Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5 end-dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5 noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.

Long non-coding RNAs (lncRNAs) are a novel class of regulatory genes that play critical roles in various processes ranging from normal development to human diseases such as cancer progression. Recent studies have shown that lncRNAs regulate the gene expression by chromatin remodelling, transcription, splicing and RNA decay control, enhancer function, and epigenetic regulation. However, little is known about translation regulation by lncRNAs. We identified a translational regulatory lncRNA (treRNA) through genomewide computational analysis. We found that treRNA is upregulated in paired clinical breast cancer primary and lymph-node metastasis samples, and that its expression stimulates tumour invasion in vitro and metastasis in vivo. Interestingly, we found that treRNA downregulates the expression of the epithelial marker E-cadherin by suppressing the translation of its mRNA. We identified a novel ribonucleoprotein (RNP) complex, consisting of RNA-binding proteins (hnRNP K, FXR1, and FXR2), PUF60 and SF3B3, that is required for this treRNA functions. Translational suppression by treRNA is dependent on the 3 0 UTR of the E-cadherin mRNA. Taken together, our study indicates a novel mechanism of gene regulation by lncRNAs in cancer progression.

Tumors must adapt to the hypoxic environment in order to grow beyond a benign microscopic mass. In addition to transcriptional activation mediated by HIF-1a, hypoxia has also been reported to inhibit translation. The degree of translational inhibition is dependent on the duration as well as the severity of the hypoxic insult. Anoxia (<0.02% O 2 ) seems to have a more rapid and dramatic effect on translation as compared to hypoxia. We show here that prolonged hypoxia dramatically and reversibly inhibits translation in PC-3 cells. We also found that mTOR is inactivated and eIF-2a is phosphorylated during hypoxic treatment but only the eIF-2a phosphorylation correlates with the translational repression. We further used polysome analysis and microarray technology to analyze the impact of this translational repression on gene expression. We found that 33 mRNAs were refractory to this translational repression and that there was no correlation between mRNA induction and the ability to recruit ribosomes during hypoxia. We also found that ribosomal protein encoding mRNAs are more sensitive to this translational repression as compared to the majority of mRNAs. Although other reports have analyzed the effect of translation inhibition on gene expression under anoxic conditions, we believe that this is the first report in hypoxic cells. Our results show that the translational repression that occurs during hypoxia does impact gene expression in the highly transformed prostate cancer cell line, PC-3.

We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl 2 , or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl 2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1a (HIF-1a) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1a on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1a in insulin-and CoCl 2 -activated leptin mRNA and protein expression was confirmed using RNA interference.

The ␣-globin poly(C)-binding proteins (␣CPs) comprise an abundant and widely expressed set of K-homolog domain RNAbinding proteins. ␣CPs regulate the expression of a number of cellular and viral mRNAs at the levels of splicing, stability, and translation. Previous surveys have identified 160 mRNAs that are bound by ␣CP in the human hematopoietic cell line, K562. To explore the functions of these ␣CP/mRNA interactions, we identified mRNAs whose levels are altered in K562 cells acutely depleted of the two major ␣CP proteins, ␣CP1 and ␣CP2. Microarray analysis identified 27 mRNAs that are down-regulated and 14 mRNAs that are up-regulated in the ␣CP1/2-codepleted cells. This ␣CP1/2 co-depletion was also noted to inhibit cell proliferation and trigger a G 1 cell cycle arrest. Tar ␣CPs,2 also known as heterogeneous nuclear ribonucleoprotein (hnRNP) E (1) or poly(C)-binding proteins (2-4), comprise a family of highly abundant and widely expressed RNA-binding proteins. There are four ␣CP loci (1, 5, 6, 7), encoding ␣CP1-␣CP4. Two major products of the ␣CP2 locus, ␣CP2 and ␣CP2-KL, arise by alternative splicing (8), and a third abundant paralog, ␣CP1, is encoded from a retrotransposed copy of a fully processed ␣CP2 transcript (5). ␣CPs are highly conserved in evolution; orthologs are encoded in the genomes of Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cervisiae (6). The abundant expression, widespread tissue distribution (1, 4, 5), and evolutionary conservation of ␣CPs suggest that they serve a basic cellular function(s).Each ␣CP isoform contains three copies of the hnRNP K homology RNA binding domain (9). ␣CPs, along with hnRNP K, are uniquely characterized by in their strong binding preference for C-rich motifs. This subset of hnRNP K homology domain proteins has been linked to post-transcriptional controls via binding to elements in 5Ј-and 3Ј-untranslated regions (UTRs) of cellular and viral mRNAs (10 -19). For example, ␣CP1 and/or ␣CP2 regulate the stability of the mRNAs encoding ␣2-globin, tyrosine hydroxylase, and ␣1(I) collagen via binding to 3Ј-UTR motifs and mediate control over the translation of specific mRNAs, including 15-lipoxygenase, CCAAT/ enhancer-binding protein ␣, folate receptor, and phosphatase 2A, by binding to either 5Ј-or 3Ј-UTR elements. In addition to regulating the expression of several cellular mRNAs, ␣CP can also regulate a number of distinct steps in viral gene expression (11, 20 -29). Taken together, these studies indicate that ␣CPs constitute key regulators in a wide spectrum of post-transcriptional controls.To develop an understanding of how ␣CPs impact on cell function, we have screened for in vivo binding targets. Microarray analysis of immunoenriched ␣CP2-mRNP complexes isolated from K562 cells (30) revealed 160 ␣CP2-associated mRNAs. These mRNAs could be clustered according to the function(s) of their encoded proteins, suggesting roles for ␣CP2 in coordination of post-transcriptional controls. One of the larger functional clusters consisted of ...

Hypoxia can regulate many cellular processes. We show that hypoxia, via hypoxia-inducible factor (HIF)-1, blocks anoikis of epithelial cells by activating signaling pathways that inhibit expression of proapoptotic proteins Bim and Bmf. Hypoxia also disrupts mammary morphogenesis and blocks anoikis associated with lumen formation in three-dimensional in vitro model of mammary acini.

Background: Oncogene HER2 (ERBB2) regulates breast cancer growth and anoikis resistance. Results: ERBB2 requires hypoxia-inducible factor (HIF)-1 for breast cancer growth in vivo and anoikis resistance in vitro. Conclusion: HIF-1 plays a critical role in ERBB2-positive breast cancers. Significance: Genes co-regulated by ERBB2 and HIF-1 may be novel therapeutic targets for treating breast cancer.