The coordinate expression of genes and production of protein is controlled at multiple steps. In addition to transcription, mRNAs are regulated at the level of pre- mRNA processing, mRNA export, translation, and stability. Post-transcriptional regulation of gene expression can be mediated by RNA-binding proteins (RBPs) and by non-coding RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). These regulatory steps are thought to function by impacting the composition or state of the messenger ribonucleoprotein particle (mRNP). Understanding the cis-acting regulatory code that directs the assembly of these combinatorial mRNP complexes is the first step towards understanding this complex process and is only beginning to be investigated systematically using genome-level tools. We have developed methods to identify with high efficiency the possible targets of RBPs in human cells. We have shown in proof-of-principal experiments that we can use RNA Immunoprecipitations (IPs) followed by microarray analysis (RIP-Chip) to identify the targets of the histone stem-loop binding protein (SLBP) with high confidence. This method has been extended by using recombinant RBPs to examine potential binding sites in purified total RNA. We have termed this method recombinant, or rRIP-chip. The rRIP-Chip method allows sampling of all possible binding sites for an RBP in a pool of total RNA, and provides a simple method to distinguish direct RNA-protein interactions from interactions that occur via a protein bridge. In this application we describe experiments to develop these methods further. Specifically, the cis-acting regulatory sequences in mRNAs bound by specific RBPs will be determined by using limited nuclease digestion and tiled mRNA arrays. We also outline experiments to extend this method to the argonaute (AGO) family of proteins that play key roles in mediating the effects of miRNAs and siRNAs. RIP-Chip analysis of RNAs associated with AGO proteins will identify which miRNAs associate with AGO proteins and which mRNAs are targeted by these miRNAs. Limited nuclease digestions will be used to identify the mRNA sequences protected by miRNAs/AGO proteins and bioinformatic analyses will be used to determine specific miRNA target sequences.The misregulation of gene expression underlies most, if not all, disease states. Understanding the regulatory code that directs the expression of genes at all levels (transcription, pre-mRNA processing, translational control and RNA stability) will provide a better understanding of this basic process. ? ? ?
