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Andrew D Redfern,
Shane M Colley,
Dianne J Beveridge,
Naoya Ikeda,
Michael R Epis,
Xia Li,
Charles E Foulds,
Lisa M Stuart, Andrew Barker,
Victoria J Russell, [......],
Simon J Kobelke,
Xiaotao Li,
Esme C Hatchell,
Christine Payne,
Keith M Giles,
Adriana Messineo,
Anne Gatignol,
Rainer B Lanz,
Bert W O'Malley,
Peter J Leedman
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ABSTRACT: The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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ABSTRACT: The enzyme deoxyhypusine hydroxylase (DOHH) catalyzes the activation of eukaryotic translation initiation factor (eIF5A), a protein essential for cell growth. Using bioinformatic predictions and reporter gene assays, we have identified a 182-nt element within the DOHH 3'-untranslated region (3'-UTR) that contains a number of target sites for miR-331-3p and miR-642-5p. Quantitative RT-PCR studies demonstrated overexpression of DOHH mRNA and underexpression of miR-331-3p and miR-642-5p in several prostate cancer cell lines compared with normal prostate epithelial cells. Transient overexpression of miR-331-3p and/or miR-642-5p in DU145 prostate cancer cells reduced DOHH mRNA and protein expression and inhibited cell proliferation. We observed synergistic growth inhibition with the combination of miR-331-3p and miR-642-5p and mimosine, a pharmacological DOHH inhibitor. Finally, we identified a significant inverse relationship between the expression of miR-331-3p or miR-642-5p and DOHH in a cohort of human prostate cancer tissues. Our results suggest a novel role for miR-331-3p and miR-642-5p in the control of prostate cancer cell growth via the regulation of DOHH expression and eIF5A activity.
Journal of Biological Chemistry 08/2012; 287(42):35251-9. · 4.77 Impact Factor
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ABSTRACT: The stability of RNAs bearing AU-rich elements in their 3'-UTRs, and thus the level of expression of their protein products, is regulated by interactions with cytoplasmic RNA-binding proteins. Binding by HuR generally leads to mRNA stabilization and increased protein production, whereas binding by AUF1 isoforms generally lead to rapid degradation of the mRNA and reduced protein production. The exact nature of the interplay between these and other RNA-binding proteins remains unclear, although recent studies have shown close interactions between them and even suggested competition between the two for binding to their cognate recognition sequences. Other recent reports have suggested that the sequences recognized by the two proteins are different. We therefore performed a detailed in vitro analysis of the binding site(s) for HuR and AUF1 present in androgen receptor mRNA to define their exact target sequences, and show that the same sequence is contacted by both proteins. Furthermore, we analysed a proposed HuR target within the 3'-UTR of MTA1 mRNA, and show that the contacted bases lie outside of the postulated motif and are a better match to a classical ARE than the postulated motif. The defining features of these HuR binding sites are their U-richness and single strandedness.
Journal of biochemistry 02/2012; 151(4):423-37. · 1.95 Impact Factor
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ABSTRACT: Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.
Nucleic Acids Research 02/2012; 40(11):5101-14. · 8.03 Impact Factor
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ABSTRACT: ERBB-2 overexpression is associated with the development and progression of cancer and mediates its resistance to therapy. It has been suggested that post-transcriptional mechanisms control the overexpression of ERBB-2 in prostate cancer (PCa). We recently demonstrated that the 3'-untranslated region (3'-UTR) of ERBB-2 mRNA contains two specific target sites for binding of the microRNA miR-331-3p and that miR-331-3p represses ERBB-2 expression and signaling in PCa cells. Here we investigate a U-rich element situated in close proximity to the distal miR-331-3p target site in the ERBB-2 3'-UTR. Specific binding of HuR to this U-rich element promotes ERBB-2 expression in PCa cells. We show that HuR antagonizes the repressive action of miR-331-3p on its distal ERBB-2 3'-UTR target site. These results support a model in which the interplay between RNA-binding proteins and microRNAs controls the post-transcriptional regulation of gene expression and suggest that both HuR and miR-331-3p participate in the overexpression of ERBB-2 observed in some PCas.
Journal of Biological Chemistry 12/2011; 286(48):41442-54. · 4.77 Impact Factor
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ABSTRACT: Aberrant expression of the epidermal growth factor receptor (EGFR) and/or human epidermal growth factor receptor 2 (HER2) is a feature of many human tumors and is associated with disease progression, treatment resistance, and poor prognosis. Protein kinase B/Akt, an important downstream effector of these receptor tyrosine kinases, induces signaling pathways that control cancer cell proliferation, invasion, angiogenesis, and apoptosis resistance. MicroRNAs (miRNAs), small noncoding RNAs that bind to the 3'-untranslated region of target mRNAs, are now recognized to play key roles in the regulation of gene expression, particularly in tumor development and metastasis. We have shown that miRNA-7 (miR-7) and miRNA-331-3p (miR-331-3p) directly regulate expression of EGFR and HER2, respectively, in glioblastoma and prostate cancer cell lines. As a consequence, miR-7 and miR-331-3p reduce Akt activity and thus have the capacity to regulate a signaling pathway critical to the development and progression of glioblastoma and prostate cancer. This chapter provides a detailed approach outlining how to confirm that a putative target of a miRNA is a direct target, and subsequent assessment of downstream signaling mediators.
