Role of hypoxia in the regulation of the microRNA-containing RNA-induced silencing complex.
plays a major role in the pathobiology of pulmonary arterial hypertension (PAH), a
disease characterized by obstructive remodeling of the small pulmonary arteries (PAs).
Acute hypoxia results in the selective constriction of PAs and elevation of pulmonary
arterial pressure, while chronic exposure to hypoxia induces structural and functional
changes to the pulmonary vasculatu... read morere. The mechanisms underlying these pulmonary vascular
changes induced by hypoxia are not entirely understood. The finding that the levels of
various microRNAs (miRNAs) are altered during the development of PAH induced by chronic
hypoxia in rats suggests that changes in miRNA expression in response to hypoxia may
play a role in PAH development. However, the mechanisms by which hypoxia regulates
miRNAs and the miRNA pathway have not been fully elucidated. In this study, we show that
hypoxia upregulates the protein levels of Argonaute2 (Ago2), a key component of the
RNA-induced silencing complex (RISC). We found that the increase in Ago2 protein levels
under hypoxia occurs post-transcriptionally and involves the hydroxylation of Ago2 by
type I collagen prolyl 4-hydroxylase (C-P4H(I)). Hydroxylation of Ago2 is also important
for its localization to stress granules and its increased association with heat shock
protein 90, whose activity is crucial for miRNA loading into the RISC, under hypoxia. We
further demonstrate that hydroxylation of Ago2 is critical for the increase in RISC
activity and the post-transcriptional elevation of miRNA levels under hypoxia. Finally,
we present evidence that similar to hypoxia, transforming growth factor-β
(TGF-β) also induces the protein levels of Ago2. The work described here reveals
that hypoxia can modulate the expression of core protein components of the miRNA pathway
and provides a novel mechanism of miRNA regulation by hypoxia through C-P4H(I)-mediated
hydroxylation of Ago2. Thus, we present an alternative mechanism for modulating gene
expression under hypoxia by post-translational modification of Ago2. In addition, given
that our lab has previously shown that the TGF-β pathway regulates miRNA
biogenesis at the transcriptional level and by facilitating Drosha-mediated miRNA
processing, the results in this thesis suggest a novel regulation of miRNA
biogenesis/activity by the TGF-β pathway via increasing Ago2 stability and
modulating RISC activity.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Biochemistry.
Advisor: Akiko Hata.
Committee: John Kyriakis, Larry Feig, Claire Moore, and Donald Bloch.
Keyword: Biochemistry.read less