Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications.

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

Regulation of fatty acid synthase (FAS) and apoptosis in estrogen-receptor positive and negative breast cancer cells by conjugated linoleic acids.

BACKGROUND: Conjugated linoleic acids (CLAs) are natural dairy food components that exhibit a unique body of potential health benefits in animals and man, including anti-cardiovascular disease and anti-cancer effects. Several studies have demonstrated that fatty acid synthase (FAS) levels (protein and mRNA) are over expressed in many carcinomas. Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate genes involved in lipid metabolism, including FAS. METHODS: Breast cancer cell lines, MCF-7 and MDA-MB-231 were treated with CLAs to investigate the regulation of SREBP-1c and FAS expression. RESULTS: In MDA-MB-231 cells, SREBP-1c and FAS were co-ordinately decreased by treatment with 25 µM CLA 9-11 and 10-12. In MCF-7 cells, the decrease in SREBP-1c and FAS expression was dependant on the concentration of CLA used. CONCLUSIONS: The data suggest a differential effect of CLAs on SREBP-1c and FAS in estrogen receptor-positive (MCF-7) compared to estrogen receptor-negative (MDA-MB-231) breast cancer cells.

Regulation of adhesion molecule expression in human endothelial and smooth muscle cells by omega-3 fatty acids and conjugated linoleic acids: involvement of the transcription factor NF-kappaB?

We previously showed conjugated linoleic acids (CLA) inhibited TNF-alpha-induced monocyte (THP-1) adhesion to human umbilical vein endothelial cells (HUVEC) in vitro which involved an increase in platelet activating factor (PAF). Here we show adhesion molecule (ADM) regulation by fatty acids and the differing role of nuclear factor kappa B (NF-kappaB) activation in HUVEC and vascular smooth muscle cells (vSMC). CLA and omega-3 long-chain polyunsaturated fatty acids (PUFA) (FA) reduced TNF-alpha-induced expression of ADMs (intercellular adhesion molecule-1 (ICAM-1); vascular cell adhesion molecule-1 (VCAM-1) but not E-selectin) on HUVEC and vSMC to different extents depending on FA type and concentration, cell type and method of analysis. IkappaBalpha phosphorylation in HUVEC and vSMC and transient transfection with NF-kappaB-luciferase reporter plasmid (HUVEC only) indicated differential NF-kappaB involvement during FA modulation (cis-9, trans-11; trans-10, cis-12 and a 50:50 mix of both CLA isomers; eicosapentaenoic acid (EPA); docosahexaenoic acid (DHA)). TNF-alpha-induced ADM expression in both cell types by 2-10-fold. In HUVEC, CLA t10, c12 and CLA mix (50:50 mixture of CLA c9, t11 and t10, c12) and EPA and DHA reduced ICAM-1 expression (15-35%) at 12.5, 25 and/or 50 microM. VCAM-1 expression was reduced by 25 microM t10, c12 isomer and mix; omega-3 PUFA and other concentrations of CLA and TNF-alpha-induced E-selectin expression were unaffected. TNFalpha-induced inhibitor kappa B (IkappaB) phosphorylation was biphasic peaking at 5 min in both cell types and 60 and 120 min in HUVEC and SMC, respectively. IkappaBalpha phosphorylation and NF-kappaB activity was reduced (29% and 30%, respectively) by 25 microM CLA mix. n-3 PUFA did not reduce IkappaBalpha phosphorylation or NF-kappaB activity but reduced ADM expression. We show that n-3 PUFA and CLA reduce expression of ADM on HUVEC and vSMC. This reflected reduced adherence of monocytes to HUVEC previously reported by our group. Reduction of ICAM-1 and VCAM-1 protein expression by n-3 PUFA was less dependent on the NF-kappaB pathway than reduction by CLA which reflected the parallel attenuation of NF-kappaB activity. This indicated involvement of other transcription factors (i.e. AP-1) in the FA regulation of ADM expression and has, to our knowledge, not been previously reported.

Fatty acid regulation of protein kinase C isoforms in prostate cancer cells.

Polyunsaturated fatty acids influence the aetiology of prostate cancer. Their effects on cellular mechanisms regulating prostate tumorigenesis are unclear. Using prostate cancer cells (LNCaP), we determined effects of n-9-OA, n-6-LA, and n-3-EPA on total PKC and its isoforms in relation to cell proliferation and PSA production. PKC-alpha, delta, gamma, iota, mu, and zeta were present in LNCaP cells; PKC-beta, epsilon, eta, and theta isoforms were not. PKC-alpha was detected only in cytosol; PKC-delta, iota, gamma, and mu were present in cytosol and in membranes. Fatty acids increased cell proliferation, total PKC activity and elicited pro-proliferative effects on specific PKC isoforms (PKC-delta and -iota). EPA and LA increased total PKC activity and reduced membrane-abundance of PKC-delta. OA reduced cytosolic and membrane PKC-delta. Only EPA reduced PKC-gamma membrane abundance. Fatty acids enhanced cytosolic PKC-iota abundance but only EPA and to a lesser extent LA increased its membrane content. Changes in PKC-delta, -iota, and -gamma did not affect PSA production.

Isolation of the human genes encoding the pyst1 and Pyst2 phosphatases: characterisation of Pyst2 as a cytosolic dual-specificity MAP kinase phosphatase and its catalytic activation by both MAP and SAP kinases.

We have isolated the human genes encoding the Pyst1 (MKP-3) and Pyst2 (MKP-X) MAP kinase phosphatases. Both genes consist of three exons interrupted by two introns and lack an intron which is conserved in all the other members of this gene family characterised to date. This reinforces the conclusion that Pyst1 and Pyst2 are members of a distinct and structurally homologous subfamily of dual-specificity (Thr/Tyr) MAP kinase phosphatases. We find that Pyst2 mRNA is constitutively expressed in a wide variety of human cell lines including those derived from ovarian, bladder and breast cancers. While there is no evidence for inducible expression of Pyst2 mRNA in human skin fibroblasts in response to cellular stress, Pyst2 mRNA levels are moderately increased in response to serum stimulation. Pyst2 protein is predominantly cytosolic when expressed in COS-1 cells. In common with Pyst1, Pyst2 shows substrate selectivity for the classical p42 (ERK2) isoform of MAP kinase both in vitro and in vivo, displaying much reduced activity towards stress activated MAP kinase isoforms such as JNK-1 and p38/RK. Pyst2 binds p42 MAP kinase in vivo and both MAP kinase binding and substrate selectivity correlate with the ability of different recombinant MAP and SAP kinases to cause catalytic activation of the Pyst2 phosphatase in vitro.

Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.

Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.