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

Abstract

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/

HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation

“…Different molecular mechanisms including conformational changes, steric hindrance, or allosteric control are employed by HEPN RNases for activity regulation. For example, the catalytic activity of Las1 ribonuclease is controlled by a conserved threonine, which alters conformation of the active site histidine (Pillon et al, 2019(Pillon et al, , 2020; Cas13a, Cas13b, and Cas13d proteins all contain regulatory a helices that sterically block HEPN active sites in apo-proteins but are rotated away upon binding of specific ssRNA (Liu et al, 2017a(Liu et al, , 2017bZhang et al, 2018). The HEPN domains in Csm6 and Csx1 proteins undergo allosteric rearrangements in response to the cyclic oligoadenylate binding to the regulatory CARF domain (Foster et al, 2020;Han et al, 2017;Kazlauskiene et al, 2017).…”

Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics

“…To analyze the masses of the RNA cleavage products produced by Nsp15, the FRET RNA substrate (0.8 μM) was incubated in the absence and presence of tagless Nsp15 variant (2.5 nM) in RNA Cleavage Buffer at 25 °C for 30 min. Mass spectrometry was performed essentially as previously described 42 with the following modifications. Buffer A was 400 mM hexafluoro-2-propanol, 3 mM triethylamine (pH 7.0) and buffer B was methanol.…”

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

“…To analyze the masses of the RNA cleavage products produced by Nsp15, the FRET RNA substrate (0.8 μM) was incubated in the absence and presence of tagless Nsp15 variant (2.5 nM) in RNA Cleavage Buffer at 25°C for 30 minutes. Mass spectrometry was performed essentially as described in ( 37 ) with the following modifications. Buffer A was 400 mM hexafluoro-2-propanol, 3 mM triethylamine (pH 7.0) and buffer B was methanol.…”