Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Conformational Switching of the Nuclear Exosome during Ribosome Biogenesis Eva Kummer and Nenad Ban* Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, Otto-Stern-Weg 5, CH-8093 Zurich, Switzerland
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Exo-10 core complex via the 3′−5′ helicase activity of Mtr4 to be digested by the processive Rrp44 nuclease at the end of the Exo-9 barrel to a length of approximately 30 nucleotides. Subsequently, the remaining 3′ extension is further shortened by the exosome cofactor nuclease Rrp6 located at the barrel entry. So far, how the nuclear exosome cofactors bind and regulate the Exo-10 core and how substrate is handed over from the helicase Mtr4 to the nuclease units of the exosome have remained unclear. Recently, Schuller et al. visualized the yeast nuclear exosome engaged with the maturing pre-60S ribosomal particle as a natural substrate using cryo-electron microscopy.2 The structure reveals how the nuclear cofactors nestle around the Exo-9 core and mediate interaction with the helicase Mtr4 that in turn promotes engagement of the substrate. The nuclear exosome undergoes large structural rearrangements upon association with the pre-60S particle. The helicase Mtr4 contacts the pre-60S at two sites on the 25S rRNA spanning approximately 90 Å. One contact is made by the helicase domain, and the other contact is established by the arch domain, a prominent insertion into the helicase core. The arch domain has been shown to regulate RNA turnover by the exosome. Besides mediating substrate engagement, Schuller et al. show that the arch domain binds to the ribosome in an extended conformation in comparison to the isolated structure, which opens the helicase channel for the incoming substrate. Moreover, the arch interacts with cofactors Rrp6 and Rrp47 to allow association of the helicase with the exosome core complex and align their substrate channels to create a continuous substrate path to feed single-stranded RNA to the Rrp44 exonuclease (Figure 1). Similar conformational changes upon ribosome binding have been observed for the helicase Ski2, which is the cytosolic counterpart of helicase Mtr4, although it interacts with a completely different site on the ribosome.3 More recently, Weick et al. have also shown that stimulation of the Mtr4 helicase activity depends not only on substrate engagement but also on association of Mtr4 with the exosome core particle, indicating that mutual crosstalk between the helicase and nuclease compartment is crucial for exosome activity control.4 Once Rrp44 has finished its processive exonucleolytic activity on the 3′ extension of 5.8S rRNA, the RNA has to be extracted from the Exo-9 channel to be handed over to Rrp6 for further trimming. In the visualized complex, the 3′ end of the rRNA is still engaged with Rrp44, a conformation that the authors speculate might represent a standby position prior to
ibosomes are large molecular RNA−protein assemblies that are responsible for the translation of genetic information into proteins in all organisms. While ribosomes produce thousands of proteins every minute to maintain cell homeostasis and support cell growth, they themselves have to be generated in a complex process that depends on an intricate network of biogenesis factors, including more than 200 proteins and 100 snoRNA species. Ribosome biogenesis proceeds in several concerted steps starting with the transcription of rRNA (rRNA) in the nucleolus in the form of extended precursor rRNAs that need to be trimmed to generate mature rRNA sequences. One of the important nucleases involved in rRNA maturation is the nuclear exosome, a macromolecular complex that is responsible for several cellular RNA surveillance processes. The exosome is an evolutionarily conserved nuclease from yeast to human and serves as the main nuclease-degrading RNA transcripts in a 3′ to 5′ direction in the nucleus as well as the cytosol. The exosome core complex (Exo-10) is composed of nine architectural subunits, homologous to bacterial PNPase and the archaeal exosome, forming a barrel-like structure (Exo9) and an associated exonuclease Rrp44, homologous to bacterial RNaseII. The RNA transcripts are threaded through the central channel of Exo-9 before they can be processed by the associated exonuclease at the bottom of the Exo-9 barrel. In yeast, the exosome possesses rather low intrinsic exoribonuclease activity that is stimulated upon association of cofactors. Cofactors are highly conserved in eukaryotes and confer substrate specificity to the exosome in the respective compartments.1 In the cytosol, Exo-10 cooperates with the Ski complex in RNA surveillance. In the nucleus, the exosome complex performs versatile tasks such as the removal of excessive or defective mRNAs, tRNAs, and noncoding RNAs as well as the maturation of small nuclear or small nucleolar RNAs [sn(o)RNAs]. The nuclear exosome is recruited to its substrates via four conserved cofactors: the 3′−5′ exoribonuclease Rrp6 and its catalytically inactive partner, Rrp47, that are constitutively bound to the exosome core and that recruit, in collaboration with the adaptor protein Mpp6, the transiently associating 3′−5′ helicase Mtr4. During ribosome biogenesis, the nuclear exosome takes part in the maturation process of the pre-35S rRNA transcript, the precursor containing the 18S, 5.8S, and 25/28S rRNAs. The nuclear exosome aids in the removal of internal transcribed spacer sequence 2 (ITS2) located between the 5.8S and 25S mature rRNAs by trimming the long 3′ extension of 5.8S rRNA after it has been cleaved from the polycistronic 35S precursor rRNA. Trimming occurs in two steps. First, single-stranded RNA is channeled into the © XXXX American Chemical Society
