Understanding the Control of mRNA-Protein Interactions During Stress and Disease


(click here for a "non-technical" abstract version)

Eukaryotic gene expression is a fundamental cellular activity that is critical for cellular identity, function, and physiology. During gene expression, a messenger RNA (mRNA) is generated by transcription and undergoes a number of different steps, including splicing and nuclear processing, nucleocytoplasmic export and localization, translation, and decay (Figure 1). These steps result in dynamic changes to the RNA sequence, structure, and the cohort of proteins bound to the mRNA. Furthermore, these changes need to occur with the proper timing and in the correct sequence to avoid aberrant expression. Therefore elaborate regulation of mRNP dynamics is required for proper gene expression.

At virtually every step in gene expression, members of a highly conserved protein family called the DEAD-box proteins are required for facilitating mRNP transitions by acting as RNA helicases and ribonucleoprotein (RNP) remodeling enzymes. Therefore, DEAD-box helicases function as master regulators of RNA and RNA-protein interactions during gene expression (and other RNA-dependent cellular processes), exerting overarching control of RNP dynamics. However, how they control different subsets of mRNAs, particularly in response to different cellular conditions, is still largely unclear.

Figure 1: An overview of gene expression and the mRNA life cycle
Figure 2: Ded1 is a critical factor in translational control. (A) Translation initiation consists of PIC assembly, scanning and recognition of the start site, then subunit joining. Ded1 has roles in PIC assembly and scanning. Ded1 also represses translation in stress granule (SG) formation and the response to cell stress.  (B) Domain structure of Ded1 with a central helicase core and N- and C-terminal regions that mediate binding to other factors. 

The DEAD-box protein Ded1 is a critical component of protein translation (Figure 2). In steady-state conditions, it promotes multiple aspects of translation initiation; however, it also plays roles in the repression of translation in stress conditions (see below for more details). The human ortholog of Ded1, DDX3X, has been linked to several different human pathologies. These include cancer, especially medulloblastoma, the most pediatric brain cancer. Mutations in DDX3X in children can also cause DDX3 Syndrome, a cognitive developmental disorder. Furthermore, as with several RNA helicases, DDX3X has roles in the replication of several viruses, including HIV and hepatitis B and C.

Our research in the Bolger lab has the dual goals of addressing fundamental biological questions and utilizing this knowledge to advance human health. We have primarily used the budding yeast Saccharomyces cerevisiae as a model system, which allows us to a synergistic combination of genetic, biochemical, and cell biology techniques in the lab. We have also expanded into using tissue culture cells for some of our work on DDX3X mutations. Current major areas of research are described below.

Figure 3: Model for Ded1/eIF4G1 during stress (TOR inactivation). In pro-growth conditions (left), Ded1 stimulates translation initiation. During stress, Ded1 causes dissociation and degradation of the translation factor eIF4G1, repressing translation.

Mechanisms of Translation Regulation

We and others have described a novel role for Ded1 in regulating translation during cellular stresses such as lack of nutrients, extreme temperatures, or oxidative stress. Interestingly, Ded1 represses translation in stress, unlike its stimulatory role in pro-growth conditions. Our working model is that Ded1 causes dissociation of its binding partner eIF4G1 from translation complexes, leading to degradation of both eIF4G1 and Ded1 (Figure 3). Current work in this area includes understanding how this stress response mechanism occurs, what the translational targets of Ded1 and its associated factors are, and whether there are differences in this mechanism between different kinds of stresses. 

Figure 4: Network cluster map of suppressors of a ded1 mutation. Known connections between the suppressors identified in an SGA screen using the yeast deletion library were mapped via STRING, followed by k-means clustering.

Interactions Between Translation and Other Pathways during Cell Stress

Many cellular pathways and processes are involved in responding to stress conditions. In order to examine how translation regulation is connected to these other pathways, we have recently conducted multiple screens. Intriguingly, we identified stress-dependent physical interactions between Ded1 and cell cycle components via mass spectrometry, suggesting a functional link between translation and the cell cycle during stress. We also identified a large number of genes that interact with ded1 mutants via a synthetic genetic array (e.g. Figure 4). We are currently working to follow up both of these results in order to better understand the cellular stress response.

DDX3X Function in Medulloblastoma

Genome sequencing studies have found frequent mutations in DDX3X in medulloblastoma, the most common pediatric brain cancer. However, it remains largely unclear how these mutations contribute to cancer biogenesis and/or progression. We have taken a broad-based approach and have analyzed the effects of a large set of the mutants in yeast. This work has indicated that the mutations cause specific changes in translation, affecting stress responses. We are now moving to a tissue culture approach in order to examine the phenotypes in medulloblastoma cell lines

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