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Gene activation by IFNs in wild type and Pias1 null BMMs

ABSTRACT: Microarray analysis was designed to study the effect of PIAS1 on interferon (IFN) signaling pathway by comparing the gene activation profiles of wild type and Pias1 null cells in response to IFN treatments. Microarray analysis was performed essentially following the manufacturer’s instructions (Affymetrix). Briefly, bone marrow-derived macrophages (BMMs) from wile type or Pias1 null littermates were either untreated, or treated with IFN-beta (500 U/ml) or IFN-gamma (10 ng/ml) for 2h or 4h. Total RNA was prepared with RNA-STAT60 (TEL-TEST) and purified with the RNeasy kit (Qiagen). Double stranded complementary DNA was synthesized from 20 ug of total RNA according to Affymetrix methodology, and purified with Phase Lock Gels (Eppendorf). Biotin-labeled RNA was synthesized with the BioArray High Yield RNA Transcript Labeling Kit (Enzo). Samples were cleaned, fragmentated and hybridized to murine genome (MGU74Av2) Genechips (Affymetrix) as instructed. GeneChips were stained with phycoerythrin-streptavidin (Molecular Probes) and scanned with a GeneChip scanner (Affymetrix). Keywords: parallel sample

ORGANISM(S): Mus musculus

PROVIDER: GSE1552 | GEO | 2004/07/16

SECONDARY ACCESSION(S): PRJNA89953

REPOSITORIES: GEO

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Project description:Interferon-induced transmembrane protein (IFITM1) plays a dual role in restriction of RNA viruses and in metastatic cancer cell growth. The signal transduction events that are orchestrated by IFITM1 are not well defined. We set out to identify IFITM1 interacting proteins to begin to define its mechanism of action. Affinity purification of SBP-tagged IFITM1, coupled to SWATH-mass spectrometry, identified significantly higher confidence interacting proteins from IFN- treated cells, relative to non-treated cells. This promoted an examination of the protein synthetic machinery in order to determine whether IFITM1 can impact on ribosomal proteome from IFN- treated cells. Isogenic ifitm1 null and ifitm1-ifitm3 null-SiHa cell panels were generated using CRISPR gRNAs to define signaling events that are linked to IFN--dependent protein synthesis. Ultracentrifugation sedimentation of cell lysates from interferon gamma treated wt and ifitm1-ifitm3 null-SiHa cells was carried out to isolate ribosomal constituents. Although SWATH-mass spectrometry of the 40S, 60S, and 80S fractions demonstrated changes in selected protein content, a significant reduction in A254 (RNA) in the 80S ribosomal fractions suggested a relatively select defect in 80S ribosomal biogenesis in ifitm1-ifitm3 null cells. The localization of IFITM1 to ribosomal protein components prompted an analysis of IFN--dependent protein synthesis using pulse SILAC. STAT1 and B2M were two dominant proteins whose synthesis increased equivalently in wt or ifitm1-ifitm3 null cells. However, MHC class I molecules and ISG15 were the most highly suppressed IFN--responsive proteins in the ifitm1-ifitm3 double null cells and this was confirmed using ifitm1 single null cells. Transient depletion of IFITM1 using targeted siRNA also depleted MHC class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1 in mediating IFN--stimulated protein synthesis and associated antigen presentation during either oncogenic or anti-viral signalling.

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Gene activation by IFNs in wild type and Pias1 null BMMs

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