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mRNA和蛋白质谱的能力。RNA干扰（RNAi）是一种基因调控机制，通过互补的microrna将Argonaute （Ago）蛋白靶向于mrna，为真核细胞提供另一种水平的转录组和蛋白质组调控。据估计，多达60%的哺乳动物蛋白质编码基因受RNAi调控。尽管人们对RNAi途径本身的组成部分以及它实现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，以确定RNAi途径的调节因子，并发现激酶，特别是受体酪氨酸激酶，在RNAi的负调节因子中显着过量。例如，激活表皮生长因子和成纤维细胞生长因子（FGF）受体可显著抑制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的受体，可能通过类似的机制下调RNAi。fgf介导的RNAi抑制与Argonaute 2 （Ago2）蛋白中丝氨酸残基磷酸化增加相关，Ago2是rna诱导沉默复合物的核心成分。该提议的基本假设是，细胞外刺激（包括生长因子和细胞因子）激活受体，通过改变miRNA谱、破坏Ago复合物的稳定性和用新生miRNA重新编程来选择性地调节mrna的翻译，从而改变细胞蛋白质组。这可能部分是由于下游蛋白激酶的激活作用于Ago2，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（FGFRs）作为我们的模型系统，但其他受体将根据需要进行研究。我们目前的研究目标将解决：1。FGFR受体信号下调RNAi活性以下子目标将继续进行。1.1 -识别连接FGFR信号传导与Ago2抑制的信号级联1.2 -定位响应FGFR激活而被磷酸化的Ago2上的残基1.3 -识别作用于Ago2的细胞激酶1.4 -研究RNAi活性降低与Ago2磷酸化的关系2。FGFR和其他受体信号传导影响miRNA谱和Ago2复合物的负载。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确定影响RNAi通路的其他细胞配体

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