Regulation of RNA interference pathways by extracellular cues

点击翻译按钮获取中文摘要

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