UC Berkeley

mRNA translation regulation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor that is involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5′ untranslated region (5′ UTR) and with the 5′ -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5′ UTR has a longer than average length, a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Chapter 2 describes our findings on the contributions of multiple 5’ UTR and start codon features in JUN translation regulation. We find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5′ UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Work presented in this chapter demonstrates additional layers of regulation for JUN translation. Chapter 3 describes isolation of a translation initiation multifactor complex (MFC) from in vitro translation reactions of a JUN 5’UTR reporter mRNA in human cell extracts. The yeast MFC is composed of eIF1, eIF2, eIF3, eIF5 and the initiator methionyl-tRNA (Met-tRNAi) and has been well-characterized. However, knowledge about the human MFC is limited and isolation of the endogenous complex from human cells hasn’t been achieved. By using the JUN mRNA as a platform for MFC binding, we present the first instance of isolation of an mRNA-bound human MFC. We also present in-depth protocol optimization and propose next steps for complex validation. Work presented in this chapter provides strong evidence for the formation of a human MFC with the presence of novel initiation factors such as eIF4A and eIF4G. Together these findings demonstrate the complexity of JUN translation regulation and establish JUN’s potential as a model transcript for understanding multiple interacting modes of translation regulation.