An Approach to Reveal Novel Translation Regulatory Mechanisms During Zebrafish Embryogenesis: Probing the Function of Non-core elF3 Subunits

Abstract

Eukaryotic translation initiation factor 3 (eIF3) is a multi-heteromeric protein that plays a central role in the initiation of translation of mRNAs in all eukaryotic cells. It consists of five core subunits that are present in both unicellular budding yeast and higher eukaryotes. However, higher eukaryotic eIF3 contains additional non-core subunits that are absent in the budding yeast. Since translational control plays a major role in early embryonic development, we investigated the role of the non-core eIF3 subunit eIF3h as a regulator of embryogenesis in zebrafish. Zebrafish eIF3h is encoded by two distinct genes -- eif3ha and eif3hb, both of which are expressed during early embryogenesis and display overlapping yet distinct and highly dynamic spatial expression patterns. Loss of function phenotypes obtained using specific morpholino oligomers against each isoform correlate with their spatial expression patterns, indicating that eif3h regulates development of the brain, heart, vasculature, and lateral line. In contrast to eIF3h, loss of a core eIF3 subunit -- eIF3c, known to be required for global protein synthesis, instead of causing any specific phenotype, show pleiotropic developmental defects. These results indicate that the non-core eIF3 subunits regulate specific developmental programs during vertebrate embryogenesis.;To investigate the molecular mechanism(s) responsible for the distinct phenotypes associated with the loss of function of each isoform, a genome-wide comparative RNA sequencing analysis of polysome-associated translationally-active mRNAs was carried out. The relative gain or loss of each specific candidate mRNA in the polysome-associated fraction relative to the total RNA pool revealed ∼ 200 translationally down-regulated mRNAs in each eif3ha and eif3hb morphants. Importantly, these groups of mRNAs are enriched in a tissue-specific manner with a marked overlap with the regions where corresponding eif3h isoforms are expressed, and also correlates strikingly with the tissues/organs that are affected by the loss either eif3h isoform. The most dramatic change due to the loss of eif3ha occurred for a group of mRNAs encoding the eye lens protein crystallins -- specifically the gamma family isoforms crygm2d that were depleted at least 10 -- 50 fold specifically in the polysome-associated translating mRNAs. This observation was further confirmed by showing that synthesis of one of the protein isoforms (Crygm2d7) from the corresponding ectopically injected mRNA was markedly inhibited in eif3ha morphants. Likewise, in eif3hb morphants, a subset of polysome-depleted mRNAs is associated with the developing heart and involved in controlling cardiac muscle contraction. Collectively, our RNA-seq data supports our hypothesis that different sets of mRNAs are regulated at the level of translation by two different eif3h genes during zebrafish embryogenesis.