Abstract:
All living systems rely on appropriate protein synthesis and therefore evolved intricate mechanisms to control their proteome. Especially, the complexity of metazoan multicellular processes requires a strict and multilayered regulation of gene expression at the level of transcription, mRNA processing, translation, mRNA stability, and protein modifications and stability. Here, I present work on two research projects that uncovered the hitherto unidentified roles of translation initiation (1) and mRNA decay factors (2) specialized in controlling unconventional mRNA translation and co-translational mRNA stability, respectively. The initiation step of translation commences with the assembly of the eukaryotic initiation factor 4F (eIF4F) complex on the mRNA 5 ́ cap structure which facilitates the recruitment of the ribosome to mediate protein synthesis. The eIF4F consists of the cap-binding protein eIF4E, the RNA helicase eIF4A and the scaffolding factor eIF4G. Apart from eIF4G, metazoan cells express other eIF4G-like proteins: eIF4G2 - a.k.a. DAP5/p97/NAT1 - and eIF4G3. Although the different homologs perform non-redundant functions in translation, we lack detailed understanding of their individual contribution to the initiation of protein synthesis. To gain insights into the function of DAP5 in translation initiation, I applied transcriptome (RNA- sequencing) and translatome (ribosome profiling) approaches to CRISPR-Cas9 engineered human HEK293T DAP5-null cells. These approaches allowed the identification of a group of mRNAs with complex 5 ́ untranslated regions (UTRs) that required the DAP5-eIF4A complex for efficient translation. In detail, the 5 ́ UTRs of DAP5 targets harbor strong secondary structures and at least one upstream open reading frame (uORF) that normally sequesters ribosomes from initiation at the main ORF (mORF). In this context, DAP5 is critical for mORF translation as it mediates re-initiation after uORF translation. Taken together, my studies on DAP5 demonstrate how cells control translation initiation using 5 ́ UTR-associated mechanisms. Regulation of translation initiation can also involve additional cap-binding proteins. The eIF4E-homologous protein (4EHP) competes with eIF4E to bind the mRNA 5 ́ cap structure. Unlike eIF4E, 4EHP does not promote translation since it is unable to recruit eIF4G and the ribosome. Instead, 4EHP represses translation and induces mRNA decay with the help of 4EHP-binding factors. The GIGYF1 and GIGYF2 proteins specifically associate with 4EHP and act as scaffolding proteins for GYF domain-associated proteins like tristetraprolin (TTP) or ZNF598, the RNA helicase and decapping activator DDX6, and NOT1 - a subunit of the CCR4-NOT deadenylase complex. When recruited to an mRNA, the 4EHP-GIGYF1/2 6 complexes thus elicit translation repression and mRNA decay. Although the molecular mechanisms have been studied in great detail, we only have sparse information on the pool of mRNAs subjected to 4EHP-GIGYF1/2 mediated repression and decay. Analysis of RNA- sequencing and ribosome profiling datasets of 4EHP- and GIGYF1/2-null cells revealed that the 4EHP-GIGYF1/2 complexes co-translationally target mRNAs with perturbed ribosome elongation or specific nascent peptide chains for decay. These include, among others, mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, a- and b-tubulin subunits and mRNAs with CAG codon repeats encoding poly-glutamine (Q) stretches. In detail, the 4EHP- GIGYF1/2 complexes are recruited to target mRNAs via the GYF domain-associated proteins and upon binding to the mRNA 5 ́ cap, and DDX6, degrade the bound mRNA. My studies show for the first time how a repressive complex, associated with neurological disorders in animal models and affected humans, specifically minimizes the protein output of a subset of mRNAs. Altogether, I identified a previously unappreciated role of the 4EHP-GIGYF1/2 complexes in maintaining protein homeostasis and preventing excessive production of potentially toxic proteins. In summary, my studies increase our understanding of how metazoan cells utilize the translation control toolbox to fine-tune their proteome and react to developmental cues, adapt to environmental changes and maintain homeostasis. The identified translational programs are fundamental for establishing a multicellular organization but at the same time allow the organism to evade cancer and neurological diseases.
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