RNA polymerase II subunit D is essential for zebrafish development

Abstract

DNA-directed RNA polymerase II (pol II) is composed of ten core and two dissociable subunits. The dissociable subcomplex is a heterodimer of Rpb4/Polr2d and Rpb7/Polr2g, which are encoded by RPB4/polr2d and RPB7/polr2g genes, respectively. Functional studies of Rpb4/Polr2d in yeast have revealed that Rpb4 plays a role primarily in pol II-mediated RNA synthesis and partly in various mRNA regulations including pre-mRNA splicing, nuclear export of mRNAs and decay of mRNAs. Although Rpb4 is evolutionally highly conserved from yeast to human, it is dispensable for survival in budding yeast S. cerevisiae, whereas it was indispensable for survival in fission yeast S. pombe, slime molds and fruit fly. To elucidate whether Rpb4/Polr2d is necessary for development and survival of vertebrate animals, we generated polr2d-deficient zebrafish. The polr2d mutant embryos exhibited progressive delay of somitogenesis at the onset of 11 h postfertilization (hpf). Mutant embryos then showed increased cell death at 15 hpf, displayed hypoplasia such as small eye and cardiac edema by 48 hpf and prematurely died by 60 hpf. In accordance with these developmental defects, our RT-qPCR revealed that expression of housekeeping and zygotic genes was diminished in mutants. Collectively, we conclude that Rpb4/Polr2d is indispensable for vertebrate development. DNA-directed RNA polymerase II, which is often referred to as Pol II, is the RNA synthesis enzyme that is composed of twelve different subunits designated as Rpb1 to Rpb12. Rpb1 is the largest subunit with respect to molecular weight, while Rpb12 is the smallest. Among them, ten subunits form the catalytic core and the remaining two subunits Rpb4 and Rpb7 form dissociable external subcomplex 1,2. The Rpb4/Rpb7 heterodimer plays an important role in promoter-dependent initiation of transcription of protein-coding genes as a cofactor of Pol II 3. In addition to this function, Rpb4/Rpb7 contributes to various RNA metabolism functions including transcription-coupled DNA repair and pre-mRNA splicing in the nucleus as well as nuclear export of mRNAs, initiation of translation and decay of mRNAs in the cytoplasm 4-7. In agreement with these functions, Rpb4/Rpb7 heterodimer shuttles between the nucleus and the cytoplasm 8. The physiological role of Rpb4/Rpb7 has been investigated in several organisms especially in yeast. In Saccharomyces cerevisiae (S. cerevisiae), Rpb4 was required for growth at high and low temperatures, but a deletion of the RPB4 gene was not lethal at regular temperature 9. A transcriptome analysis revealed that Rpb4 was involved in transcription of many genes in S. cerevisiae 10. On the other hand, Rpb4 was indispensable for cell survival in Schizosaccharomyces pombe (S. pombe) at any temperature 11. Similarly, RPB4 gene was essential for survival in slime molds (Dictyostelium dicoideum) 12. The Rpb4 protein in fruit fly (Drosophila melanogaster) was expressed with a transcriptional adaptor protein Ada2a by a bi-cistronic operon. Null fly mutants for both Rpb4 and Ada2a gene...

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