![]() ![]() ( B) Waterfall plots showing the expression change in select transcripts identified by PAR-CLIP measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The dashed vertical lines indicate the standard deviation (SD) of the median-normalized data. The same color scheme is applied to the data points in the left panels. Deviations from the central tendency are color-coded, with warm colors closer to the central tendency and cool colors towards the tail ends of the distribution. Right panels: Histograms showing the gene expression distribution in RNA-Seq experiments. ( A) Left panels: Scatter plots showing the correlation between the number of crosslinked transcript copies (per million reads) recovered by PAR-CLIP and the expression level as measured by RNA-Seq in the HEK293Trex cell line that force-expresses epitope-tagged IRE1, and under the same ER stress conditions (5 μg/ml tunicamycin). The mean expression level for each transcript (obtained from biological duplicates) was normalized to the median expression level of the entire dataset, which consisted of all transcripts for which we recovered RNA-Seq reads (FPKM > 0) in a particular condition. The dashed line indicates where the lanes from a single gel were cropped. The bracket on the left indicates the region of the gel that was excised for purification of IRE1-RNA complexes. ( F) Autoradiogram of an SDS-PAGE gel of the eluates of denaturing IPs of epitope-tagged IRE1 after radiolabeling the 5’-end of crosslinked RNAs. Bottom: Autoradiogram of a TBE-urea PAGE gel in which the RNA fragments purified from gel cutouts obtained from the gel in the top panel were resolved. The crosslinked lysates were treated with the indicated amounts of RNase T1 for 30 min at 25☌. ( E) Top: Autoradiogram of an SDS-PAGE gel in which the 5’-ends of IRE1-crosslinked RNAs were radiolabeled with polynucleotide kinase after immunoprecipitation of epitope-tagged IRE1. ER stress was induced by exposing the cells to tunicamycin. ( D) Semi-quantitative RT-PCR showing the extent of XBP1 splicing in the HEK293Trex cell line where we force-express IRE1 in the presence or absence of chemically-induced ER stress. ( C) Immunoblot showing the relative enrichment for IRE1 in native and denaturing IP conditions. ( A) Schematic of modified PAR-CLIP protocol. ( B) Immunoblots showing the enrichment for IRE1 in subcellular fractions prepared by differential detergent extraction as performed in our PAR-CLIP experiments. ‘Unknown region’ refers to transcripts associated with coding loci but that do not have a single annotated coding sequence ( i.e., alternative splicing or alternative transcription initiation sites). The diagonal dashed line indicates a slope of 1. ( F) Breakdown of mRNA regions associated with IRE1 in PAR-CLIP experiments. The histograms above and to the side of the scatter plot illustrate the frequency of transcripts that traverse the secretory pathway. ( E) Scatter plot showing the abundance of PAR-CLIP recovered transcripts in the presence or absence of chemically induced ER stress (copies per million reads: geometric mean of copy number per transcript in biological duplicates). Cardinals indicate the total or non-coding (in parenthesis) number of transcripts in each group. ( D) Venn diagrams showing the numbers of crosslinked transcripts recovered by PAR-CLIP in the presence or absence of chemically induced ER stress in biological duplicates. ( C) Breakdown of RNA classes associated with IRE1 identified by PAR-CLIP in biological duplicates. ( B) Mutation plots showing T→C transitions are the most common mutations recovered by PAR-CLIP in biological duplicates. IRE1 RNA-protein interactome cell biology co-translational protein targeting endoplasmic reticulum human ribosome unfolded protein response. Our results suggest that the ER protein translocation and targeting machineries work together with the UPR to tune the ER's protein folding load. Moreover, IRE1 binds to purified 80S ribosomes with high affinity, indicating association with ER-bound ribosomes. Crosslink sites cluster in a discrete region of the ribosome surface spanning from the A-site to the polypeptide exit tunnel. We found that IRE1 specifically crosslinks to a subset of ER-targeted mRNAs, SRP RNA, ribosomal and transfer RNAs. To understand how the UPR sensor IRE1 maintains ER homeostasis, we identified zero-length crosslinks of RNA to IRE1 with single nucleotide precision in vivo. UPR sensors monitor the ER folding status to adjust ER folding capacity according to need. The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated by a network of signaling pathways, known as the unfolded protein response (UPR).
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