Destabilization of The Ornithine Decarboxylase mRNA Transcript by the RNA-Binding Protein Tristetraprolin

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. In a normal physiological state, ODC is tightly regulated. However, during neoplastic transformation, ODC expression becomes upregulated. The studies described here show that the ODC mRNA transcript is destabilized by the RNA-binding protein tristetraprolin (TTP). We show that TTP is able to bind to the ODC mRNA transcript in both non-transformed RIE-1 cells and transformed Ras12V cells. Moreover, using mouse embryonic fibroblast cell lines that are devoid of a functional TTP protein, we demonstrate that in the absence of TTP both ODC mRNA stability and ODC enzyme activity increase when compared to wild-type cells. Finally, we show that the ODC 3′ untranslated region contains cis acting destabilizing elements that are affected by, but not solely dependent on, TTP expression. Together, these data support the hypothesis that TTP plays a role in the post-transcriptional regulation of the ODC mRNA transcript

Ornithine Decarboxylase mRNA is Stabilized in an mTORC1-dependent Manner in Ras-transformed Cells

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development

The ODC 3′-Untranslated Region and 5′-Untranslated Region Contain cis-Regulatory Elements: Implications for Carcinogenesis

It has been hypothesized that both the 3′-untranslated region (3′UTR) and the 5′-untranslated region (5′UTR) of the ornithine decarboxylase (ODC) mRNA influence the expression of the ODC protein. Here, we use luciferase expression constructs to examine the influence of both UTRs in keratinocyte derived cell lines. The ODC 5′UTR or 3′UTR was cloned into the pGL3 control vector upstream or downstream of the luciferase reporter gene, respectively, and luciferase activity was measured in both non-tumorigenic and tumorigenic mouse keratinocyte cell lines. Further analysis of the influence of the 3′UTR on luciferase activity was accomplished through site-directed mutagenesis and distal deletion analysis within this region. Insertion of either the 5′UTR or 3′UTR into a luciferase vector resulted in a decrease in luciferase activity when compared to the control vector. Deletion analysis of the 3′UTR revealed a region between bases 1969 and 2141 that was inhibitory, and mutating residues within that region increased luciferase activity. These data suggest that both the 5′UTR and 3′UTR of ODC contain cis-acting regulatory elements that control intracellular ODC protein levels

Activity‐based environmental inventory allocation

This article presents a generic method to assist product and process designers in measuring resource use and environmental discharges based on the relationships between process flow inputs and outputs and their activity. levels. It combines activity-based costing from conventional accounting with life-cycle inventories. The method is demonstrated on four electronic assembly product and process designs.The demonstration exhibits the disaggregation and allocation of costs and effluents from various manufacturing operations.This activity-based environmental allocation approach may be integrated with inventory analysis-the first step in full and streamlined life-cycle assessments, design for environment evaluation methods, environmental management activities, and new production planning models that consider environmental impacts.Journal Articl