Many cells can rapidly switch between growth and quiescence in response to environmental signals. How they orchestrate the profound reprogramming of energy metabolism and biosynthetic processes that accompanies this transformation is not well understood. This reflects an important gap in our understanding of basic growth mechanisms that are relevant to diverse aspects of biology, ranging from normal (eg. stem cell differentiation) to pathogenic (eg. tumorigenesis). Throughout eukaryotes, the mTOR signaling pathway is a master regulator of the growth/quiescence switch, and is increasingly implicated common diseases such as cancer. Central aspects of mTOR function are mediated through control of mRNA translation. Our long-term goal is to understand how mTOR control of the translation machinery orchestrates the cellular growth status in normal and disease contexts. The goal of this proposal is to establish the cellular functions and regulatory mechanisms that define two classes of mRNA targets: mRNAs with 5' terminal sequences, including the terminal oligopyrimidine (TOP) motif, that render their translation hyper-dependent on mTOR activity; and mRNAs that utilize the non-canonical translation factor EIF4G2 to maintain mTOR-resistant translation. Our hypothesis, based on preliminary data described herein, is that both classes are controlled by mechanisms that detect specific mRNA-encoded features to coordinate the production of potentially thousands of proteins to maintain the cellular growth status. To test this hypothesis, we propose in Aim 1 to use a randomized system to quantify the translation regulatory function of all possible 5' mRNA sequences. This information will be integrated with transcriptome-wide measurements of mTOR-regulated translation and endogenous mRNA 5' sequences to establish the frequency and functional scope of mRNAs whose translation is controlled through a 5'-sequence-dependent mechanism.
In Aim 2, we will employ a novel cell-free translation assay that we developed to characterize the molecular mechanism that detects and regulates this class of mRNAs. Finally, in Aim 3, we will determine the function of EIF4G2 in mTOR-independent translation, and identify its mRNA targets and the encoded features that define them. We will accomplish these goals using a multi-disciplinary approach that combines novel applications of high-throughput sequencing, bioinformatic analysis and classical biochemistry. Our results will establish the molecular features that define a post-transcriptional program of gene expression that is at the heart of cellular growth control, and add valuable insight into the underlying mechanisms that link mTOR dysfunction to disease, including cancer, autism, and metabolic disorders.

Public Health Relevance

The mTOR signaling pathway is a conserved regulator of cell growth that is deregulated in common human diseases, including cancer, diabetes, and neurologic disorders. mTOR drives these phenotypes in part through control of mRNA translation. We are seeking to determine the molecular architecture and organizing principles of this complex program of gene expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM125955-04
Application #
10000949
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Adkins, Ronald
Project Start
2017-09-15
Project End
2022-08-31
Budget Start
2020-09-01
Budget End
2021-08-31
Support Year
4
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Philippe, Lucas; Vasseur, Jean-Jacques; Debart, Fran├žoise et al. (2018) La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region. Nucleic Acids Res 46:1457-1469
Park, Yeonwoo; Reyna-Neyra, Andrea; Philippe, Lucas et al. (2017) mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4. Cell Rep 19:1083-1090