Taken together, the nanoluciferase reporter experiments uncover the and mRNA 3-UTRs are necessary and sufficient to drive a burst in translation after T cell activation. total reads mapping to the gene; T2C portion, quantity of reads with T-to-C conversions / quantity of reads; ConversionSpecificity, log (quantity of reads with T-to-C conversions / quantity of reads with other conversions); UniqueReads, reads collapsed to single copies. Also included: 5’utr/Intron/Exon/3’utr/Start_codon/Quit_codon, quantity of sites mapping to that annotation category; Junction, quantity of sites mapping to a junction between groups (coding-intron, coding-3’utr, etc.); GeneType, as explained in the gene_type category for this gene in the.gtf file used. elife-74272-supp3.xlsx (2.0M) GUID:?A308E413-F57C-4F4C-9C8C-7E227664642C Supplementary file 4: Transcriptome analysis of non-activated or Rabbit polyclonal to NOTCH1 activated Jurkat cells. Each tab lists transcript name and version, gene name, type of Talarozole transcript, length of transcript, and imply transcripts per million, calculated from two biological replicates. elife-74272-supp4.xlsx (11M) GUID:?599DC5A9-80F0-486F-A0D8-58026022F2C0 Supplementary file 5: Pathway enrichment analysis. Lists for both biological replicates of the EIF3A/C/B PAR-CLIP libraries are included (genes with 100 reads), along with associated transcript names, lengths in nts of the 5-UTR, coding Talarozole region, and 3-UTR, and reads normalized to the lengths of the transcript regions. Tabs also include the top tissue-specific pathway enrichment groups decided using the STRING Database. These list: the Gene Ontology (GO) number, GO description, observed gene count, background gene count, false discovery rate, and matching proteins in the network by Ensembl protein ID, and by gene name. elife-74272-supp5.xlsx (359K) GUID:?91E12C96-58BA-4D5E-B98F-279AA6C3B347 Supplementary file 6: Reagent information for experiments. Lists include antibodies used, PCR primers, qPCR primers, gRNA targeting sequences, and FISH probes, and DNA oligos for RNase H experiments. elife-74272-supp6.xlsx (56K) GUID:?1CA3BCA2-29FF-478B-9044-6A9F109DC73E Transparent reporting form. elife-74272-transrepform1.docx (247K) GUID:?A808F1FE-2BDD-4CFC-8C8A-378DB29380DE Source data 1: Initial western blots for main figures and figure supplements. Boxed regions are the regions shown in the figures. elife-74272-supp7.zip (31M) GUID:?9B37829A-3F7A-41FF-B07A-0D8DCA390EA9 Source data 2: Spreadsheets with numerical data used in figures and figure supplements. elife-74272-supp8.zip (589K) GUID:?9EF6B059-A512-4431-8444-63E3E2C4C7C6 Data Availability StatementSequencing data has been deposited in GEO (“type”:”entrez-geo”,”attrs”:”text”:”GSE191306″,”term_id”:”191306″GSE191306). Code used to analyze the microscopy images is available on github at https://github.com/Llamero/TCR_colocalization_analysis-macro (copy archived at https://archive.softwareheritage.org/swh:1:rev:95714d5f24259dd03aa5c184a15b9d5f1bb17b40). The following dataset was generated: De Silva D, Cate JH, Chin G. 2021. Genome-wide mapping of eIF3-RNA interactions in Jurkat cells using PAR-CLIP. NCBI Gene Expression Omnibus. GSE191306 Abstract Activation of T cells requires a quick surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific Talarozole genes remains unclear. Here, we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the and mRNA 3-untranslated regions (3-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for strong T cell activation. Use of the or 3-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) enhances the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications. and mRNAs, in activated and non-activated Jurkat cells. 5-UTR, coding sequence (CDS), and 3-UTR elements (below) along with the variable (V), joining (J) and constant (C) regions (above) for the mapped TCR genes in Jurkat cells are shown. The blue and reddish vertical lines in the plotted reads indicate the amount of T-C transitions vs other mutations, respectively for a particular nucleotide. The and mRNAs are present in both non-activated and activated Jurkat cells (Supplementary file 4). (F) FISH analysis of and mRNAs (yellow and magenta, respectively) and P body (top) marked by the location of DCP1 (cyan) and stress granules (bottom) marked by the location of G3BP1 (cyan), in activated Jurkat cells. Graphs to the right of the images indicate Pearsons correlation coefficients (PCCs) of and mRNAs localizing with each other or with P body or stress granules. TetraSpeck microsphere beads were used as a positive control for colocalization. Labels around the x axis are, B: TetraSpeck microsphere beads, C: activated Jurkat cells. (n = 5, p 0.008, for PCC values of cells relative to bead colocalization, across all the channels tested, using the Talarozole Wilcoxon rank-sum test). Images are representative of one experiment of the five impartial experiments in the graphs. Physique 1figure product 1. Open in a separate windows eIF3 PAR-CLIP experiments in activated and non-activated Jurkat cells.(A) Composition of eIF3 in.