Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex

Pillon, Monica C.; Hsu, Allen; Krahn, J.M.; Williams, Jason; Goslen, Kevin; Sobhany, Mack; Borgnia, Mario J.; Stanley, Robin E.

doi:10.1038/s41594-019-0289-8

Cited by 28 publications

(31 citation statements)

References 63 publications

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“…Our recent cryo-electron microscopy reconstructions of Chaetomium thermophilum (Ct) RNase PNK revealed the juxtaposed Las1 RHxhTH motifs within the composite nuclease active site ( Fig. 2B) (26). Each Las1 RHxhTH motif is encoded within an a-helix that lies at the interface of the Las1 HEPN homodimer.…”

Section: Resultsmentioning

confidence: 93%

“…While previous work has established that ScLas1 variants harboring R1A, H2A, or H6A mutations are non-viable in S. cerevisiae and unable to cleave the ITS2 pre-rRNA in vitro (14,26);it is unknown if any other amino acid substitutions are tolerated within this motif. To determine the requirements of the RHxhTH consensus motif, we utilized a strain of S. cerevisiae containing a tetracyclineinducible promoter (tetO7) upstream of endogenous LAS1 (tet-LAS1) (26). This strain was modified to include a 3x-Myc tag on the N-terminus of endogenous GRC3 (tet-Las1/Myc-GRC3) so that we could monitor Grc3 expression.…”

Section: Resultsmentioning

confidence: 99%

“…Each Las1 RHxhTH motif is encoded within an a-helix that lies at the interface of the Las1 HEPN homodimer. By capturing multiple conformational states of RNase PNK, we revealed that H2 is an important active site switch that toggles between nuclease active and inactive conformations (26).…”

Section: Resultsmentioning

confidence: 99%

“…Two Las1 protomers homodimerize at the axis of symmetry where the HEPN domains form the 'body' of the butterfly, while a Grc3 protomer makes up each 'wing' and holds the HEPN domains together (Fig. 1C) (26). This is reminiscent to all characterized HEPN nucleases, which require dimerization of two HEPN domains to activate nuclease activity (4,5,7,27,28).…”

Section: Textmentioning

confidence: 92%

“…Las1 adopts a canonical HEPN active site suggesting it shares a common mechanism for RNA cleavage as other HEPN nucleases. We recently solved a series of cryo-electron microscopy structures of RNase PNK, which unveiled its butterfly-like architecture (26). Two Las1 protomers homodimerize at the axis of symmetry where the HEPN domains form the 'body' of the butterfly, while a Grc3 protomer makes up each 'wing' and holds the HEPN domains together (Fig.…”

Section: Textmentioning

confidence: 99%

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It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

Pillon¹,

Goslen²,

Williams³

et al. 2019

Preprint

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Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the HEPN (higher eukaryotes and prokaryotes nucleotide-binding) nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif important for RNA cleavage. While most HEPN nucleases participate in stress activated RNA cleavage pathways, Las1 plays a fundamental role in processing the pre-ribosomal RNA. Underscoring the significance of Las1 function, mutations to the LAS1L gene have been associated with neurological dysfunction. Two juxtaposed Las1 HEPN nuclease motifs create its composite nuclease active site, however the roles of the individual HEPN residues are poorly defined. Here we show through a combination of in vivo and in vitro studies that both Las1 HEPN nuclease motifs are required for nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we define the consensus Las1 HEPN nuclease motif and uncover its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN¢ chimeras, we define the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif are necessary for nuclease specificity revealing that both HEPN motifs participate in coordinating the RNA within the active site. Taken together, our work reveals critical information about HEPN nuclease function and establishes that HEPN nucleases can be re-wired to cleave alternative RNA substrates. 185 aa (H134A) + LCT: 469-502 aa

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Textmentioning

confidence: 92%

Section: Textmentioning

confidence: 99%

See 3 more Smart Citations

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

Pillon¹,

Goslen²,

Williams³

et al. 2019

Preprint

Self Cite

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Caught in the act—Visualizing ribonucleases during eukaryotic ribosome assembly

Schneider

Bohnsack

2022

WIREs RNA

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Ribosomes are essential macromolecular machines responsible for translating the genetic information encoded in mRNAs into proteins. Ribosomes are composed of ribosomal RNAs and proteins (rRNAs and RPs) and the rRNAs fulfill both catalytic and architectural functions. Excision of the mature eukaryotic rRNAs from their precursor transcript is achieved through a complex series of endoribonucleolytic cleavages and exoribonucleolytic processing steps that are precisely coordinated with other aspects of ribosome assembly. Many ribonucleases involved in pre-rRNA processing have been identified and pre-rRNA processing pathways are relatively well defined. However, momentous advances in cryo-electron microscopy have recently enabled structural snapshots of various pre-ribosomal particles from budding yeast (Saccharomyces cerevisiae) and human cells to be captured and, excitingly, these structures not only allow pre-rRNAs to be observed before and after cleavage events, but also enable ribonucleases to be visualized on their target RNAs. These structural views of pre-rRNA processing in action allow a new layer of understanding of rRNA maturation and how it is coordinated with other aspects of ribosome assembly. They illuminate mechanisms of target recognition by the diverse ribonucleases involved and reveal how the cleavage/processing activities of these enzymes are regulated. In this review, we discuss the new insights into pre-rRNA processing gained by structural analyses and the growing understanding of the mechanisms of ribonuclease regulation.

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Transformation of Chaetomium thermophilum and Affinity Purification of Native Thermostable Protein Complexes

Kellner

Hurt

2022

Methods in Molecular Biology

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Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex

Cited by 28 publications

References 63 publications

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

Caught in the act—Visualizing ribonucleases during eukaryotic ribosome assembly

Transformation of Chaetomium thermophilum and Affinity Purification of Native Thermostable Protein Complexes

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