Activation of rnase

In fact, human IRE1 could have a substantially different tetrameric interface than yeast IRE1, given the low sequence conservation of the activation loop between the two species. Alternatively, it is possible that the activation loop dynamics may allosterically control the conformation of the RNase, but only when IRE1 is associated in a B2B dimer. Therefore, the true RNase active conformation of the protein remains elusive.

Better definition of this could aid in SAR for allosteric activators as well as inhibitors. Further studies will be needed for full mechanistic elucidation of this class of IRE1 RNase allosteric activators. This allowed us to establish that allosteric inhibitors hinder IRE1 dimerization by occupying the back pocket of the ATP-binding site. While this mechanism was proposed before 20 , our SAR studies on this scaffold show unequivocally how minor modifications can turn an allosteric activator into an inhibitor, solely by breaking the KE salt bridge and allowing the C-helix to be displaced.

This underlines the key role of the kinase activation loop conformation in the mechanism of action of G By producing a series of mutants to assess the role of each phosphorylation site, we observed that a single phosphorylation event on the IRE1 activation loop, regardless of the specific serine, does not appreciably increase RNase activity. However, two phosphorylation events are capable of significantly activating the RNase, with the combination of pS and pS being the most effective, as previously observed 8.

More specifically, we found that R, part of the catalytic HRD motif in the IRE1 kinase, is a central residue to this modulation switch: in absence of this sidechain within IREP, G class compounds can revert back to activation. This becomes even more pronounced upon substitution by alanine of both R and R located on the C-helix.

Both arginine residues interact directly with pS, the most proximal phosphorylation site to the DFG motif, whose conformation must change dramatically to enable activation by the G class. Thus, when the RpS interaction is missing, G becomes agnostic to the presence of the three phosphorylation modifications, which otherwise would rigidify the loop in an extended configuration and make conformational changes by G energetically unfavorable.

The implication around the discovery of G and its unique modulation of IRE1 is that the RNase domain of IRE1 seems to be activated via the kinase domain in two ways. In one case, phosphorylations on the kinase activation loop can stabilize an extended conformation of the loop, rigidifying the kinase and stabilizing the B2B dimer as a consequence. The stabilization of the B2B dimer by phosphorylation can only be partially enhanced by ligands such as G or pan-kinase inhibitor Staurosporine 36 , which only mildly activate IREP Fig.

This highlights how the mechanism of activation by phosphorylation and by small-molecule type I kinase inhibitors are potentially overlapping. In an alternative scenario, an upward conformation of the loop in absence of phosphorylation in a B2B dimer can increase activity of the RNase domain. We show in our work that this kind of activation can be brought upon by small molecules such as G and G, but whether this modulation is physiologically relevant remains to be seen.

In conclusion, our work diversifies the available collection of IRE1 modulators by adding a class of selective IRE1 allosteric activators while providing insight into the nature of kinase-RNase interdomain regulation within IRE1. These studies open a distinct avenue for exploring the potential therapeutic benefits of IRE1 activators in disease. RNA cleavage was measured kinetically over an hour at room temperature as an increase in fluorescence.

Samples were run in duplicate. See Supplementary Table 4 for sequences of relevant capture probes. Data analysis was carried out against an assay background control with no cell lysate no-RNA control. No Tg was used when treating cells with all other compounds. Correct clones were then sequenced.

Secondary antibody was from The Jackson Laboratory. Clear lysates were incubated with a solution of 0. Sedimentation was monitored by A Cleared supernatant was bound to Ni-NTA. Superflow beads Qiagen by gravity filtration. IRE1 eluted as a monomer. The resulting unphosphorylated sample was purified by SEC and eluted as a monomeric peak.

A distribution of three to eleven phosphorylations was observed by LC-MS. Samples were split in half for separate enzymatic digestion with 0. Digests were quenched with 0.

A binary gradient pump was used to deliver solvent A Data were collected in data-dependent mode with the precursor ions being analyzed in the FTMS and the top 15 most abundant ions being selected for fragmentation and analysis in the ITMS. Acquired mass spectral data were searched against the protein sequence using Byonic software Protein Metrics Inc.

