Biological roles of DExH RNA helicase, RHAU

In this thesis, I have described the work carried out on a single protein called
RHAU, dealing with aspects of protein localization in cells, the regulation of
global gene expression by different mechanisms, and cellular stress-responses.
RHAU, RNA helicase associated with AU-rich element, was originally identified
from the results of RNA affinity chromatography using the AU-rich element of
uPA messenger RNA. RHAU was characterized as a factor accelerating AU-rich
element-mediated mRNA degradation.
The aim of this present study was to investigate possible role(s) of RHAU in
mammalian cells. In the first part of the study, dealing with the cellular
localization of RHAU using biochemical fractionation and microscopic analysis, I
found that RHAU is predominantly localized in the nucleus, despite the fact that
mRNA degradation occurs in the cytoplasm. In HeLa cells, RHAU is localized
throughout the nucleoplasm with some concentration in nuclear speckles in a
manner dependent on ATPase activity. Furthermore, it has been shown that
transcriptional arrest changes RHAU localization to nucleolar caps, where it is
co-localized with other RNA helicases, p68 and p72. This suggests that RHAU is
involved in transcription-related RNA metabolism in the nucleus.
The discovery that RHAU is localized mainly in the nucleus prompted me to
consider the nuclear functions of RHAU, which led to a second project using
RHAU-knockdown. To see whether RHAU affects global gene expression either
transcriptionally or posttranscriptionally, microarray analysis using total RNA
prepared from RHAU-depleted HeLa cell lines was performed to measure both
the steady-state mRNA level and mRNA half-life by actinomycinD-chase. Most
transcripts whose steady-state levels were affected by RHAU knockdown showed
no change in half-life, suggesting that these transcripts were the subject of
transcriptional regulation.
In cells depleted of RHAU using shRNA, retardation of growth was observed,
especially when cells were stressed, for example, by serum-starvation. RHAU
indeed affected more genes in starved conditions, suggesting the involvement of
RHAU in cellular stress responses in mammalian cells.
Overall, the results suggest that each RNA helicase is involved in various cellular
processes. RHAU has dual functions, being involved in both the synthesis and
degradation of mRNA in different subcellular compartments. Thus, my work
presents a novel view of RNA helicases as proteins with multiple functions in
different cellular contexts.