Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach.
ABSTRACT: Employing MCT-1 oncogene mediated transformation of immortalized breast epithelial MCF10A cells; we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-à-vis cellular transformation. Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells. Significantly, we were able to demonstrate that knockdown of HuR expression using RNA interference, reduced anchorage-independent growth capacity in transformed MCF10A cells as well as decreased protein expression of a number of validated target genes. Our data demonstrate that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in post-transcriptional regulation of genes encoding proteins involved in breast epithelial cell transformation. Overall design: In this study, using a microarray approach we demonstrate that the dynamic global changes in association of two RBPs, HuR and AUF1, with cancer-related mRNAs have important influence on cell transformation in a MCT-1-mediated breast epithelial transformation model.
Project description:The aberrant expression of many genes is a common feature in the malignant transformation of cells. In mammalian cells, posttranscriptional gene regulatory processes are emerging as critical determinants controlling gene expression both in physiologic and pathologic conditions. These regulatory mechanisms are directed primarily by the interaction of mRNAs with specific RNA-binding proteins (RBP). There is an emerging body of data demonstrating that two RBPs, AUF1 and HuR, can antagonistically affect the posttranscriptional fate of target mRNAs, as well as concurrently bind to common target transcripts. Employing MCT-1 oncogene-mediated transformation of immortalized breast epithelial MCF10A cells, we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-a-vis cellular transformation. Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells. Significantly, we were able to show that knockdown of HuR expression using RNA interference reduced anchorage-independent growth capacity in transformed MCF10A cells and decreased protein expression of a number of validated target genes. Our data show that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in posttranscriptional regulation of genes encoding proteins involved in breast epithelial cell transformation. These findings further support the feasibility of using a ribonomics approach for the identification of cancer-related pathways.
Project description:Employing MCT-1 oncogene mediated transformation of immortalized breast epithelial MCF10A cells; we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-à-vis cellular transformation. Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells. Significantly, we were able to demonstrate that knockdown of HuR expression using RNA interference, reduced anchorage-independent growth capacity in transformed MCF10A cells as well as decreased protein expression of a number of validated target genes. Our data demonstrate that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in post-transcriptional regulation of genes encoding proteins involved in breast epithelial cell transformation. In this study, using a microarray approach we demonstrate that the dynamic global changes in association of two RBPs, HuR and AUF1, with cancer-related mRNAs have important influence on cell transformation in a MCT-1-mediated breast epithelial transformation model.
Project description:BACKGROUND:Sarcoidosis is characterised by up-regulation of cytokines and chemokine ligands/receptors and proteolytic enzymes. This pro-inflammatory profile is regulated post-transcriptionally by RNA-binding proteins (RBPs). We investigated in vivo expression of six RBPs (AUF1, HuR, NCL, TIA, TIAR, PCBP2) and two inhibitors of proteolytic enzymes (RECK, PTEN) in pulmonary sarcoidosis and compared it to the expression in four control groups of healthy individuals and patients with other respiratory diseases: chronic obstructive pulmonary disease (COPD), asthma and idiopathic interstitial pneumonias (IIPs). METHODS:RT-PCR was used to quantify the mRNAs in bronchoalveolar (BA) cells obtained from 50 sarcoidosis patients, 23 healthy controls, 30 COPD, 19 asthmatic and 19 IIPs patients. Flow cytometry was used to assess intracellular protein expression of AUF1 and HuR in peripheral blood T lymphocytes (PBTLs) obtained from 9 sarcoidosis patients and 6 healthy controls. RESULTS:Taking the stringent conditions for multiple comparisons into consideration, we consistently observed in the primary analysis including all patients regardless of smoking status as well as in the subsequent sub-analysis limited for never smokers that the BA mRNA expression of AUF1 (p<0.001), TIA (p<0.001), NCL (p<0.01) and RECK (p<0.05) was decreased in sarcoidosis compared to healthy controls. TIA mRNA was also decreased in sarcoidosis compared to both obstructive pulmonary diseases (COPD and asthma; p<0.001) but not compared to IIPs. There were several positive correlations between RECK mRNA and RBP mRNAs in BA cells. Also sarcoidosis CD3+, CD4+ and CD8+ PBTLs displayed lower mean fluorescence intensity of AUF1 (p?0.02) and HuR (p?0.03) proteins than control healthy PBTLs. CONCLUSION:mRNA expressions of three RBPs (AUF1, TIA and NCL) and their potential target mRNA encoding RECK in BA cells and additionally protein expression of AUF1 and HuR in PBTLs were down-regulated in our sarcoidosis patients compared to healthy individuals. Its significance, e.g. for stability of mRNAs encoding pro-inflammatory factors, should be further explored in sarcoidosis.
