The C. elegans Heterochromatin Protein 1 homologue (HPL-2) is required for the formation of a functional germline and for vulva development by acting in an Rb-related pathway.
hpl-2 arises from the alternative splicing of a single transcript that is part of an operon including the upstream gene K01G5.1, predicted to encode a RING zinc finger protein of the C3HC4 type. The AUG start codon for
hpl-2 is found 120 bp downstream from the stop codon of K01G5.1, and RT-PCR analysis confirmed the presence of an SL2 transpliced leader sequence on the
hpl-2 transcript. As there is evidence that C. elegans operons may co-regulate genes of related function, we have been studying the RING finger protein (RNG-1) in order to determine whether it is involved, directly or indirectly, in the regulation of HPL-2 activity and/or HPL-2-mediated chromatin modifications. Like HPL-2, RNG-1 corresponds to a highly conserved protein with homologues from yeast to humans. As an E3 ubiquitin ligase activity is intrinsic to the RING-finger domains, we are interested in investigating if RNG-1 is a component of the ubiquitylation pathway in C. elegans. In the light of recent findings that components of the ubiquitylation pathway are involved in gene silencing, the presence of a putative ubiquitin ligase on the same operon with HPL-2 is an attractive puzzle to decipher. To investigate for the presence of ubiquitin ligase activity, we are testing the ability or RNG-1 to assemble a polyubiquitin tail in a ubiquitylation assay. To gain an insight into the function of RNG-1, we have performed RNA interference experiments.
rng-1 (RNAi) by injection results in embryonic lethality and the few escapers show early larval arrest.
rng-1 (RNAi) by soaking and feeding yield less severe phenotypes and result in sterile worms with disorganised vulva region, abnormal oocyte development and protruding vulvas. Interestingly, these germline phenotypes resemble those obtained by
hpl-2 depletion. To study RNG-1 localisation, a RNG-1::GFP fusion protein was expressed in C. elegans. RNG-1::GFP shows a strikingly similar localisation pattern to that obtained for HPL-2 ::GFP; is ubiquitously expressed in the nucleus of both somatic and germ cells. We are now pursuing studies to determine possible alterations in the pattern of HPL-2 localisation upon depletion of RNG-1. Furthermore, we are performing a yeast two hybrid screen to identify interacting partners of RNG-1. Overall, we hope to use the data obtained in order to determine if a functional correlation between RNG-1 and HPL-2 exists.