Analysis of proteins bound to stored messenger RNA in 'Xenopus' oocytes

Regulation at the post-transcriptional level is gaining significance at a rapid pace. One example is the storage of messenger mRNA molecules in a translationally quiescent state, the so-called "masked messengers". Their existence has been known since the 1960s, but many details of their composition and structure have not yet been resolved. Masked messenger RNAs are particularly abundant in the oocytes of the African clawed toad Xenopus laevis. The aim of this study has been to examine the proteins bound to stored mRNAs in the oocytes, by focussing on the Y-box proteins which had already been identified as major components in mRNA masking, and by analyzing some of the other unidentified mRNP proteins. The YB proteins were studied in greater detail, gaining fresh information about their RNA-binding properties, defining distinct binding domains. The presence of an mRNP-associated protein kinase was confirmed, and binding assays suggested that phosphorylation influences the ability of the YB proteins to bind to mRNA. cDNA expression libraries were screened both with an RNA-binding assay and with an immunoscreening method, isolating a variety of known and novel cDNAs. Peptide sequencing of mRNP proteins revealed the presence of an RNA helicase distinct from the translation initiation factor eIF4A. It is postulated that the RNA helicase, in addition to the YB proteins, will be seen to have an important role in the formation and activity of the masked messenger RNA particles

Quantitative analysis of ERG expression and its splice isoforms in formalin-fixed, paraffin-embedded prostate cancer samples: Association with seminal vesicle invasion and biochemical recurrence

© American Society for Clinical Pathology. Objectives: The proto-oncogene ETS-related gene (ERG) is consistently overexpressed in prostate cancer. Alternatively spliced isoforms of ERG have variable biological activities; inclusion of exon 11 (72 base pairs [bp]) is associated with aggressiveness and progression of disease. Exon 10 (81 bp) has also been shown to be alternatively spliced. Within this study, we assess whether ERG protein, messenger RNA (mRNA), and ERG splice isoform mRNA expression is altered as prostate cancer progresses. Methods: Detection of the TMPRSS2-ERG fusion was done using direct methods (reverse transcription polymerase chain reaction [PCR] and fluorescence in situ hybridization) and indirect methods for ERG mRNA and protein expression using quantitative PCR and immunohistochemistry, respectively. A linear equation method was used to quantitatively determine relative proportions of ERG variants (ERG72/Δ72, ERG81/Δ81) for each sample. Results: ERG mRNA and protein expression is increased in patients with advanced prostate cancer, with higher levels of ERG expression significantly associated with seminal vesicle invasion (stage pT3b) and biochemical recurrence. Genes involved in cell migration and invasiveness (matrix metalloproteinase 7, osteopontin, and septin 9) are increased in prostate cancers that overexpress ERG. In addition, there is a clear indication of increased retention of exons 10 and 11 in prostate cancer. Conclusions: Analysis of ERG and its variants may be valuable in determining prognosis and development of prostate cancer

Role of splice variants in the metastatic progression of prostate cancer

AS (alternative splicing) and its role in disease, especially cancer, has come to forefront in research over the last few years. Alterations in the ratio of splice variants have been widely observed in cancer. Splice variants of cancer-associated genes have functions that can alter cellular phenotype, ultimately altering metastatic potential. As metastases are the cause of approximately 90% of all human cancer deaths, it is crucial to understand how AS is dysregulated in metastatic disease. We highlight some recent studies into the relationship between altered AS of key genes and the initiation of prostate cancer metastasis. ©The Authors Journal compilation ©2012 Biochemical Society

P19 H-Ras Induces G1/S Phase Delay Maintaining Cells in a Reversible Quiescence State

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family’s established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell.[Principal Findings]: We found that p19 regulates telomerase activity through its interaction with p73a/b proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state.[Significance]: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis.This work was supported by Fundacion de Investigacion Medica Mutua Madrileña Automovilista (Fundacion MMA), the Plan Nacional (MEC) BFU2005-00701 and the Fundacion Eugenio Rodriguez Pascual. M.C. was a recipient of a Fmed MMA fellowship.Peer reviewe

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

© 2018 The Author(s). Background: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. Methods: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. Results: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by >25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. Conclusions: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy

Residual ground-water levels of the neonicotinoid thiacloprid perturb chemosensing of Caenorhabditis elegans

© 2017, The Author(s). This study investigated the neurological effects of residual ground-water levels of thiaclopridon the non-target organism Caenorhabditis elegans. Nematodes treated with thiacloprid showed a dose-dependent and significantly increased twitch response at concentrations above 50 ng mL−1 that disabled their forward locomotion in liquid culture. In comparison with untreated controls, 10 ng mL−1 thiacloprid perturbed the chemosensory ability of C. elegans such that the nematodes no longer demonstrated positive chemotaxis towards a NaCl chemo-attractant, reducing their chemotaxis index from +0.48 to near to zero. Nematodes also exhibited a locomotion characteristic of those devoid of chemo-attraction, making significantly more pirouetting turns of ≥90° than the untreated controls. Compared to the untreated controls, expression of the endocytosis-associated gene, Rab-10, was also increased in C. elegans that had developed to adulthood in the presence of 10 ng mL−1 thiacloprid, suggesting their active engagement in increased recycling of affected cellular components, such as their nAChRs. Thus, even residual, low levels of this less potent neonicotinoid that may be found in field ground-water had measurable effects on a beneficial soil organism which may have environmental and ecological implications that are currently poorly understood

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance

© 2014 The Authors. The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A 165 b. Whereas flTIA-1 selectively bound VEGF-A 165 mRNA and increased translation of VEGF-A 165 b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy

The oncogene ERG: A key factor in prostate cancer

© 2016 Macmillan Publishers Limited All rights reserved. ETS-related gene (ERG) is a member of the E-26 transformation-specific (ETS) family of transcription factors with roles in development that include vasculogenesis, angiogenesis, haematopoiesis and bone development. ERG's oncogenic potential is well known because of its involvement in Ewing's sarcoma and leukaemia. However, in the past decade ERG has become highly associated with prostate cancer development, particularly as a result of a gene fusion with the promoter region of the androgen-induced TMPRRSS2 gene. We review ERG's structure and function, and its role in prostate cancer. We discuss potential new therapies that are based on targeting ERG

A report of the first international WT1 meeting, University of Manchester UK, 2008

On September 8th-9th 2008, scientists from Europe, North America and Japan gathered together at the University of Manchester (UK) at a meeting about all things WT1. The intent was to bring together recent advances in research focused on the Wilms' tumor suppressor gene WT1 from different subject areas. The WT1 gene has emerged as a key player in development and cancer in recent years. The subject areas ranged from developmental mouse genetics including kidney development, cancer cell biology, structural analysis and molecular processes and targets regulated by WT1. The meeting was jointly organized by Dr Stefan Roberts (University of Manchester, UK); Dr Michael Ladomery (University of the West of England, UK); and Prof. Nick Hastie (MRC Human Genetics Unit, UK). In this article we review the contents of the meeting, subdividing it into three broad and overlapping subject areas: new findings in the context of A) WT1's role in development; B) WT1's involvement in cancer; and C) the biochemistry of WT1 protein