Regulation of RNA interference pathways by extracellular cues

无论是原核细胞还是真核细胞，都必须能够对环境的变化做出适当的反应。这通常需要通过调节基因转录和/或mRNA的翻译来改变细胞内的mRNA和蛋白质谱的能力。RNA干扰（RNAi）是一种基因调控机制，其通过经由互补的microRNA将Argonaute（Ago）蛋白靶向至mRNA来为真核细胞提供另一水平的控制以调控转录组和蛋白质组。据估计，高达60%的哺乳动物蛋白质编码基因受到RNAi的调控。虽然对RNAi途径本身的组成部分以及它进行基因沉默的机制了解很多，但对其调控的了解相对较少。 我们已经进行了筛选以鉴定RNAi途径的调节剂，并发现激酶，特别是受体酪氨酸激酶，在RNAi的负调节剂中显著过量。例如，表皮生长因子和成纤维细胞生长因子（FGF）受体的活化显著抑制RNAi活性。初步研究结果表明，其他细胞表面受体，包括那些参与程序性细胞死亡和炎症反应下调RNAi可能通过类似的机制。FGF介导的RNAi抑制与Argonaute 2（Ago 2）蛋白中丝氨酸残基的磷酸化增加相关，Argonaute 2是RNA诱导沉默复合物的中心组分。该提议的基础假设是，通过细胞外刺激（包括生长因子和细胞因子）的受体活化通过改变miRNA谱、使Ago复合物不稳定和用新生miRNA重编程以选择性地调节mRNA的翻译来改变细胞蛋白质组。这可能部分地通过激活下游蛋白激酶而发生，所述下游蛋白激酶作用于Ago 2，影响其活性和/或与小RNA相互作用的能力。大多数研究将使用成纤维细胞生长因子受体（FGFR）作为我们的模型系统进行，但将根据需要研究其他受体。我们目前的研究目标将解决：1. FGFR受体信号转导下调RNAi活性以下子目标将被追求。1.1- 鉴定将FGFR信号传导与Ago 2的抑制联系起来的信号传导级联1.2 -对Ago 2上响应于FGFR活化而磷酸化的残基作图1.3 -鉴定作用于Ago 2的细胞激酶1.4 -研究降低的RNAi活性如何与Ago 2磷酸化相关2.FGFR和其他受体信号传导影响miRNA谱和Ago 2复合物的加载目标将被追求。2.1确定FGFR信号如何改变miRNA谱2.2确定FGFR信号如何影响RISC相关的miRNA 2.3鉴定影响RNAi途径的其他细胞配体

Cells whether they be prokaryotic or eukaryotic, must be able to respond appropriately to changes in their environments. This often entails the capacity to alter the mRNA and protein profiles within the cells by modulating gene transcription and/or translation of mRNAs. RNA interference (RNAi) is a gene regulatory mechanism that provides eukaryotic cells with yet another level of control to regulate transcriptomes and proteomes by targeting Argonaute (Ago) proteins to mRNAs via complementary microRNAs. It is estimated that up to 60% of all mammalian protein-encoding genes are regulated by RNAi. Though much is known about the components of the RNAi pathway itself, and the mechanisms by which it carries out gene-silencing, its regulation is comparatively less well understood. We have carried out screens to identify regulators of RNAi pathways and discovered that kinases, and in particular, receptor tyrosine kinases, are significantly over-represented among the negative regulators of RNAi. For example, activation of receptors for epidermal growth factor and fibroblast growth factor (FGF) significantly inhibit RNAi activity. Preliminary findings suggest that other cell surface receptors including those involved in programmed cell death and the inflammatory response downregulate RNAi potentially through a similar mechanism. FGF-mediated inhibition of RNAi was correlated with increased phosphorylation of serine residues in Argonaute 2 (Ago2) protein, a central component of the RNA-induced silencing complex. The underling hypothesis for this proposal is that receptor activation by extracellular stimuli including growth factors and cytokines alter cellular proteomes by changing the miRNA profile, destabilising Ago complexes and re-programming with nascent miRNAs to selectively regulate translation of mRNAs. This likely occurs in part by activation of downstream protein kinases that act upon Ago2 affecting its activity and/or ability to interact with small RNAs. Most of the studies will be done using fibroblast growth factor receptors (FGFRs) as our model system but other receptors will be investigated as required. Our current research objectives will address how: 1.FGFR receptor signaling downregulates RNAi activity The following sub-aims will be pursued. 1.1 -Identifying the signaling cascade(s) linking FGFR signaling to inhibition of Ago2 1.2 -Mapping residues on Ago2 that are phosphorylated in response to FGFR activation 1.3 -Identifying the cellular kinases that act on Ago2 1.4 -Investigating how decreased RNAi activity correlates to Ago2 phosphorylation 2.FGFR and other receptor signaling affects miRNA profiles and loading of Ago2 complexes The following sub-aims will be pursued. 2.1 Determine how miRNA profiles are altered by FGFR signaling 2.2 Determine how RISC-associated miRNAs are affected by FGFR signaling 2.3 Identifying other cellular ligands affecting RNAi pathway