Examination of Ornithine Decarboxylase Antizyme RNA Structure and Function for the Development of Antibiological Agents - PROJECT SUMMARY
Nearly all organisms possess the capability to synthesize the natural polyamines - putrescine, spermidine and
spermine - which are essential for cell growth and differentiation. Due to the ability of polyamines to interact
with nearly every biomolecule - DNA, RNA, phospholipids, proteins and ATP to name a few - they play many
roles within the cell in order to support cell growth. It has been well documented that polyamine levels in
mammalian cells correlate with the rate of cell growth, high polyamine concentrations have been observed in
rapidly proliferating cells and low concentrations have been measured in slow-growing or quiescent cells. Not
surprisingly, the transport and metabolism of polyamines are highly regulated by complex feedback
mechanisms. Ornithine decarboxylase (ODC) is the key regulatory enzyme in polyamine biosynthesis. ODC
homeostasis affects cell growth and cancer development. ODC over-expression has been observed in many
tumor types, including prostate, breast and skin cancers. Both ODC and cellular uptake of polyamines is
inhibited by Ornithine Decarboxylase Antizyme (OAZ). The making of Antizyme protein from OAZ mRNA
requires translational frameshifting at a highly conserved site to bypass premature termination. Mammalian
OAZ mRNAs further possess a pseudoknot (PK) RNA 3¢ to the frameshift site that stimulates +1 frameshifting.
Moreover, frameshifting is stimulated by polyamines, thus providing a feedback mechanism whereby the
accumulation of metabolic products inhibits biosynthesis. Although the role of the OAZ pseudoknot RNA
element (further designated OAZ1-PK) in polyamine-dependent frameshifting has been investigated, it has not
been examined as a distinct polyamine “sensor”. Riboswitches are elements within noncoding regions of
mRNAs that directly bind to cellular metabolites and modulate gene expression. Many riboswitches provide a
mechanism of feedback regulation for gene products within the biosynthetic pathway of the cognate
metabolite. Riboswitches are widespread among bacteria, and one class further resides in fungi and plants, but
no riboswitches have been found in animals. It is proposed that the OAZ1-PK RNA functions as a riboswitch,
and herein evidence is provided that this noncoding RNA is a polyamine sensor. This RNA element is highly
conserved among vertebrate genes required for spermine biosynthesis. Development of drugs that target
putative spermine riboswitches from different organisms might therefore be used for wide-ranging purposes
such as anticancer agents, antifungal agents, or pesticides. This proposal will examine the structure and
function of the OAZ1-PK RNA with the following specific aims: (1) examine the specificity and affinity of
polyamine binding to OAZ1-PK RNA, (2) investigate the three-dimensional structure of OAZ1-PK RNA, and (3)
explore the role of Antizyme OAZ1-PK RNA in control of gene expression.
