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

Monica C Pillon,Kevin H Goslen,Jacob Gordon,Melissa L Wells,Jason G Williams,Robin E Stanley

doi:10.1074/jbc.ra119.011193

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 (RφXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. 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.

Highlights

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
Previous work has established that the invariant arginine and histidine residues of Ruminococcus species (Rs)␾XXXH are critical to support Sus Scrofa (Ss). cerevisiae Las1 RNA cleavage in vitro and in vivo [14, 23, 24], yet little is known about the intervening residues composing this motif
This led to the discovery that beyond the first arginine (Arg-1) and last histidine (His-6) residues found in all HEPN family nucleases, the intervening residues within the Las1 R␾XXXH HEPN motif are well-conserved

Summary

Introduction

The ribosome biogenesis factor Las 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. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las plays a fundamental role in processing pre-rRNA. Using reconstituted Las HEPN-HEPN؅ chimeras, we defined the molecular requirements for RNA cleavage Both copies of the Las HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las active site. The eukaryotic HEPN nuclease Ire triggers the unfolded protein response and the bacterial HEPN nuclease SO_3166 is the toxic component of the dueling type II toxin-antitoxin system [4, 5] In contrast to these defense systems, the HEPN nuclease Las ensures the translational capacity of the cell through its role in ribosome assembly [6]. Despite the strong prevalence of the HEPN domain in critical RNA-targeting nucleases, the molecular basis for how HEPN nucleases catalyze RNA cleavage has remained elusive

Methods

Results

Conclusion