The heterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B families are nuclear RNA-binding proteins that have many cellular activities, including pre-mRNA processing, alternative pre-mRNA splicing, mRNA transportation and telomere maintenance. Zebrafish (Danio rerio) has been recognized as an alternative vertebrate model, and has been widely utilized in developmental studies, but characterization of the fish genome is still incomplete.
I used BLAST and Clustal alignment tools and found that zebrafish and other fish species have several vertebrate hnRNP A/Bs orthlogs, but an A2 ortholog was not identified. In order to confirm our bioinformatic data, we used a proteomic approach and identified zebrafish hnRNP A1, A3, AB and A0 by pull-down, using immobilized Telo4 (telomeric d(TTAGGG)4 repeats) on zebrafish embryo homogenates at different development stages (15 h-72 h post fertilization). Although specific protein bands in zebrafish isolates were not recognized using mammalian hnRNP antibodies by Western blotting, I was able to identify these hnRNP A/B proteins by mass spectrometry (MS) analysis.
Significantly, I did not observe hnRNP A2 confirming the bioinformatic data. However, I did find two hnRNP A1 sequences encoded by separate genes, which show high similarities to the minor human isoform A1b, and detected hnRNP A0s, which share some 2nd structural similarities with human A2. To characterize these two zebrafish hnRNP A1s, I performed Northern and Southern blotting of both hnRNP A1 mRNA and whole mount in situ hybridization on different development stage embryos. Both zebrafish A1 mRNAs were detected as unclear signals around 1.6 kb by Northern blotting whereas the stable expression of both A1 mRNAs were observed through young embryos to 3 days larvae by whole mount in situ hybridization. Especially, the expression of A1s mRNA was observed constantly during embryogenesis: highly expressed at animal pole cells at young stage (4 hpf-8 hpf) and much localized at brain and notochord (24 hpf) and central nervous system (CNS) area (48 hpf-72 hpf) although there was no clear distinction of expression pattern between both A1s. Furthermore, the detection of post-transcriptional modification sites of zebrafish hnRNP A0s, which have significant differences from other higher vertebrates and some second structural similarities with human A2, allow us to provide the possibilities of existence of functional counterparts of A2. The identification of zebrafish hnRNPs A1, A3, A0 and type AB will provide insight into their function.