Abstract
BackgroundThe unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis.ResultsThis analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase.ConclusionsOur data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.
Highlights
The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER)
The RNA binding site is proximal to the dynamic helix-loop element HLE, which becomes more ordered upon formation of Ire1/Ire1 interfaces in the high-order oligomer and has been proposed to serve as a part of the active site [5] (Figure 1a, Figure 1 Oligonucleotide binding to Ire1 RNase. (a) Ire1 oligomer (PDB ID 3fbv) colored by domains and RNA-binding propensity. (b) Close view of symmetric back-to-back RNase dimer from the oligomer in (a) with electron density for dCdCdGdCdAdG from the
While we recognize that the deoxy-substituted single-stranded substrate may be structurally distinct from RNA stem-loops, we surmise that its binding site on Ire1 represents that of the HP21 RNA because (i) dCdCdGdCdAdG efficiently inhibits cleavage of HP21 by Ire1KR32, and (ii) the electron density in the RNase active site directly contacts the critical catalytic residue H1061, which is involved in proton transfer and contributes ≥ 3 × 105-fold to catalysis
Summary
The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER) Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/ endoribonuclease Ire. The endoribonuclease (RNase) domain of Ire initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The unfolded protein response (UPR) is an intracellular signaling pathway that provides homeostatic feedback regulation between the endoplasmic reticulum (ER) and the gene expression program in the nucleus. To this end, the UPR senses the conditions inside the ER, detecting an imbalance between newly made proteins and the protein folding capacity in the ER, and activates a corrective response. Intron removal allows for the production of the UPR transcription activators Hac and XBP1, respectively, which upregulate UPR target genes
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