Dissecting the differences between mRNA translation on the ER and in the cytoplasm in human cells

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My lab aims to understand the differences between the translation of mRNAs in the cytosol and on the ER. We plan to uncover how mRNAs are partitioned between these two pools and to determine how they are distinctly regulated. Approximately 30% of all human protein-coding genes, code for membrane and secretory proteins. Although ER-targeting of mRNA is thought to be co-translational in nature, over the past decade, my lab, along with other groups, have demonstrated that a second RNA-based targeting system also exists. In particular my lab discovered an mRNA receptor, p180, which recruits and then maintains mRNAs on the surface of the ER. To gain further insight into the sorting of mRNA to the ER and the the cytosol, we purified mRNAs from these two compartments and analyzed their protein constituents by mass spectrometry. Interestingly, we identified a large number of RNA-binding proteins associate preferentially with ER-bound mRNAs including many translation initiation factors (including the eIF3 complex). Intriguingly, we also discovered that glycolysis enzymes are associated almost exclusively with cytosolic-associated mRNAs. In this grant I outline how we will follow up on our mass spectrometry data in order to better understand how mRNAs are partitioned to the ER and cytosol. 1) Determining how mRNAs are anchored to the ER by the p180-dependent pathway. Only a subset of mRNAs utilize the p180-dependent ER-targeting pathway. However, since p180 likely recognizes mRNAs in a sequence-independent manner, additional proteins which recognize particular motifs must be involved. In addition, other mRNA receptors likely exist, although their identity remains mysterious. In this aim we outline how we will identify these putative accessory proteins and alternative receptors. In preliminary data we show that one of the proteins identified in our mass spec data is required for p180-dependent ER-anchoring of mRNAs. 2) Gaining insight into the association of glycolysis enzymes with mRNAs. In this aim we will investigate whether glycolysis enzymes are either preventing mRNAs from targeting to the ER, or regulating the translation of mRNAs that encode predominantly cytosolic proteins. We then investigate whether these enzymes couple translation regulation with the metabolic status of the cell. 3) Determining the mechanism by which translation initiation factors are enriched in the ER. In this aim we will determine whether translation initiation factors are directly tethered to the ER or are preferentially bound to mRNAs that are translated on the ER. We then will determine whether their distribution is altered by cellular stress. By pursuing this research program we will gain insight into how mRNAs on the ER are regulated distinctly from their cytosolic counterparts. Our findings will provide a deeper understanding into how secretion is regulated at the level of mRNA localization.