R35GM139480

Mechanisms of RNA Localization and Translational Regulation on the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is the subcellular site of secretory and membrane protein synthesis and performs critical functions in secretory/membrane protein biogenesis and cellular proteostasis. In addition to its established role in secretory/membrane protein synthesis, recent studies examining the mRNA transcriptomes of cytosolic and ER-bound ribosomes reveal that cytosolic protein transcripts are broadly represented on the ER, with ribosome footprinting analyses demonstrating translation of cytosolic protein mRNAs on ER-associated ribosomes. These findings identify an unexpected mRNA transcriptome-wide function for the ER in proteome expression and reopen fundamental questions regarding the mechanisms regulating mRNA localization and translation on the ER.

Principally, where current models posit that mRNA localization to the ER is co-translational and signal sequence-dependent, the abundant presence and translation of cytosolic protein mRNAs on the ER indicates that either alternative and/or multiple pathways mediate mRNA localization to the ER. As well, and although SRP pathway function in protein translocation is well established, the question of SRP pathway function in mRNA localization to the ER remains largely unexplored. Also of significance, the recent findings that a number of translocon-associated proteins, including SEC61A, SS, TRAPA, RIBOPHORIN I, and P180, are mRNA binding proteins (RBPs) suggest previously unappreciated roles for ER resident RBPs in the biology of RNA localization and translation on the ER.

This proposal merges three primary research themes of our laboratory: (i) SRP pathway function in mRNA and ribosome localization to the ER; (ii) ER-localized translation initiation as a mechanism of localized protein synthesis, and (iii) RNA binding protein function in RNA localization and translational regulation, to address new questions regarding cellular mechanisms of mRNA and ribosome localization to the ER. Building on the past decade and a half of our research into RNA localization and translational regulation on the ER, including founding evidence identifying an mRNA transcriptome-wide role for the ER in cellular proteome expression, the proposed research will utilize mammalian tissue culture cell systems, gene editing and silencing approaches, RNA-Seq and Ribo-Seq transcriptome analyses, HITS-CLIP and PAR-CLIP studies of ER RNA binding proteins and their RNA interactomes, and biochemical analyses of the subcellular organization of the translation machinery, to obtain new insights into the cellular organization and regulation of proteome expression.

This research is expected to advance understanding into the systems and pathways governing post-transcriptional gene expression in the cell. By emphasizing in vivo analyses and native biosynthetic approaches to the study of RNA and ribosome trafficking dynamics, this research is significant in its efforts to rigorously test existing paradigms and advance understanding of cellular mechanisms of localized protein synthesis.

Duke University

THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES (NIGMS) SUPPORTS BASIC RESEARCH THAT INCREASES OUR UNDERSTANDING OF BIOLOGICAL PROCESSES AND LAYS THE FOUNDATION FOR ADVANCES IN DISEASE DIAGNOSIS, TREATMENT, AND PREVENTION. NIGMS ALSO SUPPORTS RESEARCH IN SPECIFIC CLINICAL AREAS THAT AFFECT MULTIPLE ORGAN SYSTEMS: ANESTHESIOLOGY AND PERI-OPERATIVE PAIN, CLINICAL PHARMACOLOGY ?COMMON TO MULTIPLE DRUGS AND TREATMENTS, AND INJURY, CRITICAL ILLNESS, SEPSIS, AND WOUND HEALING.? NIGMS-FUNDED SCIENTISTS INVESTIGATE HOW LIVING SYSTEMS WORK AT A RANGE OF LEVELSFROM MOLECULES AND CELLS TO TISSUES AND ORGANSIN RESEARCH ORGANISMS, HUMANS, AND POPULATIONS. ADDITIONALLY, TO ENSURE THE VITALITY AND CONTINUED PRODUCTIVITY OF THE RESEARCH ENTERPRISE, NIGMS PROVIDES LEADERSHIP IN SUPPORTING THE TRAINING OF THE NEXT GENERATION OF SCIENTISTS, ENHANCING THE DIVERSITY OF THE SCIENTIFIC WORKFORCE, AND DEVELOPING RESEARCH CAPACITY THROUGHOUT THE COUNTRY.

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

Durham, North Carolina 27705 United States

Single Zip Code

Maximizing Investigators' Research Award (R35 - Clinical Trial Optional) (PAR-19-367)

Amendment Since initial award the total obligations have increased 428% from $641,023 to $3,385,121.