Project description:BACKGROUND & AIMS:Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine are tightly regulated by 2 genes: methionine adenosyltransferase 1A (MAT1A) and methionine adenosyltransferase 2A (MAT2A). MAT1A is expressed in the adult liver, whereas MAT2A expression primarily is extrahepatic and is associated strongly with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. METHODS:In silico analysis of the 3' untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AU-rich RNA binding factor 1 (AUF1) and HuR, respectively. We investigated the posttranscriptional regulation of MAT1A and MAT2A by AUF1, HuR, and methyl-HuR in the aforementioned biological processes. RESULTS:During hepatic de-differentiation, the switch between MAT1A and MAT2A coincided with an increase in HuR and AUF1 expression. S-adenosylmethionine treatment altered this homeostasis by shifting the balance of AUF1 and methyl-HuR/HuR, which was identified as an inhibitor of MAT2A messenger RNA (mRNA) stability. We also observed a similar temporal distribution and a functional link between HuR, methyl-HuR, AUF1, and MAT1A and MAT2A during fetal liver development. Immunofluorescent analysis revealed increased levels of HuR and AUF1, and a decrease in methyl-HuR levels in human livers with hepatocellular carcinoma (HCC). CONCLUSIONS:Our data strongly support a role for AUF1 and HuR/methyl-HuR in liver de-differentiation, development, and human HCC progression through the posttranslational regulation of MAT1A and MAT2A mRNAs.
Project description:Post-transcriptional mRNA regulation by RNA binding proteins (RBPs) associated with AU-rich elements (AREs) present in the 3' untranslated region (3'UTR) of specific mRNAs modulates transcript stability and translation in eukaryotic cells. Here we have functionally characterised the importance of the AREs present within the Bcl2 3'UTR in order to maintain Bcl2 expression. Gene targeting deletion of 300 nucleotides of the Bcl2 3'UTR rich in AREs diminishes Bcl2 mRNA stability and protein levels in primary B cells, decreasing cell lifespan. Generation of chimeric mice indicates that Bcl2-ARE?/? B cells have an intrinsic competitive disadvantage compared to wild type cells. Biochemical assays and predictions using a bioinformatics approach show that several RBPs bind to the Bcl2 AREs, including AUF1 and HuR proteins. Altogether, association of RBPs to Bcl2 AREs contributes to Bcl2 protein expression by stabilizing Bcl2 mRNA and promotes B cell maintenance.
Project description:RNA-binding proteins (RBPs) that associate with specific mRNA sequences and function as mRNA turnover and translation regulatory (TTR) RBPs are emerging as pivotal posttranscriptional regulators of gene expression. However, little is known about the mechanisms that govern the expression of TTR-RBPs. Here, we employed human cervical carcinoma HeLa cells to test the hypothesis that TTR-RBP expression is influenced posttranscriptionally by TTR-RBPs themselves. Systematic testing of the TTR-RBPs AUF1, HuR, KSRP, NF90, TIA-1, and TIAR led to three key discoveries. First, each TTR-RBP was found to associate with its cognate mRNA and with several other TTR-RBP-encoding mRNAs, as determined by testing both endogenous and biotinylated transcripts. Second, silencing of individual TTR-RBPs influenced the expression of other TTR-RBPs at the mRNA and/or protein level. Third, further analysis of two specific ribonucleoprotein (RNP) complexes revealed that TIA-1 expression was controlled via HuR-enhanced mRNA stabilization and TIAR-repressed translation. Together, our findings underscore the notion that TTR-RBP expression is controlled, at least in part, at the posttranscriptional level through a complex circuitry of self- and cross-regulatory RNP interactions.
Project description:Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms such as the repression of growth-inhibitory proteins, including JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polyamines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3' untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. The depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of HuR-JunD mRNA complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in intestinal epithelial cells through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.
Project description:Gene expression patterns vary dramatically in a tissue-specific and age-dependent manner. RNA-binding proteins that regulate mRNA turnover and/or translation (TTR-RBPs) critically affect the subsets of expressed proteins. However, very little is known regarding the tissue- and age-dependent expression of TTR-RBPs in humans. Here, we use human tissue arrays containing a panel of organ biopsies from donors of different ages, to study the distribution and abundance of four TTR-RBPs: HuR, AUF1, TIA-1, and TTP. HuR and AUF1 were expressed with remarkably similar patterns. Both TTR-RBPs were present in high percentages of cells and displayed elevated intensities in many age groups and tissues, most notably in the gastrointestinal and reproductive systems; they were moderately expressed in the urinary and immune systems, and were almost undetectable in muscle and brain. TIA-1 was also abundant in many tissues and age groups; TIA-1 was expressed at high levels in the gastrointestinal, immune, urinary, and reproductive systems, and at low levels in brain and muscle. By contrast, TTP-expressing cells, as well as TTP signal intensities declined with advancing age, particularly in the immune, nervous, and muscular systems; however, TTP levels remained elevated in the gastrointestinal tract. The widespread abundance of HuR, AUF1, and TIA-1 throughout the body and in all age groups was in stark contrast with their declining levels in human diploid fibroblasts (HDFs) undergoing replicative senescence, a cultured-cell model of aging. Conversely, TTP levels increased in senescent HDFs, while TTP levels decreased with advancing age. Our studies provide a framework for the study of human TTR-RBP function in different tissues, throughout the human life span.
Project description:BACKGROUND: The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs). METHODS: The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. RESULTS: We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. CONCLUSION: This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.
Project description:Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.