Methods in molecular biology (Clifton, N.J.) 01/2011; 676:147-63.
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ABSTRACT: Recent years have seen a massive expansion in our understanding of the biology of microRNAs (miRNAs) in cancer, through the identification of miRNAs with aberrant expression in specific cancers and the functional validation of their critical target molecules and cellular effects. In parallel, targeted therapeutic agents to block signalling pathways critical to tumour growth and progression have been developed but have yielded disappointing clinical results. The discovery of miRNAs that regulate ErbB signalling in cancer cells brings new hope that in the future these oncogenic pathways can be more effectively inhibited to improve patient outcomes.
Current Opinion in Pharmacology 12/2010; 10(6):655-61. · 6.86 Impact Factor
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Vicki Athanasopoulos, Andrew Barker,
Di Yu,
Andy H-M Tan,
Monika Srivastava,
Nelida Contreras,
Jianbin Wang,
Kong-Peng Lam,
Simon H J Brown,
Christopher C Goodnow,
Nicholas E Dixon,
Peter J Leedman,
Robert Saint,
Carola G Vinuesa
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ABSTRACT: Roquin is an E3 ubiquitin ligase with a poorly understood but essential role in preventing T-cell-mediated autoimmune disease and in microRNA-mediated repression of inducible costimulator (Icos) mRNA. Roquin and its mammalian paralogue membrane-associated nucleic acid binding protein (MNAB) define a protein family distinguished by an approximately 200 amino acid domain of unknown function, ROQ, that is highly conserved from mammals to invertebrates and is flanked by a RING-1 zinc finger and a CCCH zinc finger. Here we show that human, Drosophila and Caenorhabditis elegans Roquin and human MNAB localize to the cytoplasm and upon stress are concentrated in stress granules, where stalled mRNA translation complexes are stored. The ROQ domain is necessary and sufficient for localization to arsenite-induced stress granules and to induce these structures upon overexpression, and is required to trigger Icos mRNA decay. Gel-shift, SPR and footprinting studies show that an N-terminal fragment centred on the ROQ domain binds RNA from the Icos 3'-untranslated region comprising the minimal sequence for Roquin-mediated repression, adjacent to the miR-101 sequence complementarity. These findings identify Roquin as an RNA-binding protein and establish a specific function for the ROQ protein domain in mRNA homeostasis. Structured digital abstract * MINT-7711163: TIA-1 (uniprotkb:P31483) and Roquin (uniprotkb:Q4VGL6) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711475: RLE-1 (uniprotkb:O45962) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711487: DmRoquin (uniprotkb:Q9VV48) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711447, MINT-7711460: MNAB (uniprotkb:Q9HBD1) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711176: eIF3 (uniprotkb:P55884) and Roquin (uniprotkb:Q4VGL6) colocalize (MI:0403) by fluorescence microscopy (MI:0416) * MINT-7711192: DCP1A (uniprotkb:Q9NPI6) and TIA-1 (uniprotkb:P31483) colocalize (MI:0403) by fluorescence microscopy (MI:0416).
FEBS Journal 05/2010; 277(9):2109-27. · 3.79 Impact Factor
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ABSTRACT: Androgen receptor (AR)-mediated pathways play a critical role in the development and progression of prostate cancer. However, little is known about the regulation of AR mRNA stability and translation, two central processes that control AR expression. The ErbB3 binding protein 1 (EBP1), an AR corepressor, negatively regulates crosstalk between ErbB3 ligand heregulin (HRG)-triggered signaling and the AR axis, affecting biological properties of prostate cancer cells. EBP1 protein expression is also decreased in clinical prostate cancer. We previously demonstrated that EBP1 overexpression results in decreased AR protein levels by affecting AR promoter activity. However, EBP1 has recently been demonstrated to be an RNA binding protein. We therefore examined the ability of EBP1 to regulate AR post-transcriptionally. Here we show that EBP1 promoted AR mRNA decay through physical interaction with a conserved UC-rich motif within the 3'-UTR of AR. The ability of EBP1 to accelerate AR mRNA decay was further enhanced by HRG treatment. EBP1 also bound to a CAG-formed stem-loop in the 5' coding region of AR mRNA and was able to inhibit AR translation. Thus, decreases of EBP1 in prostate cancer could be important for the post-transcriptional up-regulation of AR contributing to aberrant AR expression and disease progression.