Byonic search results were analyzed in the Byologic software Protein Metrics Inc. Peptide identification was confirmed by MS2 peptide fragmentation. Phosphorylated peptides were label-free quantified relative to its unmodified form by AUC integration of their extracted ion chromatograms in Thermo Xcalibur Qual Browser software Thermo Fisher Scientific.

The full results of the phosphorylation mapping experiments are reported in Supplementary Data 5. Protein crystals were grown as reported below. Data reduction statistics are reported in Table 1 , which includes resolution estimates derived from both isotropic and anisotropic scaling.

Crystallization was done in 24 well Linbro plates, hanging drop. Crystals appeared after 2 days and grew to the final size in 4 days. Data collection was done at the Advanced Light Source, beamline 5. Crystals were flash frozen in liquid nitrogen. Each sample was run in duplicate. Peptides were identified using Byonic version 3. Peptides were charge-state-averaged, and deuterium uptake information extracted using algorithms described exactly in chapter 4. Changes in protection factors were extracted using an empirical algorithm In a manner largely consistent with standards set forward recently for the dissemination of HX-MS data 51 , we have included plots of every peptide measured and used in this work see Supplementary Data 2 and listed the computed peptide protection factors individually for each condition tested along with other useful experimental details See Supplementary Data 3.

We have not included a list of deuterium uptake values for each time point for every peptide included in this work because the peptide protection factor with range of observations given in Supplementary Data 3 reduce this information into a more useful format.

Additional information can also be found in Supplementary Data 4. Note: PDB files use residues numbers that are substantially offset from the sequential numbering used in the peptide plots and tables.

Further information on research design is available in the Nature Research Reporting Summary linked to this article. The authors declare that all other data supporting the findings of this study are available within the article and its Supplementary Data files, or from the corresponding authors on request.

Source data are provided with this paper. Walter, P. The unfolded protein response: from stress pathway to homeostatic regulation. Science , — Mori, K. Signalling pathways in the unfolded protein response: development from yeast to mammals. An unfolded protein-induced conformational switch activates mammalian IRE1. Elife 6 , e Gardner, B. Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response.

Lee, K. Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing. Cell , 89— Korennykh, A. Structural and functional basis for RNA cleavage by Ire1. BMC Biol. Li, H. Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering. Natl Acad.

Prischi, F. Phosphoregulation of Ire1 RNase splicing activity. Google Scholar. Sanches, M. Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors. The unfolded protein response signals through high-order assembly of Ire1. Nature , — Lu, Y. Cell 55 , — Acosta-Alvear, D. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks.

Cell 27 , 53—66 Hollien, J. Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response. Lu, M. Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis. Science , 98— Chang, T. Coordination between two branches of the unfolded protein response determines apoptotic cell fate. Cell 71 , — Bae, D. Cell Biol. Wang, M. Protein misfolding in the endoplasmic reticulum as a conduit to human disease.

The impact of the endoplasmic reticulum protein-folding environment on cancer development. Cancer 14 , — Wang, L. Feldman, H. ACS Chem. Mendez, A. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic. Elife 4 , e So, J.

Cell Metab. Wang, J. Inositol-requiring enzyme 1 facilitates diabetic wound healing through modulating MicroRNAs. Diabetes 66 , — Rashid, H. Bujisic, B. Blood , — Su, A. Viruses 9 , Zhang, P. Pirog, K. A binary gradient pump was used to deliver solvent A Data were collected in data-dependent mode with the precursor ions being analyzed in the FTMS and the top 15 most abundant ions being selected for fragmentation and analysis in the ITMS.

Acquired mass spectral data were searched against the protein sequence using Byonic software Protein Metrics Inc. Byonic search results were analyzed in the Byologic software Protein Metrics Inc. Peptide identification was confirmed by MS2 peptide fragmentation. Phosphorylated peptides were label-free quantified relative to its unmodified form by AUC integration of their extracted ion chromatograms in Thermo Xcalibur Qual Browser software Thermo Fisher Scientific.

