Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM

Liu, Jun-Jie; Bratkowski, Matthew; Liu, Xueqi; Niu, Chao; Ke, Ailong; Wang, Hongwei

doi:10.1038/nsmb.2736

Cited by 54 publications

(102 citation statements)

References 44 publications

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“…Both complexes were mono-disperse under EM and hence were suitable for single-particle two-dimensional (2D) classification analysis. As expected, the 2D class averages of the Exo10 complex showed classical Exo10 structures, as previously observed [13,17]. In contrast, the 2D class averages of the Exo10-Ski7 complex revealed a prominent additional density attached to the top of the Exo10 core ( Figure 1C).…”

Section: Purification Of the Endogenous Exo10-ski7 Complexsupporting

confidence: 52%

“…The solvent exposure of the Rrp44 active site in the apo structure of Exo10 also suggests another pathway where RNA substrates reach the Rrp44 active site without passing through the central channel of the core. This direct-access pathway does not require RNA substrates to have very long single-stranded 3′ overhangs and enables Exo10 to process RNAs with a tertiary structure [17]. Interestingly, our previous structural studies have indicated that Exo10 adopts distinct conformations in the through-core and direct-access pathways, of which the latter is very similar to the apostate of the complex [17].…”

Section: Introductionmentioning

confidence: 99%

“…In our previous studies, we purified the endogenous Exo10 complex from an RRP6-knockout S. cerevisiae strain with TAP-tagged Rrp46 [13,17]. Using the same protocol except for a minor modification at the washing step of the tandem affinity purification procedure, we recovered the exosome complex after elution from the IgG beads.…”

Section: Purification Of the Endogenous Exo10-ski7 Complexmentioning

confidence: 99%

“…This direct-access pathway does not require RNA substrates to have very long single-stranded 3′ overhangs and enables Exo10 to process RNAs with a tertiary structure [17]. Interestingly, our previous structural studies have indicated that Exo10 adopts distinct conformations in the through-core and direct-access pathways, of which the latter is very similar to the apostate of the complex [17]. Our previous studies have also indicated that the through-core conformation of Exo10 is very likely to be induced by the RNA substrate within the central channel.…”

Section: Introductionmentioning

confidence: 99%

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CryoEM structure of yeast cytoplasmic exosome complex

Liu

Niu

et al. 2016

Cell Res

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The eukaryotic multi-subunit RNA exosome complex plays crucial roles in 3′-to-5′ RNA processing and decay. Rrp6 and Ski7 are the major cofactors for the nuclear and cytoplasmic exosomes, respectively. In the cytoplasm, Ski7 helps the exosome to target mRNAs for degradation and turnover via a through-core pathway. However, the interaction between Ski7 and the exosome complex has remained unclear. The transaction of RNA substrates within the exosome is also elusive. In this work, we used single-particle cryo-electron microscopy to solve the structures of the Ski7-exosome complex in RNA-free and RNA-bound forms at resolutions of 4.2 Å and 5.8 Å, respectively. These structures reveal that the N-terminal domain of Ski7 adopts a structural arrangement and interacts with the exosome in a similar fashion to the C-terminal domain of nuclear Rrp6. Further structural analysis of exosomes with RNA substrates harboring 3′ overhangs of different length suggests a switch mechanism of RNA-induced exosome activation in the through-core pathway of RNA processing.

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Section: Purification Of the Endogenous Exo10-ski7 Complexsupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

Section: Purification Of the Endogenous Exo10-ski7 Complexmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CryoEM structure of yeast cytoplasmic exosome complex

Liu

Niu

et al. 2016

Cell Res

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“…Thus different temperatures, solution conditions, and biochemical reaction time-points will yield thermodynamics and kinetics information for bio-macromolecule complexes correlated with structural information. Overall, these data lead to a deeper insight into molecular mechanisms [13,14].…”

Section: Inhomogeneous Samplesmentioning

confidence: 99%

Cryo-electron microscopy for structural biology: current status and future perspectives

Wang

2015

Sci. China Life Sci.

