NSF/MCB-BSF:Transfer of mRNA between cells through membrane nanotubes

Albert Einstein College Of Medicine, Bronx NY

Investigators

Abstract

Messenger RNAs (mRNAs) are molecules that convey genetic information from genes (DNA) in the cell nucleus to the cell body, where they act as the blueprints for the synthesis of proteins encoded by the genes. mRNA levels are tightly controlled by their rate of synthesis in the nucleus and degradation in the cell body. The regulation of these two processes modulates the number of mRNA molecules and, hence, protein levels within the cell. Therefore, modulating mRNA levels directly affects both cellular structure and behavior. Recently, a third process that affects mRNA levels was discovered, i.e. that cells can transfer and share mRNA molecules between themselves. However, the implications of intercellular mRNA transfer upon neighbor cells are still poorly understood. Thus, there is an immediate need for a quantitative analysis to better understand this novel process. Characterization of this process is performed using state-of-the-art microscopy and biochemical techniques. The main goals of this project are: 1) to develop tools for live imaging of this process, 2) to determine the scope of this process, 3) to decipher the molecular mechanism that performs and regulates this process and 4) to define the physiological consequences of this process. It was found that mRNA molecules transfer between cells, through structures that are called membrane nanotubes (i.e. long, thin tubes that connect cells). The quantitative analysis suggests that 1-5% of the mRNA molecules in cells actually come from "donor" cells. However, there is much that is still unknown. In this project, a live imaging system will be developed to visualize mRNA transfer for the first time in living cells. Second, the scope of this process will be determined, e.g. how many RNAs undergo transfer and what is the efficiency thereof? This will be performed by co-culturing human "donor" and murine "acceptor" cells, followed by separating them by flow cytometry. After the cells are separated, their mRNA content will be analyzed by RNA-seq techniques and the scope of human mRNAs that transferred to murine cells will be revealed. In addition, we will verify the scope of RNA transfer by fluorescence microscopy using multiplex single-molecule Fluorescent In Situ Hybridization (smFISH). Proteins involved in mRNA transfer will determined using RNA affinity purification to isolate those that bind the transferred mRNA and by assessing the effects of mutation of the suspected candidates in cells. Last, but most important, a series of biochemical, imaging and cell biology assays will be performed in order to determine if the transferred mRNAs are being translated into proteins in the acceptor cells, and affect their physiology. Deciphering the process of mRNA transfer through membrane nanotubes and its consequences will further our understanding of its role in controlling local cellular environments with respect to tissue development and maintenance along with the cellular responses to stress. We will also gain an understanding of how membrane nanotubes are formed and deliver materials, and our aim is to also develop new tools and techniques for mRNA imaging and to establish an open database of transferred RNAs. This research was funded by a joint Israeli and United State program in which the NSF provides funding for the US laboratory, located at Albert-Einstein School of Medicine in New York, while the Israeli BSF provides funds for the Israeli lab at the Weizmann Institute of Science in Rehovot.