The Exosome: A Conserved Eukaryotic RNA Processing Complex Containing Multiple 3′→5′ Exoribonucleases , 1997, Cell.

[12]  C. Lima,et al.  Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. , 2000, Molecular cell.

[13]  B. Luisi,et al.  A duplicated fold is the structural basis for polynucleotide phosphorylase catalytic activity, processivity, and regulation. , 2000, Structure.

[14]  Don C. Wiley,et al.  Determining the structure of an unliganded and fully glycosylated SIV gp120 envelope glycoprotein. , 2005 .

[15]  P. Mitchell,et al.  Functions of the exosome in rRNA, snoRNA and snRNA synthesis , 1999, The EMBO journal.

[16]  B. Séraphin,et al.  A single subunit, Dis3, is essentially responsible for yeast exosome core activity , 2007, Nature Structural &Molecular Biology.

[17]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[18]  M. Kiledjian,et al.  Functional Link between the Mammalian Exosome and mRNA Decapping , 2001, Cell.

[19]  G. Pruijn,et al.  The exosome, a molecular machine for controlled RNA degradation in both nucleus and cytoplasm. , 2004, European journal of cell biology.

[20]  K. Hopfner,et al.  Structural framework for the mechanism of archaeal exosomes in RNA processing. , 2005, Molecular cell.

[21]  Roy Parker,et al.  Degradation of mRNA in eukaryotes , 1995, Cell.

[22]  Axel T Brunger,et al.  Considerations for the refinement of low-resolution crystal structures. , 2006, Acta crystallographica. Section D, Biological crystallography.

[23]  M. Deutscher,et al.  An important role for RNase R in mRNA decay. , 2005, Molecular cell.

[24]  J. Butler,et al.  A Nuclear 3′-5′ Exonuclease Involved in mRNA Degradation Interacts with Poly(A) Polymerase and the hnRNA Protein Npl3p , 2000, Molecular and Cellular Biology.

[25]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[26]  Thomas C Terwilliger,et al.  SOLVE and RESOLVE: automated structure solution and density modification. , 2003, Methods in enzymology.

[27]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[28]  S. Altman,et al.  A protein subunit of human RNase P, Rpp14, and its interacting partner, OIP2, have 3′→5′ exoribonuclease activity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  E. Conti,et al.  The archaeal exosome core is a hexameric ring structure with three catalytic subunits , 2005, Nature Structural &Molecular Biology.

[30]  C. Lima,et al.  Structure-based analysis of catalysis and substrate definition in the HIT protein family. , 1997, Science.

[31]  Roy Parker,et al.  Eukaryotic mRNA decapping. , 2004, Annual review of biochemistry.

[32]  M. Mann,et al.  AU Binding Proteins Recruit the Exosome to Degrade ARE-Containing mRNAs , 2001, Cell.

[33]  E. Andrulis,et al.  Differential distribution of exosome subunits at the nuclear lamina and in cytoplasmic foci. , 2005, Molecular biology of the cell.

[34]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[35]  E. Petfalski,et al.  RNA Degradation by the Exosome Is Promoted by a Nuclear Polyadenylation Complex , 2005, Cell.

[36]  R. Parker,et al.  Mechanisms of mRNA surveillance in eukaryotes. , 1999, Annual review of genetics.

[37]  W. Keller,et al.  A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control , 2005, PLoS biology.

[38]  Ben Lehner,et al.  The Roles of Intersubunit Interactions in Exosome Stability* , 2003, Journal of Biological Chemistry.

[39]  R. Parker,et al.  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 , 1998, The EMBO journal.

[40]  M. Deutscher,et al.  Purification and Characterization of the Escherichia coli Exoribonuclease RNase R , 2002, The Journal of Biological Chemistry.

[41]  G. Pruijn,et al.  Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring. , 2002, Journal of molecular biology.

[42]  Elena Conti,et al.  The Exosome and the Proteasome: Nano-Compartments for Degradation , 2006, Cell.

[43]  E. Conti,et al.  Structural basis of 3' end RNA recognition and exoribonucleolytic cleavage by an exosome RNase PH core. , 2005, Molecular cell.

[44]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[45]  J. Wilusz,et al.  Sequence-specific RNA binding mediated by the RNase PH domain of components of the exosome. , 2006, RNA.