Similarly to how chromatin remodeling plays an important role in transcriptional regulation, messenger ribonucleoprotein (mRNP) remodeling is an important mechanism to regulate mRNA function and stability. In eukaryotes, actively translating mRNAs in the cytoplasm are associated with a 5'cap, the cap binding translation initiation complex eIF4F, and a polyA tail. mRNPs that accumulate in complex with decapping factors are known to assemble into cytoplasmic mRNP granules named processing bodies (PBs). PBs contain mRNA decay factors and lack many components of the eIF4F complex as well as PABP. Several PB factors have been shown to be members of a multisubunit complex that acts to enhance mRNA decapping. However, which of these factors are important for decapping and the mechanism by which they stimulate decapping is unknown. Lsm4, a PB factor and a subunit of the Lsm1-7 complex, contains a C- terminal RGG domain of unknown function. Recent work has shown that several proteins with an RGG domain can act to inhibit translation in yeast. The goal of this proposal is to understand how the human decapping machinery remodels and disassembles the eIF4F cap-binding complex to gain access to the mRNA cap. Furthermore this proposal aims to identify the function of the C-terminal RGG domain of the Lsm4 subunit of the human Lsm1-7 complex with the primary hypothesis that it functions in repression of translation initiation via interaction with eIF4G. A secondary goal is t develop the technical and professional skills of the applicant so that he may become an independent, critical thinking research scientist.
The first aim i s to determine which cofactors interact with Lsm4 in an RGG domain dependent manner, and the effect of the RGG domain on translational repression, RNA stability, and PB formation. This will be done by using existing cell lines that stably express WT Lsm4 or RGG domain mutants to perform various biochemical assays including co-immunoprecipitations (co-IPs) followed by Western blotting and mass spectrometry (MS) to identify interacting partners and pulse-chase experiments combined with Northern blotting and polysome profiling to determine mRNA stability and translational activity.
The second aim i s to determine the role of post-translational modifications (PTMs) in mRNP remodeling. Studies have shown that the arginines of the RGG domain undergo dimethylation. The factor responsible for methylating Lsm4 will be identified by depleting cells of factors known to perform dimethylations and observing the effects on Lsm4 PTMs. The effect this has on RGG domain function as seen in Aim 1 will then be tested. MS experiments have identified that the RGG domain interacts with the cytoplasmic acetyl transferase comlex, Hat1-Rbbp7. This points to the role PTMs may have in mRNP regulation. The targets of the Hat1-Rbbp7 complex will be identified and the role of their acetylation in the mRNP decay process will be studied through mutational analysis and Hat1-Rbbp7 depletion.
The proposed work is basic research designed to identify the factors involved in mRNP remodeling as part of translation inhibition and mRNA decay. The misregulation of mRNA decay is implicated in a wide range of human diseases, yet the mechanism of mRNA decay remains poorly understood. A better understanding of mRNP remodeling and its importance in mRNA decay will give us further insight into these diseases, and potentially novel ways to provide treatment.