Worm Breeder's Gazette 16(5): 37 (February 1, 2001)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

RNAi screen and functional analysis of genes required for germline development

Momoyo Hanazawa1, Makoto Mochii2, Naoto Ueno3, Yuji Kohara4, Yuichi Iino1

1 Molecular Genetics Research Laboratory, University of Tokyo, Tokyo 113-0033, Japan.
2 Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.Present address: Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo 678-1297, Japan.
3 Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
4 Genome Biology Laboratory, National Institute of Genetics, Mishima 411-8540, Japan.

We are interested in germline development in C. elegans. In order to identify genes involved in germline development efficiently, we employed the Suppression Subtractive Hybridization (SSH) technique to generate subtractive cDNA pools either enriched for or deprived of the cDNAs from germline tissues. We then performed differential hybridization using these subtracted cDNA pools as probes on the high-density cDNA grid, on which about 7,600 non-overlapping EST clones were spotted, to identify a set of genes specifically expressed in the germline. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, fifteen clones showed sterility with a variety of defects in germline development, and other 21 clones showed embryonic and larval lethality.

We focused on two of the clones that showed the sterility phenotype. RNAi using one clone, yk445a8, caused sterility with underproliferative germ cells. This clone is derived from the T05G5.10 gene, which encodes a homologue of eIF5A. eIF5A was originally isolated as a candidate translation initiation factor, but recent studies have suggested a role in nuclear export of RNA. More recent studies show that it interacts with exportin 4 and has putative RNA binding domains, which suggests a function as an export adapter. C. elegans has another eIF5A homologue, F54C9.1. RNAi with a cDNA clone for this gene caused a larval arrest phenotype. In situ hybridization and Northern analysis showed the germline-specific expression pattern of T05G5.10 and the ubiquitous expression pattern of F54C9.1. We speculate that the germline specific eIF5A may recognize a set of mRNAs essential for the proliferation of germ nuclei and helps their export to the cytoplasm.

Another clone in which we are interested is yk519f1. It corresponds to the F35G12.10 gene, which encodes a homologue of the ATP synthase b subunit. The RNAi worms had many germ nuclei arrested at the pachytene stage and spermatocyte-like nuclei were often also observed. F35G12.10 shows a strong similarity with another C. elegans gene, F02E8.1. Northern analysis of staged worms shows that F35G12.10 is expressed in later larval stages and in adults and F02E8.1 is expressed at all stages. Now we intend to determine if the germline-specific ATP synthase b subunit regulates the function of mitochondria, which might in turn control the fates of germ nuclei.