Project description:We used microarrays to develop gene signatures for XBP1 and IRE1 in myeloma cells to explore the role of this UPR/differentiation pathway in proteasome inhibitor resistance. RPMI-8226 and JJN3 human myeloma cells were infected with lentivirus expressing non-targeted (NT) shRNA, shIRE1 or shXBP1 and processed for RNA extraction and hybridization on Affymetrix microarrays. Cells without lentiviral infection were processed as controls for gene expression changes related to the cellular response to lentivirus.
Project description:We used microarrays to develop gene signatures for XBP1 and IRE1 in myeloma cells to explore the role of this UPR/differentiation pathway in proteasome inhibitor resistance.
Project description:We developed a bioinformatics approach called the Read-Split-Walk (RSW) pipeline, and evaluated it using two Ire1? heterozygous and two Ire1?-null samples. The 26nt non-canonical splice site in Xbp1 was detected as the top hit by our RSW pipeline in heterozygous samples but not in the negative control Ire1? knockout samples. We compared the Xbp1 results from our approach with results using the alignment program BWA, STAR, Exonerate and the Unix “grep” command. We then applied our RSW pipeline to RNA-Seq data from the SKBR3 human breast cancer cell line. RSW reported a large number of non-canonical spliced regions for 108 genes in chromosome 17, which were identified by an independent study. Identification of non-canonical spliced regions for mouse MEF samples (two Ire1? heterozygous and two Ire1?-null samples)
Project description:The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring-enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency (DKO) of IRE1 and its downstream transcription factor XBP1 in NKp46 + NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1 sufficient Ly49H + NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that cell-intrinsic IRE1/XBP1 activation is required for NK cell proliferation early upon viral infection, as part of a canonical UPR response.
Project description:The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring-enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency (DKO) of IRE1 and its downstream transcription factor XBP1 in NKp46 + NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1 sufficient Ly49H + NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that cell-intrinsic IRE1/XBP1 activation is required for NK cell proliferation early upon viral infection, as part of a canonical UPR response.
Project description:This study is to examine the role of IRE1-XBP1 in regulation the activation of hexosamine biosynthetic pathway and N-glycosylation of extracellular matrix in RSV infection
Project description:IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD), which cleaves RNA at an XBP1-like consensus sequence (CUGCAG) accompanied by a stem-loop structure. MM cells are known to exhibit an elevated level of baseline ER stress, but RIDD activity has not been well studied in this disease. To investigate novel RIDD targets of possible relevance to the survival/proliferation of MM cells we combined in vitro cleavage assay with RNA sequencing. Bioinformatic analysis revealed hundreds of putative IRE1 substrates, 32 of which were chosen for validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. We show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. Our results demonstrate for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.