Protein-nucleic acid interactions play key roles in both transcriptional and translational regulation, not only in the direct interaction of proteins with DA and mRNA to regulate these processes, but also the interactions among proteins, lncRNAs, and DNAs. Understanding these interactions requires knowledge of the molecules that are involved. lncRNAs interact with proteins to achieve several consequences in gene regulation. First, lncRNAs can act as a molecular sink for proteins that may interact with DNA or RNA. Their interaction with the lncRNA thus diverts the protein from binding to its primary target. lncRNAs can also act as guides for proteins, leading them to their DNA targets to either repress or activate transcription. Finally, lncRNAs can act as platforms upon which molecules can congregate to perform a function, such as histone modification, as a team at a specific location and time. Each of these functions requires the interaction of lncRNAs with a diverse set of proteins. Knowledge of the proteins bound to a specific lncRNA will allow for determination of the mechanisms by which that lncRNA affects gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of proteins. In this proposal we focus on the particular problem of identifying the proteins that are bound to specific lncRNA molecules, their relationship to the progression of prostate cancer and their potential as markers of the disease and its progression. We describe an RNA-centric approach to discovery of the lncRNA-binding proteins that is innovative compared with existing technologies in two major ways. First, crosslinking of proteins to lncRNAs is done in vivo under normal cellular conditions. Therefore, proteins and lncRNAs will be folded normally, will be present at their normal cellular concentrations, and will be in their normal cellular locations. Second, sequence-specific capture of the lncRNA is used to extract the RNA of interest along with its crosslinked proteins. This is a universal capture strategy that does not rely on the availability of any other capture reagent (e.g. antibody or aptamer). Following enrichment by sequence-specific capture, mass spectrometry will be utilized to identify and quantify the associated proteins.

Public Health Relevance

The aim of this proposal is to provide scientists with tools to comprehensively determine the proteins associated with any individual lncRNA molecule. This information will enable better understanding of the effects of lncRNAs on the development and progression of prostate cancer, as well as identifying a new class of potential disease biomarkers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA193481-01
Application #
8857740
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Knowlton, John R
Project Start
2015-03-04
Project End
2018-02-28
Budget Start
2015-03-04
Budget End
2016-02-29
Support Year
1
Fiscal Year
2015
Total Cost
$338,724
Indirect Cost
$109,974
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Rolfs, Zach; Solntsev, Stefan K; Shortreed, Michael R et al. (2018) Global Identification of Post-Translationally Spliced Peptides with Neo-Fusion. J Proteome Res :
Damodaran, Shivashankar; Kyriakopoulos, Christos E; Jarrard, David F (2017) Newly Diagnosed Metastatic Prostate Cancer: Has the Paradigm Changed? Urol Clin North Am 44:611-621
Sheynkman, Gloria M; Shortreed, Michael R; Cesnik, Anthony J et al. (2016) Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation. Annu Rev Anal Chem (Palo Alto Calif) 9:521-45