Principal Investigator: Kazuya Nomura
Grant Title: Study on the roles of glycogenes involved in cell division of C. elegans
AbstractC. elegans is an ideal model organism for studying functions of glycoconjugates in development,differentiation, and morphogenesis. The inhibition of gene function via RNAi and deletion
mutagenesis is quick and easy, and abnormal phenotypes can be observed in vivo by using
time-lapse microscopy and by using various transgenic worms expressing fluorescent protein-taggedproteins. By using this model organism, we have been studying functional roles of glycogenes. We
previously reported that chondroitin synthesis, GlcCer synthesis and GPI-anchor synthesis areindispensable for oogenesis and/or early embryonic cell division.
To identify glycogenes involved in cell cycle progression, we listed human glycogene orthologs in
the worm genome by bioinformatics, and carried out RNAi of all the selected human glycogene
orthologs. Since oocyte formation and early embryonic cell division can be observed in vivo easily,
we paid special attention to the germline phenotypes and early embryonic cell division. Besides
abnormalities in oogenesis and early embryonic cell division, we also examined whether unfolded
protein response (UPR) is induced when each glycogene is knocked down with RNAi. For thispurpose, worms expressing Phst-4::gfp gene product (GFP gene linked to the promoter sequence of
hsp-4, a worm BiP ortholog) were RNAi-treated and the expression of the GFP was monitored byusing COPASTM Biosort (a laser fluorescence worm sorter).
By knocking down all the 156 glycogenes selected, we confirmed most of the previously reported
RNAi phenotypes, and found novel RNAi-phenotypes. RNAi of chondroitin synthesis resulted inabnormalities of early embryonic cell division (sqv-5 and mig-22 RNAi) while oogenesis wasseverely affected in mig-22 RNAi-treated animals. RNAi of sulfotransferases that are essential forneuronal wiring (hst-2 and hst-6) also resulted in malformation of the germline. RNAi of hst-3.1
resulted in abnormal oogenesis. The results indicate that sulfotransferases are essential for oogenesis.
RNAi of glycogenes involved in N-glycosylation were also extensively tested. RNAi of genes
involved in the early phase of LLO (lipid-linked oligosaccharides) synthesis resulted in severe
germline phenotypes in addition to UPR, while RNAi of genes involved in the later phase of LLO
synthesis resulted in no abnormalities. Inhibition of oligosaccharyltransferase genes (stt-3, dad-1,ostb-1, ribo-1 etc.) resulted in severe germline phenotypes and UPR. The results strongly indicatethat N-glycosylation (LLO synthesis and attachment of the LLO to proteins) is essential for normal
oogenesis to occur. Phenotypes observed for the 156 tested glycogenes are compiled in our newly
constructed database C. elegans GlycoGene DataBase (CGGDB) at AIST, Tsukuba, Japan. In
addition to the results of our RNAi experiments, information on each C. elegans glycogene including
DNA sequence, amino acid sequence, human orthologs, BLAST results, links to WormBase and insitu database, phenotypes observed (RNAi and gene variant), and references are concisely compiled.
We hope our database is useful for the researchers who want to begin their analysis of any C. elegans
glycogenes of their interest.