The full results of the phosphorylation mapping experiments are reported in Supplementary Data 5. Protein crystals were grown as reported below. Data reduction statistics are reported in Table 1 , which includes resolution estimates derived from both isotropic and anisotropic scaling.

Crystallization was done in 24 well Linbro plates, hanging drop. Crystals appeared after 2 days and grew to the final size in 4 days. Data collection was done at the Advanced Light Source, beamline 5. Crystals were flash frozen in liquid nitrogen. Each sample was run in duplicate. Peptides were identified using Byonic version 3. Peptides were charge-state-averaged, and deuterium uptake information extracted using algorithms described exactly in chapter 4. Changes in protection factors were extracted using an empirical algorithm In a manner largely consistent with standards set forward recently for the dissemination of HX-MS data 51 , we have included plots of every peptide measured and used in this work see Supplementary Data 2 and listed the computed peptide protection factors individually for each condition tested along with other useful experimental details See Supplementary Data 3.

We have not included a list of deuterium uptake values for each time point for every peptide included in this work because the peptide protection factor with range of observations given in Supplementary Data 3 reduce this information into a more useful format.

Additional information can also be found in Supplementary Data 4. Note: PDB files use residues numbers that are substantially offset from the sequential numbering used in the peptide plots and tables. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

This paper is dedicated to the memory of our late co-author Susan E. We thank the members of the laboratory of Prof. Source data 8. W and W. P and P. L performed the phosphorylation mapping experiments. All authors discussed results and commented on the manuscript. Peer review information Nature Communications thanks John Burke and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Joachim Rudolph, Email: moc. Weiru Wang, Email: moc. Supplementary information is available for this paper at National Center for Biotechnology Information , U. Nat Commun. Published online Dec Day , 4 Benjamin T. Walters , 5 Susan E. Clark , 5 Maureen H. Eric S. Day 4 Pharmaceutical Development, Genentech, Inc. Benjamin T. Susan E. Kevin R. Maureen H. Yung-Chia A. Chen 3 Cancer Immunology, Genentech, Inc. Peter S. Author information Article notes Copyright and License information Disclaimer.

Corresponding author. Received Mar 10; Accepted Nov 9. This article has been cited by other articles in PMC. Description of Additional Supplementary Files. Subject terms: Kinases, Endoplasmic reticulum, Stress signalling, Small molecules, X-ray crystallography. Introduction The Unfolded Protein Response UPR is a highly regulated intracellular network that alleviates endoplasmic reticulum ER stress caused by elevated levels of misfolded proteins in the ER lumen 1.

Open in a separate window. Characterization of IRE1 kinase inhibitors. Table 1 X-ray crystallography data collection and refinement statistics. The nature of the tail substituent determines the degree of RNase activation. G modifies the conformation of the IRE1 kinase activation loop. G allosterically inhibits the RNase activity of phosphorylated IRE1 Our results suggest that the activation loop conformation is critical to the mechanism of allosteric RNase modulation by G G activates pre-associated IRE1 in a cellular context Although G seems to effect conformational changes of the kinase activation loop of IREP, it is unclear precisely how this leads to RNase activation.

G acts on pre-associated IRE1. Cell treatment with compounds and western blots of crosslinked lysates KMS and MDA-MB cells were obtained from ATCC, authenticated by short tandem repeat profiles, and tested to ensure they were mycoplasma-free within 3 months of use. X-ray crystallography Protein crystals were grown as reported below. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Supplementary information Supplementary Information 8. Peer Review File 1. Description of Additional Supplementary Files 13K, docx. Supplementary Data 1 18K, xlsx. Supplementary Data 2 8. Supplementary Data 3 95K, xlsx. Supplementary Data 4 K, xlsx. Supplementary Data 5 9. Reporting Summary 98K, pdf. Acknowledgements This paper is dedicated to the memory of our late co-author Susan E.

Source data Source data 8. Author contributions E. Competing interests E. Footnotes Peer review information Nature Communications thanks John Burke and the other, anonymous, reviewers for their contribution to the peer review of this work. Contributor Information Joachim Rudolph, Email: moc. Supplementary information Supplementary information is available for this paper at References 1. Walter P, Ron D.