Nucleic Acids Research 02/2010; 38(11):3619-31. · 8.03 Impact Factor
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ABSTRACT: Hypospadias is a urogenital birth defect affecting infant boys. Periconceptual parental occupational exposure to endocrine disrupting chemicals (EDCs) with oestrogenic or anti-androgenic properties may adversely affect male genital development in utero. The aim of this study was to investigate the association between both maternal and paternal occupational exposures to EDCs and hypospadias.
This registry-based case-control study considered 1202 cases of hypospadias in children born in Western Australia between 1980 and 2000 and 2583 male controls randomly selected from birth records for whom information regarding parental occupation was available from the Western Australian Maternal and Child Health Research Database. Occupational exposures to seven groups of potential EDCs were independently coded by two researchers according to a validated job-exposure matrix.
Multivariable analysis showed a strong association with potential maternal occupational exposure to heavy metals with an over twofold increased risk of hypospadias (OR 2.6; 95% CI 1.3 to 5.2), and women exposed to phthalates were more likely to have an affected son (OR 1.2; 95% CI 0.8 to 1.7). Compared with mild or isolated cases, the risks of moderate-severe hypospadias or multiple defects were increased up to two- and fivefold, respectively, with maternal exposure to most types of EDCs. Paternal occupational exposures to polychlorinated organic (OR 1.3; 95% CI 1.0 to 1.8) and bi-phenolic (OR 1.6; 95% CI 1.0 to 2.6) compounds were also possible risk factors.
Our findings provide preliminary evidence of an association between exposure to EDCs with oestrogenic or anti-androgenic properties and increased risk of hypospadias.
Occupational and environmental medicine 11/2009; 67(9):585-9. · 3.64 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and are aberrantly expressed in human cancer. The ERBB-2 tyrosine kinase receptor is frequently overexpressed in prostate cancer and is associated with disease progression and poor survival. We have identified two specific miR-331-3p target sites within the ERBB-2 mRNA 3'-untranslated region and show that miR-331-3p expression is decreased in prostate cancer tissue relative to normal adjacent prostate tissue. Transfection of multiple prostate cancer cell lines with miR-331-3p reduced ERBB-2 mRNA and protein expression and blocked downstream phosphatidylinositol 3-kinase/AKT signaling. Furthermore, miR-331-3p transfection blocked the androgen receptor signaling pathway in prostate cancer cells, reducing activity of an androgen-stimulated prostate-specific antigen promoter and blocking prostate-specific antigen expression. Our findings provide insight into the regulation of ERBB-2 expression in cancer and suggest that miR-331-3p has the capacity to regulate signaling pathways critical to the development and progression of prostate cancer cells.
Journal of Biological Chemistry 08/2009; 284(37):24696-704. · 4.77 Impact Factor
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ABSTRACT: Hypospadias, a common birth defect, has shown widespread variation in reported rates and temporal trends across countries over the last 30 years. The aim of this study was to determine the prevalence and trends of hypospadias in an Australian population.
Population-based study of all male infants born in Western Australia (WA) between 1980 and 2000 diagnosed with hypospadias and notified to the WA Birth Defects Registry.
Prevalence of hypospadias, birth outcome and association with other congenital anomalies, stratified by degree-of-severity.
1788 cases of hypospadias were registered in WA in 1980-2000 with an overall prevalence of 34.8 (95% confidence interval (CI): 33.2 to 36.4) cases per 10 000 births. The prevalence increased by 2.0% per annum (95% CI: 1.2% to 2.8%) from 27.9 in 1980 to 43.2 per 10 000 births in 2000 (p<0.001). Hypospadias was mild in 84% of cases, moderate-severe in 11% and unspecified in 5%, with the number of moderate-severe hypospadias almost doubling over time (p<0.01). There were 1465 (82%) cases of isolated hypospadias and 323 (18%) had co-existing anomalies. Infants with co-existing genital (relative risk (RR) 4.5; 95% CI: 3.3 to 6.1) or non-genital (RR 1.5; 95% CI: 1.0 to 2.2) anomalies were more likely to have moderate-severe hypospadias compared with isolated cases.
Hypospadias affects one in 231 births and has been reported to have increased significantly over the last 20 years. Future investigation of the aetiology of hypospadias is important to identify potentially modifiable risk factors and ensure optimal male reproductive health in the future.
Archives of Disease in Childhood 07/2007; 92(7):580-4. · 2.88 Impact Factor
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Esme C Hatchell,
Shane M Colley,
Dianne J Beveridge,
Michael R Epis,
Lisa M Stuart,
Keith M Giles,
Andrew D Redfern,
Lauren E C Miles, Andrew Barker,
Louisa M MacDonald,
Peter G Arthur,
James C K Lui,
Jemma L Golding,
Ross K McCulloch,
Cecily B Metcalf,
Jackie A Wilce,
Matthew C J Wilce,
Rainer B Lanz,
Bert W O'Malley,
Peter J Leedman
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ABSTRACT: Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.
Molecular Cell 07/2006; 22(5):657-68. · 14.18 Impact Factor
