RNA degradation paths in a 12-subunit nuclear exosome complex

The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3′–5′ RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6–Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.

[1]  D. Libri,et al.  RNA quality control in the nucleus: the Angels' share of RNA. , 2013, Biochimica et biophysica acta.

[2]  Quansheng Liu,et al.  Reconstitution, Activities, and Structure of the Eukaryotic RNA Exosome , 2006, Cell.

[3]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[4]  Randy J. Read,et al.  Electronic Reprint Biological Crystallography Decision-making in Structure Solution Using Bayesian Estimates of Map Quality: the Phenix Autosol Wizard Biological Crystallography Decision-making in Structure Solution Using Bayesian Estimates of Map Quality: the Phenix Autosol Wizard , 2022 .

[5]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[6]  Sergey Melnikov,et al.  The Structure of the Eukaryotic Ribosome at 3.0 Å Resolution , 2011, Science.

[7]  Mikkel H. Schierup,et al.  RNA Exosome Depletion Reveals Transcription Upstream of Active Human Promoters , 2008, Science.

[8]  Jun-Jie Liu,et al.  Visualization of Distinct Substrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy , 2013, Nature Structural &Molecular Biology.

[9]  C. Lima,et al.  Exo- and endoribonucleolytic activities of yeast cytoplasmic and nuclear RNA exosomes are dependent on the noncatalytic core and central channel. , 2012, Molecular cell.

[10]  M. Deutscher,et al.  Substrate Recognition and Catalysis by the Exoribonuclease RNase R* , 2006, Journal of Biological Chemistry.

[11]  T. Jensen,et al.  RNA decay machines: the exosome. , 2013, Biochimica et biophysica acta.

[12]  Clemens Vonrhein,et al.  Data processing and analysis with the autoPROC toolbox , 2011, Acta crystallographica. Section D, Biological crystallography.

[13]  D. Tollervey,et al.  Transcriptome-wide Analysis of Exosome Targets , 2012, Molecular cell.

[14]  B. Séraphin,et al.  Intracellular ribonucleases involved in transcript processing and decay: precision tools for RNA. , 2013, Biochimica et biophysica acta.

[15]  Kevin Cowtan,et al.  Fitting molecular fragments into electron density , 2007, Acta crystallographica. Section D, Biological crystallography.

[16]  P. Mitchell,et al.  Rrp6, rrp47 and cofactors of the nuclear exosome. , 2011, Advances in experimental medicine and biology.

[17]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[18]  Randy J. Read,et al.  Phenix - a comprehensive python-based system for macromolecular structure solution , 2012 .

[19]  Elena Conti,et al.  Crystal structure of an RNA-bound 11-subunit eukaryotic exosome complex , 2013, Nature.

[20]  Michael W. Briggs,et al.  Rrp6p, the Yeast Homologue of the Human PM-Scl 100-kDa Autoantigen, Is Essential for Efficient 5.8 S rRNA 3′ End Formation* , 1998, The Journal of Biological Chemistry.

[21]  Anette Thyssen Jonstrup,et al.  Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain , 2006, Proceedings of the National Academy of Sciences.

[22]  P. Zwart,et al.  Towards automated crystallographic structure refinement with phenix.refine , 2012, Acta crystallographica. Section D, Biological crystallography.

[23]  C. Lima,et al.  Structure of an Rrp6-RNA exosome complex bound to polyA RNA , 2014, Nature.

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

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

[26]  P. Mitchell,et al.  The exosome‐binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase , 2014, The EMBO journal.

[27]  M. Topf,et al.  RNA channelling by the eukaryotic exosome , 2010, EMBO Reports.

[28]  E. Conti,et al.  The RNA exosome and proteasome: common principles of degradation control , 2013, Nature Reviews Molecular Cell Biology.

[29]  Wolfgang Kabsch,et al.  Integration, scaling, space-group assignment and post-refinement , 2010, Acta crystallographica. Section D, Biological crystallography.

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

[31]  J. Butler,et al.  Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p , 2008, Nucleic acids research.

[32]  C. Lima,et al.  The eukaryotic RNA exosome. , 2014, Current opinion in structural biology.

[33]  F. Bonneau,et al.  The Yeast Exosome Functions as a Macromolecular Cage to Channel RNA Substrates for Degradation , 2009, Cell.

[34]  Quansheng Liu,et al.  Activities of human RRP6 and structure of the human RRP6 catalytic domain. , 2011, RNA.

[35]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[36]  E. Conti,et al.  Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family. , 2008, Molecular cell.

[37]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[38]  A. Dziembowski,et al.  The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro , 2013, Nucleic acids research.

[39]  N. Ban,et al.  Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6 , 2011, Science.

[40]  A. van Hoof,et al.  Functions of the cytoplasmic exosome. , 2011, Advances in experimental medicine and biology.

[41]  Philip R. Evans,et al.  How good are my data and what is the resolution? , 2013, Acta crystallographica. Section D, Biological crystallography.

[42]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

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