Tristetraprolin (TTP) is a RNA-binding protein that inhibits the expression of pro-inflammatory cytokines and invasiveness-associated genes. TTP levels are decreased in many different cancer types and it has been proposed that this protein could be used as a prognostic factor in breast cancer. Here, using publicly available DNA microarray datasets, "serial analysis of gene expression" libraries and qRT-PCR analysis, we determined that TTP mRNA is present in normal breast cells and its levels are significantly decreased in all breast cancer subtypes. In addition, by immunostaining, we found that TTP expression is higher in normal breast tissue and benign lesions than in infiltrating carcinomas. Among these, lower grade tumors showed increased TTP expression compared to higher grade cancers. Therefore, these data indicate that TTP protein levels would provide a better negative correlation with breast cancer invasiveness than TTP transcript levels. In mice, we found that TTP mRNA and protein expression is also diminished in mammary tumors. Interestingly, a strong positive association of TTP expression and mammary differentiation was identified in normal and tumor cells. In fact, TTP expression is highly increased during lactation, showing good correlation with various mammary differentiation factors. TTP expression was also induced in mammary HC11 cells treated with lactogenic hormones, mainly by prolactin, through Stat5A activation. The effect of this hormone was highly dependent on mammary differentiation status, as prolactin was unable to elicit a similar response in proliferating or neoplastic mammary cells. In summary, these studies show that TTP expression is strongly linked to the mammary differentiation program in human and mice, suggesting that this protein might play specific and relevant roles in the normal physiology of the gland.

The regulation of gene expression is a fundamental cellular process that is controlled at multiple levels. Abnormal regulation of gene expression has been directly implicated in the pathogenesis of some diseases of animals and humans and may contribute to the disease process in unrecognized ways in many others. Furthermore, novel treatment strategies for a number of different diseases may hinge upon our ability to exploit mechanisms that normally alter the expression of endogenous genes. While the study of gene regulation has traditionally focused on transcription as a major regulator of gene expression, it has recently become apparent that the post-transcriptional control of gene expression may play an equally important role. In particular, rapid, context-specific regulation of the stability of mRNA transcripts encoding highly active proteins, such as cytokines, growth factors, oncogenes and cell-cycle regulators, appears to play a key role in the control of these molecules and the processes they mediate. Many of the known regulatory pathways for mRNA stability involve proteins that interact with specific AU-rich elements in the 3'-untranslated region of the transcript. This review will address some important aspects of the normal regulation of mRNA stability and known or potential contributions of RNA stability regulation to health and disease.

Thrombospondin-1 (TSP-1) is a matricellular protein that participates in numerous normal and pathological tissue processes and is rapidly modulated by different stimuli. The presence of 8 highly-conserved AU rich elements (AREs) within the 3'-untranslated region (3'UTR) of the TSP-1 mRNA suggests that post-transcriptional regulation is likely to represent one mechanism by which TSP-1 gene expression is regulated. We investigated the roles of these AREs, and proteins which bind to them, in the control of TSP-1 mRNA stability. The endogenous TSP-1 mRNA half-life is approximately 2.0 hours in HEK293 cells. Luciferase reporter mRNAs containing the TSP-1 3'UTR show a similar rate of decay, suggesting that the 3'UTR influences the decay rate. Site-directed mutagenesis of individual and adjacent AREs prolonged reporter mRNA halflife to between 2.2 and 4.4 hours. Mutation of all AREs increased mRNA half life to 8.8 hours, suggesting that all AREs have some effect, but that specific AREs may have key roles in stability regulation. A labeled RNA oligonucleotide derived from the most influential ARE was utilized to purify TSP-1 AREbinding proteins. The AU-binding protein AUF1 was shown to associate with this motif. These studies reveal that AREs in the 3'UTR control TSP-1 mRNA stability and that the RNA binding protein AUF1 participates in this control. These studies suggest that ARE-dependent control of TSP-1 mRNA stability may represent an important component in the control of TSP-1 gene expression.

