The availability of the human genome sequence has shown that there are many genes
of completely unknown function; one of these is C20ORF149, or CLWD. We
initially studied this gene because of its proximity to the deletion in wasted mice, but
have found that whilst it is not involved in the phenotype of these animals it is
interesting in its own right.
The aim of my PhD is to investigate the function(s) of CLWD by characterising its
location in cells and in tissues, as well as the phenotype of cells where CLWD is
knocked down using siRNA. Results from indirect immunofluoresence suggest that
CLWD may be a new component in the mitotic spindle. CLWD localises to
centrosomes in prometaphase and moves to mitotic spindles in metaphase. Results
from immunohistochemistry show that CLWD is expressed in a wide range of tissues
with strong expression in the epithelial areas of tissues. In addition, CLWD is
located on human chromosome 20ql3.3 and this region is known to be amplified in
several cancers. Real-Time PCR analysis shows that CLWD is however not
overexpressed in the breast and ovarian tumours analysed. Like 10% of the genes
present in human genome, CLWD has an upstream open reading frame (uORF)
present in its 5'UTR. Using the luciferase reporter system, I showed that the uORF
in CLWD has an inhibitory effect on CLWD translation. I also described some
preliminary studies to assess the circadian rhythm regulated nature of Clwd in mouse
liver and SCN.
Results from RNAi studies suggest that CLWD has a vital role in cell survival. Cells
where CLWD is knocked down using siRNA show a decrease in cell proliferation
rate and an increase in mitotic index, suggesting CLWD is important for cell growth.
CLIFD-depleted cells also show a striking phenotype in nuclei where the nuclei are
irregular/lobular in shape (nuclear blebbing). Micronuclei, lagging chromosomes,
and multinuclear nuclei are also seen in siClwd transfected cells, suggesting CLWD
may play an important role in both mitosis and cytokinesis.
Together, I have provided evidence suggesting CLWD may have a crucial role in cell
growth and mitosis. Further studies of CLWD, including the identification of its
interacting proteins will provide more insights into its function.