Program of the 2001 International Worm Meeting - Sternberg Lab ...

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555. CHARACTERIZATION OF
RNF-5, A RING FINGER PROTEIN
WITH E3 LIGASE ACTIVITY WHICH
CONTRIBUTES TO CELL
PROLIFERATION AND
DEVELOPMENT IN C. ELEGANS.
Limor Broday 1 , Thomas
Buschmann 1 , Stuart Milstein 2 , Lila
Pirkkaka 1 , Michael Hengartner 2 ,
Ze’ev Ronai 1
1 Ruttenberg Cancer Center, Mount Sinai
School of Medicine, New York NY 10029
2 Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY 11724
In searching for RING finger proteins in C.
elegans that resemble mammalian RINGs
known to exhibit E3 ligase activities, we
characterized rnf-5 (C16C10.7), the nematode
homolog of RNF5, an unknown protein that
shares features within the RING domain with
BRCA1, Cbl and Mdm2. Three different spliced
transcripts of rnf-5 were expressed during
development, with the highest expression in
young adults. Interestingly, L2d pre-dauer
larvae preferentially expressed an rnf-5 mRNA
that lacks the RING domain. We used RNAi to
determine the function of rnf-5 during C.
elegans development. We found that
rnf-5(RNAi)-treated worm populations showed
an increased fraction of dauer and dauer-like
larvae. Adult progeny of animals treated with
rnf-5 dsRNA had an overcrowed germ line
containing a higher density of germ cell nuclei.
We also noticed a weak egg-laying defect in
these animals. Over-expression of rnf-5 resulted
in slower growth. In vitro ubiquitination assays
revealed that RNF-5 exhibits E3 ligase activity,
as reflected by efficient self-ubiquitination upon
the addition of E1, E2 and ubiquitin. The E3
ligase activity of RNF-5 was confirmed in 293T
cells, which exhibit a high basal level of
ubiquitination under normal growth conditions.
A marked decrease in RNF-5 ubiquitination was
noted following UV-irradiation, suggesting that
stress inhibits the E3 ligase activity of the
protein. PI3K p110 or AKT efficiently inhibit
RNF-5 E3 ligase activity, suggesting that
RNF-5 might be regulated by the insulin
pathway. Taken together, our results identify a
new RING finger protein that exhibits E3 ligase
activity and is likely important to the regulation
of cell proliferation and development in C.
elegans.
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