Received: May 26, 2018
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DOI: 10.1021/acs.biochem.8b00593 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Mechanism of exosome activation. In the nucleus, the exosome core particle (composed of Exo-9 and the exonuclease Rrp44) is constitutively associated with its cofactors Rrp6, Rrp47, and Mpp6. These cofactors establish transient binding of the helicase Mtr4, which in turn promotes substrate engagement. Mtr4 is bound such that the helicase and exosome form a continuous substrate path to directly feed the substrate into the exonucleolytic site of the exosome. Bulky substrates (such as the late pre-60S ribosomal particle) cause conformational changes in the arch domain of the Mtr4 helicase, thereby opening the helicase site toward the substrate. Mtr4 binding additionally leads to displacement of the nuclease core of the associated Rrp6 from the cap of the Exp-9 barrel. How substrates are handed over between the nucleolytic sites of processive Rrp4 and distributive Rrp6 remains unresolved.
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handover. Therefore, how the substrate is switching from the Exo-10 core to the associated Rrp6 and whether this requires participation of other components of the complex remain unclear. In the absence of Mtr4, Rrp6 has been shown to bind to the cap of the Exo-9 barrel, thereby closing the lid of the nuclear exosome in the absence of the substrate.5 Strikingly, Mtr4 binds an overlapping site on the Exo-9 cap. Therefore, Mtr4 binding displaces the Rrp6 nuclease domain upon engaging the exosome. Rrp6 consequently undergoes the transition from a closed cap-bound form to an open flexible form with only its C-terminal domain remaining attached to side of the Exo-9 barrel (Figure 1). The structure of the late nuclear pre-60S particle containing ITS2 and associated biogenesis factors has been recently determined by Wu et al.6 In the structure reported by Schuller et al., ITS2 has been processed by the exosome and is therefore missing. Furthermore, this processing induces dissociation of most biogenesis factors previously associated with pre-60S ITS2 and may additionally cause conformational changes in the pre-60S particle that lead to the next step of ribosome biogenesis. Schuller et al. show beautifully how large conformational changes in the exosome are required for substrate engagement and nuclease activation. Although previous studies have revealed structural features of the exosome core in isolation either with artificial substrates or in complex with some of its cofactors, this is the first example of the complete exosome, including all nuclear cofactors and the specificity-conferring helicase in an active substrate-engaged conformation. This structure also contributes to our understanding of the complex process of ribosome biogenesis. Some mechanistic questions remain unresolved and will surely be the subject of future investigations.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Eva Kummer: 0000-0003-0644-7877 Funding
Our work was supported by Swiss National Science Foundation Grant 310030B_163478 and National Centre of Excellence in RNA and Disease Project 138262 to N.B. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. Simone Mattei for critical reading of the manuscript. REFERENCES
(1) Houseley, J., LaCava, J., and Tollervey, D. (2006) RNA-quality control by the exosome. Nat. Rev. Mol. Cell Biol. 7, 529−539. (2) Schuller, J. M., Falk, S., Fromm, L., Hurt, E., and Conti, E. (2018) Structure of the nuclear exosome captured on a maturing preribosome. Science 360, 219−222. (3) Schmidt, C., et al. (2016) The cryo-EM structure of a ribosomeSki2-Ski3-Ski8 helicase complex. Science 354, 1431−1433. (4) Weick, Puno, M. R., Januszyk, K., Zinder, J. C., DiMattia, M. A., and Lima, C. D. (2018) Helicase-dependent RNA decay illuminated by a cryo-EM structure of a human nuclear RNA exosome-Mtr4 complex. Cell 173, 1663−1677.e21. (5) Makino, D. L., Schuch, B., Stegmann, E., Baumgärtner, M., Basquin, C., and Conti, E. (2015) RNA degradation paths in a 12subunit nuclear exosome complex. Nature 524, 54−58. (6) Wu, S., et al. (2016) Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes. Nature 534, 133−137.
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DOI: 10.1021/acs.biochem.8b00593 Biochemistry XXXX, XXX, XXX−XXX
