DAP5 is a translation initiation factor of the eIF4G family, which directs a non-canonical cap-independent mode of protein translation. It provides a mechanism to maintain translation of itself and of specific target mRNAs in several cellular contexts. In somatic cells subjected to stress conditions, when overall cap-dependent translation is compromised, it contributes to the regulation of a cell’s life-or-death decisions. In embryonic stem cells, it directs the translation of a specific set of proteins that are critical for the transition from pluripotency to differentiation.
Studying DAP5-mediated translation in cell based and cell free systems
Several novel DAP5 mRNA targets have been identified in our laboratory, including the anti-apoptotic Bcl-2 and Bcl-XL, and the M-phase kinase CDK1, translation of which suppresses cell death during mitosis. More recently, we identified the N-terminal truncated form of p53, known as Δ40p53, as a translation target of DAP5; an IRES located within the coding sequence drives translation of this alternative p53 form in response to ER stress.
Using in vitro translation assays of reporter plasmids carrying various IRES sequences, we have proven that DAP5 mediates IRES-dependent, cap-independent translation of several proposed target mRNAs, and that DAP5’s interactions with specific translation initiation factors are critical for its activity. Moreover, in vitro RNA binding assays proved that DAP5 can directly bind its target mRNA.
DAP5 and hESC Differentiation
A major direction in our lab is to understand how DAP5’s function as a cap-independent translation factor regulates human embryonic stem cell (hESC) differentiation. DAP5 KD hESCs fail to differentiate towards various cell lineages, and form disorganized embryoid bodies (EBs) that continue to express pluripotency genes.
We are using a two-pronged approach involving RNA-seq of (1) actively translated polysomal mRNAs, comparing DAP5 KD and WT hESCs, and (2) mRNAs that co-IP with DAP5 in hESCs, to identify direct DAP5 target mRNAs. So far we have identified:
- The chromatin modifier HMGN3 as an IRES-dependent target of DAP5 whose function is necessary for hESC differentiation.
- Oxidative Phosphorylation and mitochondrial transport proteins. Mitochondrial function, which is necessary for differentiation, is defective in DAP5 KD hESCS.
- Ribosomal proteins, suggesting a hierarchy in which DAP5 directly drives cap-independent translation of ribosomal proteins, to prepare the cells for the strong increase in global protein translation upon the onset of differentiation.
IRES-dependent translation of various DAP5 target mRNAs leads to different cell fate decisions, during mitosis, ER stress and hESC differentitation.