ABSTRACT
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.
Subject(s)
Exosome Multienzyme Ribonuclease Complex/chemistry , Exosome Multienzyme Ribonuclease Complex/metabolism , RNA Stability , Saccharomyces cerevisiae/enzymology , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Models, Molecular , Movement , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA/chemistry , RNA/metabolism , RNA, Ribosomal, 5.8S/chemistry , RNA, Ribosomal, 5.8S/genetics , RNA, Ribosomal, 5.8S/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3' degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Substitution , Cytoskeletal Proteins/chemistry , Exosome Multienzyme Ribonuclease Complex/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Substitution/genetics , Conserved Sequence/genetics , Crystallography, X-Ray , Cytoskeletal Proteins/genetics , Evolution, Molecular , Exosome Multienzyme Ribonuclease Complex/genetics , Humans , Hydrolysis , Models, Molecular , Protein Subunits/chemistry , Protein Subunits/genetics , RNA Precursors/chemistry , RNA Processing, Post-Transcriptional , RNA, Long Noncoding/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Substrate Specificity/geneticsABSTRACT
The exosome is the major 3'-5' RNA-degradation complex in eukaryotes. The ubiquitous core of the yeast exosome (Exo-10) is formed by nine catalytically inert subunits (Exo-9) and a single active RNase, Rrp44. In the nucleus, the Exo-10 core recruits another nuclease, Rrp6. Here we crystallized an approximately 440-kilodalton complex of Saccharomyces cerevisiae Exo-10 bound to a carboxy-terminal region of Rrp6 and to an RNA duplex with a 3'-overhang of 31 ribonucleotides. The 2.8 Å resolution structure shows how RNA is funnelled into the Exo-9 channel in a single-stranded conformation by an unwinding pore. Rrp44 adopts a closed conformation and captures the RNA 3'-end that exits from the side of Exo-9. Exo-9 subunits bind RNA with sequence-unspecific interactions reminiscent of archaeal exosomes. The substrate binding and channelling mechanisms of 3'-5' RNA degradation complexes are conserved in all kingdoms of life.
Subject(s)
Exosome Multienzyme Ribonuclease Complex/chemistry , Exosome Multienzyme Ribonuclease Complex/metabolism , Protein Subunits/chemistry , RNA/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Subunits/metabolism , RNA/chemistry , Saccharomyces cerevisiae/geneticsABSTRACT
Membrane-bound Ras is activated by translocation of the Son of Sevenless (SOS) protein to the plasma membrane. SOS is inactive unless Ras is bound to an allosteric site on SOS, and the Dbl homology (DH) and Pleckstrin homology (PH) domains of SOS (the DH-PH unit) block allosteric Ras binding. We showed previously that the activity of SOS at the membrane increases with the density of PIP(2) and the local concentration of Ras-GTP, which synergize to release the DH-PH unit. Here we present a new crystal structure of SOS that contains the N-terminal histone domain in addition to the DH-PH unit and the catalytic unit (SOS(HDFC), residues 1-1049). The structure reveals that the histone domain plays a dual role in occluding the allosteric site and in stabilizing the autoinhibitory conformation of the DH-PH unit. Additional insight is provided by kinetic analysis of the activation of membrane-bound Ras by mutant forms of SOS that contain mutations in the histone and the PH domains (E108K, C441Y, and E433K) that are associated with Noonan syndrome, a disease caused by hyperactive Ras signaling. Our results indicate that the histone domain and the DH-PH unit are conformationally coupled, and that the simultaneous engagement of the membrane by a PH domain PIP(2)-binding interaction and electrostatic interactions between a conserved positively charged patch on the histone domain and the negatively charged membrane coincides with a productive reorientation of SOS at the membrane and increased accessibility of both Ras binding sites on SOS.
