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首页> 外文期刊>Molecular and Cellular Biology >CARHSP1 Is Required for Effective Tumor Necrosis Factor Alpha mRNA Stabilization and Localizes to Processing Bodies and Exosomes
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CARHSP1 Is Required for Effective Tumor Necrosis Factor Alpha mRNA Stabilization and Localizes to Processing Bodies and Exosomes

机译：CARHSP1是有效的肿瘤坏死因子αmRNA稳定所必需的，并定位于加工体和外泌体

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Tumor necrosis factor alpha (TNF-α) is a critical mediator of inflammation, and its production is tightly regulated, with control points operating at nearly every step of its biosynthesis. We sought to identify uncharacterized TNF-α 3′ untranslated region (3′UTR)-interacting proteins utilizing a novel screen, termed the RNA capture assay. We identified CARHSP1, a cold-shock domain-containing protein. Knockdown of CARHSP1 inhibits TNF-α protein production in lipopolysaccharide (LPS)-stimulated cells and reduces the level of TNF-α mRNA in both resting and LPS-stimulated cells. mRNA stability assays demonstrate that CARHSP1 knockdown decreases TNF-α mRNA stability from a half-life (t1/2) of 49 min to a t1/2 of 22 min in LPS-stimulated cells and from a t1/2 of 29 min to a t1/2 of 24 min in resting cells. Transfecting CARHSP1 into RAW264.7 cells results in an increase in TNF-α 3′UTR luciferase expression in resting cells and CARHSP1 knockdown LPS-stimulated cells. We examined the functional effect of inhibiting Akt, calcineurin, and protein phosphatase 2A and established that inhibition of Akt or calcineurin but not PP2A inhibits CARHSP1 function. Subcellular analysis establishes CARHSP1 as a cytoplasmic protein localizing to processing bodies and exosomes but not on translating mRNAs. We conclude CARHSP1 is a TNF-α mRNA stability enhancer required for effective TNF-α production, demonstrating the importance of both stabilization and destabilization pathways in regulating the TNF-α mRNA half-life.
机译：肿瘤坏死因子α（TNF-α）是炎症的关键介质，其产生受到严格调节，控制点几乎在其生物合成的每个步骤都起作用。我们试图利用一种称为RNA捕获测定法的新型筛选方法来鉴定未表征的TNF-α3'非翻译区（3'UTR）相互作用蛋白。我们确定了CARHSP1，一种含有冷休克结构域的蛋白质。抑制CARHSP1可以抑制脂多糖（LPS）刺激的细胞中TNF-α蛋白的产生，并降低静息和LPS刺激的细胞中TNF-αmRNA的水平。 mRNA稳定性测定表明，敲低CARHSP1可使TNF-αmRNA的稳定性从49分钟的半衰期（ t 1/2 ）降至 t 22的> 1/2 ，从29分钟的 t 1/2 到 t < / em> 1/2 在静息细胞中持续24分钟。将CARHSP1转染到RAW264.7细胞中会导致静息细胞和CARHSP1抑制LPS刺激的细胞中TNF-α3'UTR荧光素酶表达的增加。我们检查了抑制Akt，钙调磷酸酶和蛋白磷酸酶2A的功能作用，并确定抑制Akt或钙调磷酸酶而不抑制PP2A抑制CARHSP1功能。亚细胞分析将CARHSP1建立为一种定位于加工体和外泌体的细胞质蛋白，而不是翻译的mRNA。我们得出结论，CARHSP1是有效产生TNF-α所需的TNF-αmRNA稳定性增强剂，表明稳定和去稳定途径在调节TNF-αmRNA半衰期中都非常重要。

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