Asymmetrical localization and specific protein binding patterns of mRNA play a key role in many cellular pro- cesses, and aberrant mRNA localization has been observed in cancer and several neurological diseases. Gain- ing a deeper understanding of mRNA localization patterns and the corresponding mechanisms of mRNA transport would provide valuable information regarding disease progression and potential therapeutic ap- proaches. However, the RNA labeling methods currently available suffer from various challenges and limitations, creating an ongoing demand for improved methods for labeling and imaging of specific mRNA sequences in living cells. Harnessing the power of molecular recognition between RNA and small molecules, we have devel- oped ribozyme sequences that are capable of self-alkylation with an electrophilic fluorescein analogue, and can be fused to an mRNA of interest and expressed in cells. These ribozymes are anticipated to serve the dual purpose of enabling fluorescence-based visualization of mRNA and providing a handle for immunoprecipitation of proteins bound to specific RNA sequences. Compared with other RNA labeling technologies, the proposed ribozyme-based approach offers the benefits of smaller fusion size, potential for use with a broad palette of small-molecule fluorophores, and reduction of background signal by removal of excess fluorophore. Additionally, the proposed ribozyme-based approach enables new applications that are not possible with other RNA labeling technologies, such as pulse-chase labeling and transcript-specific immunoprecipitation.
The specific aims of this project are to: (1) utilize the ribozymes to isolate and identify transcript-specific RNA-binding proteins; (2) fluorescently label and visualize mRNAs in living cells and monitor time-resolved mRNA dynamics; (3) utilize our RNA labeling and immunoprecipitation methods to gain insight into the mechanism and specific localization pat- terns of non-canonical ER-localized mRNAs. This research is anticipated to provide powerful tools for studying the localization and transport mechanisms of mRNA in living cells, and will put these tools to immediate use to answer biological questions regarding ER-localized RNAs. This is in turn expected to further our understanding of fundamental cellular processes and offer new insights into the mechanisms and treatment of disease.

Public Health Relevance

A large fraction of mRNAs localize to particular sites within the cell, and disruptions in these mRNA locali- zation patterns have been observed in neurological diseases and cancer. The goal of this application is to develop tools to better understand the patterns and mechanisms of RNA localization. This is anticipated to provide insight into the mechanisms of disease and illuminate potential new targets for therapeutic drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM116991-01A1S1
Application #
9431631
Study Section
Program Officer
Fabian, Miles
Project Start
2016-08-01
Project End
2021-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
1
Fiscal Year
2017
Total Cost
$7,951
Indirect Cost
$2,694
Name
University of Utah
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Ellipilli, Satheesh; Phillips, John D; Heemstra, Jennifer M (2018) Synthesis of comb-shaped DNA using a non-nucleosidic branching phosphoramidite. Org Biomol Chem 16:4659-4664