Structure of the nuclear exosome captured on a maturing preribosome

Schuller, Jan M.; Falk, Sebastian; Fromm, Lisa; Hurt, Ed; Conti, Elena

doi:10.1126/science.aar5428

Cited by 102 publications

(155 citation statements)

References 35 publications

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“…The remaining nine proteins (Exo-9) are forming a barrel-like complex in which RNA can be channelled through before it is degraded by Rrp44, but Exo-9 proteins do not degrade or modify the RNA itself (50,51). Still, all ten subunits are positioned to directly interact with RNA and multiple interactions with the individual subunits have been demonstrated in high resolution structure studies (52)(53)(54)(55). Hence, all ten subunits are amenable to UV-crosslinking and, as a result, 9 out of the 10 subunits were identified by PTex ( Fig.…”

Section: A Global Snapshot Of Human Rna-protein Complexesmentioning

confidence: 99%

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Urdaneta

Vieira-Vieira

Hick

et al. 2018

Preprint

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Recent methodological advances allowed the identification of an increasing number of RNAbinding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to nonadenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

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Section: A Global Snapshot Of Human Rna-protein Complexesmentioning

confidence: 99%

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Urdaneta

Vieira-Vieira

Hick

et al. 2018

Preprint

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“…In Saccharomyces cerevisiae more than 200 proteins were identified as necessary for efficient assembly of the 40S and 60S ribosomal subunits. Over the past years, major advances in the field of ribosome biogenesis have been achieved (reviewed in [1][2][3][4]), including the threedimensional structures of pre-ribosome complexes obtained by cryo-electron microscopy, which revealed molecular details of the ribosome assembly pathway [5][6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Trypanosoma brucei RRP44 is involved in an early stage of large ribosomal subunit RNA maturation

et al. 2019

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Ribosomal RNA precursors undergo a series of structural and chemical modifications to generate matured RNA molecules that will comprise ribosomes. This maturation process involves a large set of accessory proteins as well as ribonucleases, responsible for removal of the external and internal transcribed spacers from the pre-rRNA. Early-diverging eukaryotes belonging to the Kinetoplastida class display several unique characteristics, in particular in terms of RNA synthesis and maturation. These peculiarities include the rRNA biogenesis and the extensive fragmentation of the large ribosomal subunit (LSU) rRNA. The role of specific endo-and exonucleases in the maturation of the unusual rRNA precursor of trypanosomatids remains largely unknown. One of the nucleases involved in rRNA processing is Rrp44, an exosome associated ribonuclease in yeast, which is involved in several metabolic RNA pathways. Here, we investigated the function of Trypanosoma brucei RRP44 orthologue (TbRRP44) in rRNA processing. Our results revealed that TbRRP44 depletion causes unusual polysome profile and accumulation of the complete LSU rRNA precursor, in addition to 5.8S maturation impairment. We also determined the crystal structure of TbRRP44 endonucleolytic domain. Structural comparison with Saccharomyces cerevisiae Rrp44 revealed differences in the catalytic site and substitutions of surface residues, which could provide molecular bases for the lack of interaction of RRP44 with the exosome complex in T. brucei. ARTICLE HISTORY

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“…This raises the speculation that the pleiotropic effect of PfCRT mutations may not be the sole cause for changes in antimalarial drug sensitivity. We did find PPQ resistant isolates with exo mutations and single copy of PM2; P. falciparum exonuclease (PF3D7_1362500) has 25.62%, 17.92%, and 17.07% amino acid sequence identity to exosome complex exonuclease RRP6 [51], exonuclease 3′→5′ domain containing protein 2 (EXD2) [52], and exonuclease 3′→5′ domain containing protein 1 (EXD1) [53], respectively. The eukaryotic RNA exosome is a 3′→5′ degradation machinery involved in the processing of coding and noncoding RNAs [54].…”

Section: Discussionmentioning

confidence: 95%

Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Boonyalai

Vesely

Thamnurak

et al. 2020

Preprint

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Background: High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. It is essential to establish sensitive and reliable markers of PPQ resistance which can be adopted for monitoring the prevalence of drug resistance and guide drug policy change. Several genetic markers have been proposed such as copy numbers of P. falciparum multidrug resistance 1 ( pfmdr1 ) and plasmepsin 2 (PM2), and mutations in exonuclease (exo-E415G) and P. falciparum chloroquine resistance transporter (PfCRT) genes.Methods: To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia and the presence of exo-E415G and PfCRT mutations as well as PM2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. The efficacy of several drug combinations between standard clones and newly cloned P. falciparum Cambodian isolates was also measured.Results: The characterization of culture-adapted isolates revealed that exo-E415G and novel PfCRT mutations can confer PPQ-resistance, in the absence of PM2 amplification. In vitro testing of PPQ resistant parasites showed bimodal dose-response phenotype as previously reported in both Cambodian isolates and genome-edited Dd2 strains. Our finding is the first PPQ resistant clinical isolate with documented swollen digestive vacuole phenotype. From the field isolates, we were able to clone a new parasite line, 14-B5, which is sensitive to arteminsinin and piperaquine but resistant to chloroquine. Most of the drug combinations tested revealed antagonistic interaction against the 14-B5 line, indicating its different genetic background from other P. falciparum laboratory reference lines.Conclusions: Surveillance for PPQ resistance in regions that rely on DHA-PPQ as the first line treatment should depend on the monitoring of the reflective molecular markers. Bimodal dose response curves and swelling of the food vacuole can be used as PPQ resistant phenotype. The use of currently circulating parasite isolates is necessary for relevant drug combination development against current P. falciparum resistance profiles.

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Structure of the nuclear exosome captured on a maturing preribosome

Cited by 102 publications

References 35 publications

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Trypanosoma brucei RRP44 is involved in an early stage of large ribosomal subunit RNA maturation

Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

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