Expression analysis of the rap55 homolog in the zebrafish germline

Abstract

The zebrafish is a model organism that reproduces sexually, then requires to produce specialized germ cells. Oocytes are loaded with ribonucleo-proteins and mRNA of maternal origin clustered in germ granules. During the first 10 cell cycles in early zebrafish development, transcription is absent and maternal mRNA clustered at the germ granules, is the main source for protein translation. In zebrafish some germ granules components have been described such as the protein or mRNA of the genes vasa, gcl, dead end and nanos. Recently the protein Rap55/Car1/Trailer hitch/Lsm14/Rap55 was found to be another germ granule component in organisms like Drosophila melanogaster, Caenorhabditis elegans and Xenopus laevis. This is the Scd6 homolog described in Saccharomyces cerevisiae, that is located in P-bodies (Processing bodies), therefore Rap55 is possible involved in mRNA processing. We have found three zebrafish homologous genes for Rap55 located in chromosomes 13, 23 and 25 respectively. These genes have highly conserved domains, as the Lsm domain in the amino-terminal region and the FDF domains: DFDF, FFD and TFG. They contain also RGG boxes located in glycine-rich regions in the c-termini. RGG boxes are RNA binding domains. RT-PCR analysis showed that the three homologs were expressed in all developmental stages from two cells to 5 days post-fertilization. We are currently studying the expression pattern of a GFP-fusion protein from one isoform that express in ovary, besides analyzing the effect of blocking its expression by morpholino antisense injections.