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Recently, significant technical breakthroughs in both hardware equipment and software algorithms have enabled cryo-electron microscopy (cryo-EM) to become one of the most important techniques in biological structural analysis. The technical aspects of cryo-EM define its unique advantages and the direction of development. As a rapidly emerging field, cryo-EM has benefitted from highly interdisciplinary research efforts. Here we review the current status of cryo-EM in the context of structural biology and discuss the technical challenges. It may eventually merge structural and cell biology at multiple scales.cryo-electron microscopy, structural biology, cell biology, three-dimensional reconstruction Citation:Wang HW. Cryo-electron microscopy for structural biology: current status and future perspectives. Sci China Life Sci, 2015Sci, , 58: 750 -756, doi: 10.1007 Since the 1980s, structural biology has become a booming area in life sciences. By determining the three-dimensional (3-D) structures of proteins, nucleic acids, and their complexes, structural biology has given us accurate insights into concerning the shapes of complicated bio-macromolecules, the arrangements of atoms and molecules, and physical properties such as electro-potential distributions and hydrophobicity. These structures provide a crucial understanding of how bio-macromolecules execute specific biological functions. As techniques of structural biology become more mature in the 21st century, they assume more significant roles in various sub-disciplines of life sciences. Of the more than 10 5 protein structures that have been deposited in the Worldwide Protein Data Bank, most were solved by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy ( Figure 1A). Recent technical progress has stimulated the use of new structural biology tools. In December 2013, two landmark Nature articles revealed the atomic-resolution structure of the TRPV1 ion channel in membranes that was imaged by cryo-EM [1,2]. With cryo-EM, Yifan Cheng and David Julius et al. [1,2] at the University of California, San Francisco, imaged for the first time the 3-D structure of the membrane channel protein TRVP1 with a resolution of 3.4 Å, and constructed an atomic model. In addition, several nearly atomic models of viruses, proteasomes, and ribosomes have been resolved by cryo-EM in the past few years [36]. The significance of the work was to obtain high-resolution structural information from a very small amount of sample, without requiring protein crystallization, and to simultaneously obtain the structures of the protein complex in different states over a relatively short time. In only one year, cryo-EM has received extensive attention as a means to directly obtain atomic structures of bio-macromolecules.Electron microscopy has been used in structural biology for many years. Since the 1950s, it has revealed cellular, sub-cellular, and bio-macromolecular structures. Based on completely different principles from X-ray crystallography and NMR spectroscopy in so...

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Comparison of preribosomal RNA processing pathways in yeast, plant and human cells – focus on coordinated action of endo‐ and exoribonucleases

2017

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Edited by Michael IbbaProper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo-and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans.Keywords: endoribonuclease; exoribonuclease; pre-rRNA processing; ribosome biogenesis; RNA quality control; RNA trimming Ribosome biosynthesis is a multistep process that includes several tightly controlled events -ribosomal DNA (rDNA) transcription, processing of rRNA precursors into mature molecules, accompanied by binding of ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs), and the final assembly of all Abbreviations CD, catalytic domain; dsRBD, double-stranded RNA-binding domain; ETS, external transcribed spacer; ITS, internal transcribed spacer; LSU, large ribosome subunit (60S); miRNA, microRNA; mRNA, messenger RNA; MRP RNA, noncoding RNA component of the RNase MRP; mTOR, mammalian target of rapamycin; nt(s), nucleotide(s); PIN domain, PilT N-terminal domain; Pol I/II/III, RNA polymerase I/II/III; prerRNA, ribosomal RNA precursor; RBF, ribosome biogenesis factor; RMRP, RNase MRP (mitochondrial RNA processing ribonuclease); RNase, ribonuclease; RNB domain, RNase II domain; RNP, ribonucleoprotein; RP, ribosomal protein; rRNA, ribosomal RNA; siRNA, short interfering RNA; snoRNA, small nucleolar RNA; snoRNP, small nucleolar ribonucleoprotein; snRNA, small nuclear RNA; SSU, small ribosome subunit (40S); TRAMP4 or TRAMP5, Trf4/Air/Mtr4 or Trf5/Air/Mtr4 polyadenylation complex; tRNA, transfer RNA.

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Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM

Cited by 54 publications

References 44 publications

CryoEM structure of yeast cytoplasmic exosome complex

CryoEM structure of yeast cytoplasmic exosome complex

Cryo-electron microscopy for structural biology: current status and future perspectives

Comparison of preribosomal RNA processing pathways in yeast, plant and human cells – focus on coordinated action of endo‐ and exoribonucleases

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