NF-kappa beta in mRNA Stability and Cell Differentiation

描述（由申请人提供）：诱导转录因子NF-kappaB是一种具有良好特征的正调控因子，可调节编码细胞因子、细胞因子受体、抗凋亡蛋白和细胞周期调节蛋白的基因表达。令人惊讶的是，我们发现（gutridge et al., Science 289, 2363-2366） NF-kappaB表达导致编码MyoD的mRNA急剧和快速丢失，MyoD是通过明显的转录后控制机制调节肌肉形成的所谓“总开关”。因此，我们能够将MyoD的大约400个核苷酸区域转移到GAPDH Mrna上，从而使该杂交Mrna具有NF-（）和细胞因子敏感性。在新的数据中，我们已经确定了MyoD mRNA编码区域的133个核苷酸片段是NF-kappaB调节的RNA破坏途径的特定靶点。在这个133个核苷酸的破坏片段中有两个序列5‘- ACTACAGTG-3’和另一个5- attacagcg -3'的拷贝。体外转录的MyoD mRNA在tnf处理的细胞提取物中被降解，但在tnf处理的细胞提取物中不被降解，这是由于nf - κ b活性被阻断。有趣的是，我们发现另一个参与分化的关键转录因子，即Sox9的mRNA，在细胞因子或NF-kappaB表达时被靶向丢失。Sox9对慢细胞的分化至关重要。重要的是，我们在Sox9 mRNA中发现了一个与MyoD重复高度相关的元件的3个拷贝。其中一个序列重复的突变克服了NF-kappaB依赖的损失机制。现在已经确定了其他含有重复元件副本的mrna，并提出了初步证据，表明在鳞状细胞癌中下调的一组基因在类似的RNA序列元件中富集。本提案的目标是通过nf - kappab依赖性mRNA衰变的分子表征，并通过鉴定RNA序列和潜在因子需求，充分表征这种不寻常的nf - kappab依赖性转录后基因沉默过程。此外，我们建议通过分析可能的基因敲除模型、成骨分化模型和癌细胞生长和分化模型来分析nf - kappab依赖性基因沉默机制的生理和疾病相关性。这些实验将对NF-kappaB控制的调控途径进行表征，NF-kappaB在转录后水平上起到沉默基因表达的作用，并可能通过抑制分化途径和控制重要的调控途径参与重要的生理和疾病特异性机制。

DESCRIPTION (provided by applicant): The inducible transcription factor NF-kappaB is a well-characterized positive regulator of expression of genes encoding cytokines, cytokine receptors, anti-apoptotic proteins and cell-cycle regulatory proteins. Surprisingly we found (Guttridge et al., Science 289, 2363-2366) that NF-kappaB expression leads to the dramatic and rapid loss of the mRNA encoding MyoD, the so-called "master switch" regulator of myogenesis through an apparent post-transcriptional control mechanism. Thus, we were able to transfer an approximate 400 nucleotide region of MyoD onto the GAPDH Mrna which conferred NF-(( and cytokine sensitivity on that hybrid mRNA. In new data, we have identified a 133 nucleotide fragment of the coding region of the MyoD mRNA as being a specific target of an NF-kappaB - regulated RNA destruction pathway. Within this 133 nucleotide destruction fragment are two copies of the sequence 5'- ACTACAGTG-3' and another of 5-AtTACAGcG-3'. In vitro transcribed MyoD mRNA is degraded in extracts of TNF-treated cells but not in extracts from TNF-treated cells, which are blocked for NF-kappaB activity. Interestingly, we find that the mRNA for another key transcription factor involved in differentiation, namely Sox9, is targeted for loss when cytokines or NF-kappaB is expressed. Sox9 is essential for the differentiation of chrondrocytes. Importantly, we find in the Sox9 mRNA 3 copies of an element highly related to the MyoD repeat. Mutation of one of these sequence repeats overcomes the NF-kappaB -dependent loss mechanism. Other mRNAs have now been identified which contain copies of the repeat element and preliminary evidence is presented that a gene set downregulated in squamous cell carcinoma is enriched in similar RNA sequence elements. The goals of this proposal are to fully characterize this unusual, NF-kappaB-dependent post-transcriptional gene silencing process through the molecular characterization of the NF-kappaB-dependent mRNA decay and by identifying RNA sequence and potential factor requirements. Additionally, we propose to analyze the physiological and disease relevance of the NF-kappaB-dependent gene silencing mechanism by analyzing possible gene knockout models, models for osteogenic differentiation, and models for cancer cell growth and differentiation. These experiments will characterize the regulatory pathway controlled by NF-kappaB, which functions to silence gene expression at the post-transcriptional level and which is likely to be involved in important physiological and disease specific mechanisms through suppression of differentiation pathways and control of important regulatory pathways.