The unfolded protein response: from stress pathway to homeostatic regulation. Mori K. Signalling pathways in the unfolded protein response: development from yeast to mammals. An unfolded protein-induced conformational switch activates mammalian IRE1. Gardner BM, Walter P. Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Lee KPK, et al. Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing.

Korennykh AV, et al. Structural and functional basis for RNA cleavage by Ire1. BMC Biol. Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering. Natl Acad. Phosphoregulation of Ire1 RNase splicing activity. Sanches M, et al. Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors.

The unfolded protein response signals through high-order assembly of Ire1. Acosta-Alvear D, et al. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Hollien J, Weissman JS. Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response.

Lu M, et al. Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis. Chang T-K, et al. Coordination between two branches of the unfolded protein response determines apoptotic cell fate. Cell Biol. Wang M, Kaufman RJ. Activation of RNase L is known to be important in the defense against viral infections, yet it remains mostly unclear what the effects of mRNA degradation caused by this activation have on the host.

We investigated how altered translation of the modified transcriptome might contribute to this function by performing ribosome profiling experiments on RNase L activated cells. This finding disfavors models where translation termination and recycling are directly modulated and instead points to the importance of mRNA fragmentation in explaining this phenomenon. In addition, we showed that data from others 29 demonstrate that these mRNA fragments are stable enough to be detected in cells.

This model also raises the possibility that mRNA fragments generated by other endonucleases may be translated, such as those created by the RNase L paralog Ire1 that is activated by ER stress or those facilitated by the human coronavirus CoV Nsp1 protein 74— These observations raise the question of how these fragments are stabilized to avoid rapid degradation.

Xrn1 was already shown to be an important regulator of the innate immune response due to its ability to degrade viral dsRNA fragments and thereby tune the host's ability to detect the virus via other dsRNA sensors 77 , Thus, inhibition of Xrn1 could play a role in setting the stability of both host and viral RNA fragments in the cell during RNase L activation. Another question raised by this model is how 40S ribosomal subunits are recruited to these fragments in order to initiate translation.

Additionally, it is conceivable that there would be an overabundance of free ribosomes during RNase L activation because most mRNAs are degraded, freeing up ribosomes that would normally be engaged in translating conventional capped mRNAs.

For example, it is conceivable that RNase L cleavage might damage ribosomal RNA rRNA to an extent that alters the fidelity of translation initiation in a way that permits these damaged ribosomes to translate regions outside of the main ORF.

It is also possible that changes in the abundance of initiation factors during the broad reprogramming of the transcriptome, or activation of stress pathways as a result of mRNA loss, could facilitate translation of altORFs. Thus, it is possible that RNase L activation could also modulate this process.

Another hypothesis is that ribosomes on altORFs are usually present at low levels and that RNA decay triggered by RNase L increases their relative proportion because they offer some protection against degradation.

Our observation also raises the question of whether translation of these altORFs affects the innate immune response and offers any benefit to the host in clearing virus-infected cells.

This highlights a potential role of ribosome rescue factors during RNase L activation in maintaining a pool of free ribosomes. Such a model could account for the observation that JNK is critical to RNase-L mediated apoptosis 92 , a process thought to be beneficial for eliminating cells infected by viruses. The peptide products that are produced by altORF translation during RNase L activation could also have functional roles.

It is therefore conceivable that peptides generated from translation of RNase L cleavage fragments could also be presented on HLA-I and benefit the host, acting as additional danger signals. However, given the short time frame of RNase L activation, detection of cryptic peptides in RNase L activated cells has proven to be difficult data not shown. Thus, methodology development is needed to assess whether altORF-derived peptides are stable and functional.

In addition, it is possible that translation of the mRNA decay intermediates generated by RNase L could serve other roles. For example, they could sequester ribosomes and thereby prevent translation of viral transcripts. On the other hand, translation of fragments could be detrimental to the host since ribosomes melt mRNA secondary structure 97 and this structure in the fragments was suggested to be important for activating other dsRNA sensors that trigger interferon production and assembly of stress granules 4 , 31 , This suggests that altORF translation is triggered by at least some viral infections.

