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Proteomic analysis of T-lymphocytes, WT and after TTN gene silencing

ABSTRACT: LTQ folder: It cointains 16 folders (8 CTRL and 8 siRNA TTN; 4 biological replicates x 2 technical replicates per condition) Q EXA folder: It cointains 16 folders (8 CTRL and 8 siRNA TTN; 4 biological replicates x 2 technical replicates per condition) LCQ DECA XP folder: It cointains 2 folders corresponding to the analysis of proteins from pooled raft fractions. About LTQ and LCQ DECA XP, peptide mixtures were first separated by means of ion-exchange chromatography using seven steps of increasing ammonium chloride concentration (0, 50, 100, 150, 200, 300, and 600 mM). Each salt step was directly loaded onto the reversed phase column and separated with an acetonitrile gradient: eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile; the gradient profile was 5% B for 3 min followed by 5 to 50% B within 40 min.

INSTRUMENT(S): LTQ, LCQ Deca XP Plus, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Pierluigi Mauri Dario Di Silvestre

PROVIDER: MSV000087338 | MassIVE | Thu Apr 29 09:13:00 BST 2021

REPOSITORIES: MassIVE

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Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. HTA 2.0 microarray results of total from a primary CD4 T cell culture after treatment with siRNAs (Control, U2AF1, SRRM2, SYNCRIP, and ILF2) and 24 hours after anti-CD3/CD28 bead activation Please note that the Series supplementary 'TotalRNA_HTA2_all_siRNA_100414.xslx' file has multiple worksheets containing: 1) Differentially expressed genes in ctrl siRNA vs. U2AF1 siRNA; 2) Differntially spliced genes in ctrl siRNA vs. U2AF1 siRNA; 3) Differentially spliced exons in ctrl siRNA vs. U2AF1 siRNA; 4) Differentially expressed genes in ctrl siRNA vs. SRRM2 siRNA; 5) Differntially spliced genes in ctrl siRNA vs. SRRM2 siRNA; 6) Differentially spliced exons in ctrl siRNA vs. SRRM2 siRNA; 7) Differentially expressed genes in ctrl siRNA vs. SYNCRIP siRNA; 8) Differntially spliced genes in ctrl siRNA vs. SYNCRIP siRNA; 9) Differentially spliced exons in ctrl siRNA vs. SYNCRIP siRNA; 10) Differentially expressed genes in ctrl siRNA vs. ILF2 siRNA; 11) Differntially spliced genes in ctrl siRNA vs. ILF2 siRNA; 12) Differentially spliced exons in ctrl siRNA vs. ILF2 siRNA

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Proteomic analysis of T-lymphocytes, WT and after TTN gene silencing

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