Evaluating posttranscriptional regulation of cytokine genes.
Evaluating posttranscriptional regulation of cytokine genes.
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评估细胞因子基因的转录后调控。
DOI：
10.1007/978-1-61779-439-1_5
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Bohjanen,PaulR
中科院分区：
文献类型：
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作者：
Rattenbacher,Bernd;Bohjanen,PaulR
多种细胞因子对于多细胞生物体中的细胞间通讯是必需的，并且细胞因子失调具有有害作用，导致疾病状态。因此，必须严格控制细胞因子的表达。细胞因子基因表达的调节发生在不同的水平，包括转录和转录后水平。最终，细胞因子转录物的稳态水平由该mRNA的转录和降解的平衡决定。细胞因子mRNA的降解速率可以通过用放线菌素D阻断转录，在不同时间点收集RNA，并通过北方印迹评估mRNA水平随时间的变化来测量。在这些转录物的3 '非翻译区（UTR）中发现了介导许多细胞因子转录物快速降解的顺式作用元件，包括富含AU的元件（战神）。顺式调控元件（ARE）通过与反式作用蛋白（如tristetraprolin或HuR）结合来调控细胞因子mRNA的降解。基于这些RNA结合蛋白与放射性标记的RNA序列的结合，可以使用电迁移率变动测定或紫外交联测定来可视化这些RNA结合蛋白。调节细胞因子mRNA衰变的RNA结合蛋白可以使用RNA亲和方法纯化，使用其靶RNA序列作为诱饵。在本章中，我们回顾了测量细胞因子mRNA衰减的方法和表征调节细胞因子mRNA衰减的顺式作用元件和反式作用因子的方法。
A wide variety of cytokines are necessary for cell–cell communication in multicellular organisms, and cytokine dysregulation has detrimental effects, leading to disease states. Thus, it is a necessity that the expression of cytokines is tightly controlled. Regulation of cytokine gene expression takes place at different levels, including transcriptional and posttranscriptional levels. Ultimately, the steady-state levels of cytokine transcripts are determined by the equilibrium of transcription and degradation of this mRNA. Degradation rates of cytokine mRNAs can be measured in cells by blocking transcription with actinomycin D, harvesting RNA after different time points, and evaluating mRNA levels over time by northern blot.Cis-acting elements that mediate the rapid decay of numerous cytokine transcripts, including AU-rich elements (AREs), are found in the 3′ untranslated region (UTR) of these transcripts. Putative regulatorycis-elements can be cloned into the 3′ UTR of a reporter transcript in order to assess their function in regulating mRNA decay.Cis-elements, such as AREs, regulate cytokine mRNA decay by binding totrans-acting proteins, such as tristetraprolin or HuR. These RNA-binding proteins can be visualized using electromobility shift assays or UV crosslinking assays based on their binding to radioactively labeled RNA sequences. RNA-binding proteins that regulate cytokine mRNA decay can be purified using an RNA affinity method, using their target RNA sequence as the bait. In this chapter, we review the methods for measuring cytokine mRNA decay and methods for characterizing thecis-acting elements andtrans-acting factors that regulate cytokine mRNA decay.