Structure of the Dual Enzyme Ire1 Reveals the Basis for Catalysis and Regulation in Nonconventional RNA Splicing

Abstract

Ire1 is an ancient transmembrane sensor of ER stress with dual protein kinase and ribonuclease activities. In response to ER stress, Ire1 catalyzes the splicing of target mRNAs in a spliceosome-independent manner. We have determined the crystal structure of the dual catalytic region of Ire1at 2.4 A resolution, revealing the fusion of a domain, which we term the KEN domain, to the protein kinase domain. Dimerization of the kinase domain composes a large catalytic surface on the KEN domain which carries out ribonuclease function. We further show that signal induced trans-autophosphorylation of the kinase domain permits unfettered binding of nucleotide, which in turn promotes dimerization to compose the ribonuclease active site. Comparison of Ire1 to a topologically disparate ribonuclease reveals the convergent evolution of their catalytic mechanism. These findings provide a basis for understanding the mechanism of action of RNaseL and other pseudokinases, which represent 10% of the human kinome.

HSPC111 Governs Breast Cancer Growth by Regulating Ribosomal Biogenesis

“…RTCD1 was previously identified in the HeLa cell extract, and it is an enzyme that catalyzes conversion of a 3 0 -phosphate group into the 2 0 ,3 0 -cyclic phosphodiester at the 3 0 end of RNAs (also named RNA 2,3-cyclic phosphate) in an ATP-dependent manner (23,24). RNA 2,3-cyclic phosphate ends are crucially important in ribosome assembly through regulating RNA-protein binding and RNA stability (27,(29)(30)(31). Previous studies have validated that RTCD1-mediated conversion of 2,3-cyclic phosphate ends on rRNAs is indispensable to 18S rRNA biogenesis (32,33).…”

Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis

“…Upon activation, the IRE1 dimer undergoes autotransphosphorylation in which one monomer phosphorylates the other (11). Through the analysis of the structure of the cytosolic domain of IRE1, Lee et al (12) found that dimerization brings together the kinase domains in a face-to-face manner that would seemingly facilitate autotransphosphorylation.…”

Stress du réticulum endoplasmique

“…Le domaine cytosolique L'oligomérisation du domaine luminal d'IRE1 permet la juxtaposition des domaines cytosoliques et facilite l'autophosphorylation dans le domaine kinase et l'activation du domaine RNase [5]. La structure du domaine cytosolique d'Ire1p (S. cerevisiae) suggère, tout comme celle du domaine luminal, que cette protéine pourrait exister à l'état de dimère en l'absence de stress [5] ( Figure 2 ; partie inférieure du schéma).…”

“…La structure du domaine cytosolique d'Ire1p (S. cerevisiae) suggère, tout comme celle du domaine luminal, que cette protéine pourrait exister à l'état de dimère en l'absence de stress [5] ( Figure 2 ; partie inférieure du schéma). L'activation pourrait alors résulter de changements conformationnels.…”

“…L'activité RNase de ces deux enzymes est régulée par la dimérisation ainsi que par la liaison à un nucléotide [7] de type adénosine di ou tri-phosphate [8]. Le nucléotide, en se fixant sur le site kinase, permet un changement conformationnel induisant l'activité RNase [5].…”