All mRNA molecules are subject to posttranscriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. In recent years, a paradigm shift has occurred in our understanding of PTGR, triggered by discoveries of regulatory RNAs and extensive RNA processing including alternative polyadenylation and splicing. These developments have led to the realization that virtually every human gene is subject to some degree of PTGR. Understanding the mechanisms of target RNA recognition, and the function of the hundreds of mRNA-binding proteins encod- ed in the human genome, is therefore an open challenge that requires the joint effort of multidisciplinary teams. The recent introduction of deep sequencing technologies has enabled the development of new methods for precisely mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites in human cells. It is only now possible to resolve interdependencies and redundancies of binding of RBPs and ribonucleoprotein particles (RNPs) to mRNA molecules and evaluate their contribution to gene regulation in the context of organismal development or normal and disease states. The broad aim of this application is to identify and characterize the interaction networ of mRNA-binding proteins at the sequence, structural and functional level, with a particular focus on transport and shuttling - crucial PTGR mechanisms that have received little attention. A driving concept is whether larger """"""""chromatin""""""""- like packaging of RNPs facilitates transport and translational regulation.
The specific aims i nclude: (1) Global identification of target RNA site for a comprehensive panel of nucleocytoplasmic transport and shuttling proteins by Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR- CLIP) involving deep sequencing of libraries for all proteins and computational analysis. (2) Integrated annotation of binding sites on transcripts across libraries, using a probabilistic graphical modeling approach to identify prevalent site configurations from heterogenenous data. PAR-CLIP data, complemented by expression as well as sequence and RNA structural features, will allow for the identification of targets for combinatorial and redundant regulation. (3) Development of experimental systems for assessment of the phenotypic outcomes of the perturbation of the transporter interaction network and elucidation of its role in normal and disease states. (4) Perform biophysical and structural studies substantiating the biochemical and computational findings. Natural, as well as designed RNA-recognition element (RRE)-representing RNA ligands, will by co-crystallized with their respective recombinant proteins placing emphasis on obtaining larger RNP structures. The proposed work will thus establish the first theoretical and experimental models that relate global maps of protein-RNA interactions to RNA transport, setting the stage for future systems-wide studies of PTGR.

Public Health Relevance

The control of gene expression at the mRNA level plays a larger role than previously anticipated. This proposal aims to resolve the joint contribution of dozens of mRNA-binding proteins shuttling between nucleus and cytoplasm, as well of proteins participating in nucleocytoplasmic transport. This approach will identify numerous important regulatory regions in mRNAs, as well as uncover mRNAs and RNA-binding proteins particularly vulnerable to mutation and deregulation in disease states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104962-03
Application #
8668118
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (51))
Program Officer
Bender, Michael T
Project Start
2012-09-30
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
3
Fiscal Year
2014
Total Cost
$876,432
Indirect Cost
$128,770
Name
Rockefeller University
Department
Genetics
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Vincent, Jessica; Adura, Carolina; Gao, Pu et al. (2017) Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice. Nat Commun 8:750
Yang, Hui; Patel, Dinshaw J (2017) Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9. Mol Cell 67:117-127.e5
Garzia, Aitor; Meyer, Cindy; Morozov, Pavel et al. (2017) Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins. Methods 118-119:24-40
Yamaji, Masashi; Jishage, Miki; Meyer, Cindy et al. (2017) DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs. Nature 543:568-572
Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui et al. (2017) Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex. Cell 171:414-426.e12
Meyer, Cindy; Garzia, Aitor; Tuschl, Thomas (2017) Simultaneous detection of the subcellular localization of RNAs and proteins in cultured cells by combined multicolor RNA-FISH and IF. Methods 118-119:101-110
Höfer, Katharina; Li, Sisi; Abele, Florian et al. (2016) Structure and function of the bacterial decapping enzyme NudC. Nat Chem Biol 12:730-4
Mobin, Mehrpouya B; Gerstberger, Stefanie; Teupser, Daniel et al. (2016) The RNA-binding protein vigilin regulates VLDL secretion through modulation of Apob mRNA translation. Nat Commun 7:12848
Murn, Jernej; Teplova, Marianna; Zarnack, Kathi et al. (2016) Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt. Nat Struct Mol Biol 23:16-23
Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R et al. (2016) PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease. Cell 167:1814-1828.e12

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