In most mammals, the Zfp36 gene family consists of three conserved members, with a fourth member, Zfp36l3, present only in rodents. The ZFP36 proteins regulate post-transcriptional gene expression at the level of mRNA stability in organisms from humans to yeasts, and appear to be expressed in all major groups of eukaryotes. In Mus musculus, Zfp36l3 expression is limited to the placenta and yolk sac, and is important for overall fecundity. We sequenced the Zfp36l3 gene from more than 20 representative species, from members of the Muridae, Cricetidae and Nesomyidae families. Zfp36l3 was not present in Dipodidae, or any families that branched earlier, indicating that this gene is exclusive to the Muroidea superfamily. We provide evidence that Zfp36l3 arose by retrotransposition of an mRNA encoded by a related gene, Zfp36l2 into an ancestral rodent X chromosome. Zfp36l3 has evolved rapidly since its origin, and numerous modifications have developed, including variations in start codon utilization, de novo intron formation by mechanisms including a nested retrotransposition, and the insertion of distinct repetitive regions. One of these repeat regions, a long alanine rich-sequence, is responsible for the full-time cytoplasmic localization of Mus musculus ZFP36L3. In contrast, this repeat sequence is lacking in Peromyscus maniculatus ZFP36L3, and this protein contains a novel nuclear export sequence that controls shuttling between the nucleus and cytosol. Zfp36l3 is an example of a recently acquired, rapidly evolving gene, and its various orthologues illustrate several different mechanisms by which new genes emerge and evolve.

Abstract. Thrombospondin-1 (TSP-1) is a large extracellular matrix-associated protein that is important for normal follicular development, is rapidly modulated during follicular growth and plays important roles in cellular proliferation and angiogenesis. TSP-1 mRNA is post-transcriptionally regulated, although the underlying mechanisms are largely unknown. Insulin-like growth factor-1 is a potent signalling molecule that participates in folliculogenesis. We hypothesized that IGF-1 modulates TSP-1 expression in granulosa cells, and that such modulation requires rapid turnover of the TSP-1 mRNA and protein. Spontaneously-immortalized rat granulosa cells (SIGC) were cultured in the presence or absence of IGF-1, after which the expression and turnover of TSP-1 mRNA and protein was evaluated by western blot and quantitative PCR. RNA stability reporter constructs were prepared in which wild-type and mutated AU-rich elements from the TSP-1 3'UTR were cloned downstream of the luciferase gene in a mammalian expression vector. These were transfected into SIGC cells in order to characterize mRNA elements that regulate the stability of the TSP-1 mRNA. TSP-1 expression decreased rapidly at the mRNA and protein levels in IGF-1 treated cultures. Following 12 h of IGF-I treatment, TSP-1 protein decreased by 25% and was 73% lower than in untreated cultures. The half-life of endogenous TSP-1 mRNA in SIGC was 2.0 h. This was not changed in the presence of IGF-1, however, transcription of new TSP-1 mRNA was inhibited. Reporter mRNAs with mutated AU-rich elements demonstrated a longer half-life than mRNAs in which the wild type AU-rich elements were present. These studies reveal that IGF-1 rapidly inhibits TSP-1 expression at the protein and mRNA levels in cultured granulosa cells through apparent inhibition of TSP-1 transcription. The decrease depends on an intrinsically short half-life of TSP-1 mRNA and protein. The short mRNA half life is due, at least in part, to AU-rich elements in the 3'UTR of the TSP-1 mRNA. Key words: Extracellular matrix, Gene expression, mRNA stability, Ovary, Post-transcriptional (J. Reprod. Dev. 57: [76][77][78][79][80][81][82][83] 2011) uccessful mammalian folliculogenesis is dependent on the rapid and precise regulation of specific gene expression during various stages of follicle development in response to endocrine, paracrine, and autocrine factors that vary through the estrous cycle (reviewed in [1,2]). The ability of follicular cells to respond appropriately to such stimuli is one key factor in the progression of any individual follicle to dominance, and failure to do so is one key reason for follicular atresia [3]. The complex mechanisms that control follicular gene expression in granulosa and other ovarian cells are poorly understood.Although gonadotrophin-mediated signalling pathways are clearly indispensable for most aspects of follicle growth, the importance of other soluble and local factors in promoting follicular "success" is increasingly apparent. One such factor is Insulin-like growth ...