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. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
1R01HG004499-01
Application #
7347325
Study Section
Special Emphasis Panel (ZRG1-GGG-J (10))
Program Officer
Good, Peter J
Project Start
2008-05-01
Project End
2011-04-30
Budget Start
2008-05-01
Budget End
2009-04-30
Support Year
1
Fiscal Year
2008
Total Cost
$360,000
Indirect Cost
Name
Dartmouth College
Department
Genetics
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
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Amacher, Jeanine F; Cushing, Patrick R; Brooks 3rd, Lionel et al. (2014) Stereochemical preferences modulate affinity and selectivity among five PDZ domains that bind CFTR: comparative structural and sequence analyses. Structure 22:82-93
Grant, Gavin D; Brooks 3rd, Lionel; Zhang, Xiaoyang et al. (2013) Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors. Mol Biol Cell 24:3634-50
Cheng, Chao; Ung, Matthew; Grant, Gavin D et al. (2013) Transcription factor binding profiles reveal cyclic expression of human protein-coding genes and non-coding RNAs. PLoS Comput Biol 9:e1003132
Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor et al. (2012) Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control. Mol Biol Cell 23:3079-93