Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Pillon, Monica C.; Sobhany, Mack; Stanley, Robin E.

doi:10.1261/rna.065037.117

Cited by 18 publications

(27 citation statements)

References 57 publications

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“…Yeast Spotting Assays and Growth Curves. Yeast spotting assays and growth curves were performed as described previously (24) with minor modifications. Transformed LAS1 yeast strains were pre-incubated in YPD that was supplemented with 40 µg/ml doxycycline for 24 hours at 22°C prior to performing assays.…”

Section: Generation Of Las1 Yeast Strainsmentioning

confidence: 99%

“…Western Blots. Western blotting was performed as described previously (24) with minor modifications.…”

Section: Generation Of Las1 Yeast Strainsmentioning

confidence: 99%

“…C2 pre-rRNA Cleavage and Phosphorylation Assays. C2 RNA cleavage assays were performed as described previously(23,24) with minor changes. Full-length ScLas1-Grc3 and chimericScLas1 HEPN|HEPN¢+LCT -Grc3 variants (0-3.2 µM) were incubated with 1 mM EDTA and 50 or 100 nM 3¢fluorescein labeled C2 RNA substrate (5¢-GUCGUUUUAGGUUUUACCAACUGCGGC/36-FAM/-3¢) in the absence and presence of 2 mM nucleotide (ATP or ADPnP).…”

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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: Generation Of Las1 Yeast Strainsmentioning

confidence: 99%

“…Western Blots. Western blotting was performed as described previously (24) with minor modifications.…”

Section: Generation Of Las1 Yeast Strainsmentioning

confidence: 99%

mentioning

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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“…Further distinguishing Las1 from other HEPN nucleases is Las1's dependence on its binding partner, the Grc3 polynucleotide kinase for nuclease activation (23). While no other HEPN nuclease is known to require an auxiliary protein for HEPN activation, Las1 and Grc3 are reliant on one another for protein stability and enzyme activation (15,23,24). Together the ribonuclease (RNase) Las1 and polynucleotide kinase (PNK) Grc3 assemble into a tetrameric complex, known as RNase PNK.…”

mentioning

confidence: 99%

“…Together the ribonuclease (RNase) Las1 and polynucleotide kinase (PNK) Grc3 assemble into a tetrameric complex, known as RNase PNK. Through this higher-order assembly, RNase PNK coordinates its dual enzymatic functions through a poorly understood mechanism of molecular cross-talk that ensures efficient processing of the pre-rRNA (23)(24)(25).…”

mentioning

confidence: 99%

It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly

Pillon

Goslen

Gordon

et al. 2020

Journal of Biological Chemistry

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The ribosome biogenesis factor, Las1 is an essential endoribonuclease that is well conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RfxxxH) important for RNA cleavage. While most HEPN nucleases participate in stress-activated RNA cleavage pathways, Las1 plays a fundamental role in processing pre-ribosomal RNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN HEPN¢ chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage.Endoribonucleases play critical roles in the processing, maturation, and destruction of https://www.jbc.org/cgi/

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Ribosomal RNA Processing

Dalal

Lyons²

2023

Encyclopedia of Cell Biology

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Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex

Cited by 18 publications

References 57 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

It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly

Ribosomal RNA Processing

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