Regulation of RNA interference pathways by extracellular cues

细胞，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mRNAs的翻译来改变细胞内的mRNA和蛋白质谱。RNA干扰(RNAi)是一种基因调控机制，它通过互补的microRNAs将精氨酸(AGO)蛋白靶向mRNAs，从而为真核细胞提供另一种水平的转录调控和蛋白质组调控。据估计，多达60%的哺乳动物蛋白质编码基因受RNAi调控。虽然人们对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，但对它的调控相对较少了解。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的负调节因子中明显过度表达。例如，表皮生长因子和成纤维细胞生长因子受体的激活显著抑制了RNAi的活性。初步发现，其他细胞表面受体，包括那些参与细胞程序性死亡和炎症反应的受体，可能通过类似的机制下调RNAi。成纤维细胞生长因子介导的RNAi抑制与RNA诱导沉默复合体的中心成分ArgAerte 2(Ago2)蛋白中丝氨酸残基的磷酸化增加有关。这一提议的基本假设是，包括生长因子和细胞因子在内的细胞外刺激激活受体，通过改变miRNA图谱，破坏AGO复合体的稳定，并用新生的miRNAs重新编程，选择性地调节mRNAs的翻译，从而改变细胞蛋白质组。这可能部分是通过激活作用于Ago2的下游蛋白激酶来影响其活性和/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如何：1.FGFR受体信号下调RNAi活性将追求以下子目标。1.1-确定将FGFR信号与Ago2的抑制联系起来的信号级联(S)1.2-响应FGFR激活而磷酸化的Ago2上的映射残基1.3-确定作用于Ago2的细胞激酶1.4-研究RNAi活性降低如何与Ago2磷酸化有关2.FGFR和其他受体信号影响miRNA图谱和Ago2复合体的加载将追求以下子目标。2.1确定FGFR信号如何改变miRNA图谱2.2确定FGFR信号如何影响RISC相关miRNAs 2.3确定影响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