However, since many viruses are known to suppress activation of RNase L, the potential role of altORF translation in the clearance of the viruses is likely to vary. Therefore, more targeted studies are needed to investigate the role of RNase L in altORF translation in the context of viruses. Nevertheless, our findings reveal that widespread translation outside of coding sequences is a component of the innate immune response and add to the growing body of examples of alternative translation events.

We believe further study of this phenomenon has the potential to identify new mechanistic targets for therapeutic intervention. We are thankful to Dr Bret Hassel for providing 2—5A for initial experiments. National Center for Biotechnology Information , U. Journal List Nucleic Acids Res v. Nucleic Acids Res. Published online Feb 9. Author information Article notes Copyright and License information Disclaimer.

To whom correspondence should be addressed. This article has been cited by other articles in PMC. Abstract Ribonuclease L RNase L is activated as part of the innate immune response and plays an important role in the clearance of viral infections. Graphical Abstract. Open in a separate window. Synthesis and purification of 2—5A 2—5A was synthesized enzymatically in vitro by recombinant human OAS1.

Computational analysis Read processing The fastq files were de-barcoded by the core facility and footprints of 25—34 nucleotides were sorted by internal 5 nucleotide sample barcode and trimmed of linkers by Cutadapt Metagene analysis We created average ribosome footprint density metagene plots by using Plastid's metagene function.

Differential translation analysis To determine changes in ribosome footprint distribution in RNase L activated cells we analyzed two complete and independent Ribo-seq datasets replicates 1 and 2. Reduced transcriptome analysis For more detailed analysis, we implemented a more stringent transcriptome-only analysis approach with a single transcript isoform per gene to enable higher precision mapping and eliminate spurious reads. Figure 3. Figure 7. Quantification and statistical information.

Figure 6. Figure 1. Figure 2. Figure 4. Figure 5. Catalytic activity of RNase L is required for alternative translation events Having established that activation of RNase L increases the relative translation of altORFs, we further investigated the mechanism of this phenomenon by testing whether the catalytic activity of RNase L was required.

Conflict of interest statement. None declared. Yoneyama M. Kang D. Expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda a novel type I interferon-responsive apoptosis-inducing gene. Hartmann G. Nucleic acid immunity. Malathi K. Jr, Silverman R. Han Y. Floyd-Smith G. Interferon action. Zhou A. Interferon Cytokine Res.

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Impact of RNase L overexpression on viral and cellular growth and death. Hassel B. A dominant negative mutant of 2—5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. Banerjee S. RNase L is a negative regulator of cell migration. Ireland D.

RNase L mediated protection from virus induced demyelination. PLoS Pathog. Yakub I. Mashimo T. Lucas M. Cell Biol. An essential role for the antiviral endoribonuclease, RNase-L, in antibacterial immunity. Long T. RNase-L deficiency exacerbates experimental colitis and colitis-associated cancer. Bowel Dis. Carpten J. Germline mutations in the ribonuclease L gene in families showing linkage with HPC1.

Casey G. Wang L. Lee T. Regulation of human RNase-L by the miR family reveals a novel oncogenic role in chronic myelogenous leukemia. Rath S. Donovan J. Burke J. Andersen J. RNA biology. Manivannan P. RNase L amplifies interferon signaling by inducing protein kinase R-mediated antiviral stress granules.

Bisbal C. Cloning and characterization of a RNAse L inhibitor. A new component of the interferon-regulated 2—5A pathway. Khoshnevis S. The iron-sulphur protein RNase L inhibitor functions in translation termination. EMBO Rep. Pisarev A. The role of ABCE1 in eukaryotic posttermination ribosomal recycling.

Young D. Le Roy F. A newly discovered function for RNase L in regulating translation termination. Mol Biol. Studier F. Protein production by auto-induction in high density shaking cultures. Protein Expr. McGlincy N. Transcriptome-wide measurement of translation by ribosome profiling.