The MEX3 protein family of RNA binding E3 ubiquitin ligases - a switch for controlling rapid mRNA decay
Messenger RNA (mRNA) decay is an important mechanism to control gene expression. It ensures a dynamic and rapid translatome remodeling response upon developmental and environmental cues. The canonical mRNA decay pathway starts with the removal of the 3´ poly(A) tail by deadenylases, such as the CCR4-NOT complex, which acts in both, bulk and targeted mRNA decay. In the latter, adaptor proteins recognize a specific sequence or feature in the target mRNA and recruit CCR4-NOT. A subgroup of these RNA binding proteins (RBP) contains in addition to their RNA binding functionality an E3 ubiquitin ligase domain (RBE3L). Their unique architecture allows them to bridge RNA and protein metabolism and to act as a molecular switch upon certain stimuli. An example of an RBE3L is the muscle excess 3 (MEX3) protein family (MEX3 A-D). Best characterized is MEX3C, which acts as a molecular switch in the immune system, foremost in natural killer (NK) cells. Resting NK cells are kept inactive by the intracellular interaction of major histocompatibility complex I (MHC-I) with inhibitory receptors on the NK cell surface. Upon inflammation, surface MHC-I levels need to be reduced in a rapid manner to switch NK cells to an activated state. MEX3C enables that switch by a novel mechanism: It specifically recognizes the MHC-I mRNA and recruits and ubiquitinates the CNOT7/8 deadenylase of the CCR4-NOT complex. This results in a rapid decay of MHC-I mRNA and NK cell activation.
A similar mechanism has been proposed for MEX3A and MEX3B in the evasion of the immune system by cancer cells. The molecular details of how MEX3 proteins recruit CCR4-NOT and how CNOT7/8 ubiquitination stimulates mRNA decay are elusive to date. Both CCR4-NOT and MEX3 interact functionally with the translation machinery, raising the question if mRNA decay occurs co-translationally. To answer the outstanding questions, we will recombinantly express human CCR4-NOT complex, MEX3 proteins and variants thereof and perform interaction studies to decipher their interaction network. Recombinant proteins will be also used for functional in vitro studies, such as deadenylation-, ubiquitination- and translation assays and combinations thereof. In addition, we will employ an integrative structural biology approach (cryoEM, macromolecular crystallography, crosslinking mass-spectrometry, NMR) to obtain structural information at the atomic level. We will use in cellulo studies, to test results from the in vitro assays and to identify novel interaction and ubiquitination targets of MEX3 proteins. Together, our goal is to understand the molecular details of the ubiquitin-meditated on switch in mRNA decay by the MEX3 protein family, with a focus on MEX3C in MHC-I mRNA decay.