Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis

SMfinder: Small molecules finder for metabolomics and lipidomics analysis

Metabolomics and lipidomics studies are becoming increasingly popular but available tools for automated data analysis are still limited. The major issue in untargeted metabolomics is linked to the lack of efficient ranking methods allowing accurate identification of metabolites. Herein, we provide a user-friendly open-source software, named SMfinder, for the robust identification and quantification of small molecules. The software introduces an MS2 false discovery rate approach, which is based on single spectral permutation and increases identification accuracy. SMfinder can be efficiently applied to shotgun and targeted analysis in metabolomics and lipidomics without requiring extensive in-house acquisition of standards as it provides accurate identification by using available MS2 libraries in instrument independent manner. The software, downloadable at www.ifom.eu/SMfinder, is suitable for untargeted, targeted, and flux analysis

Proteomic characterization of grapevine resistance against downy mildew activated by Trichoderma harzianum T39

The host-specific oomycete Plasmopara viticola is the causal agent of downy mildew, one of the most important grapevine diseases. In recent years, several molecules able to induce a plant-mediated resistance against P. viticola have been described. Some inducers, i.e. benzothiaziadole (BTH), activate a direct resistance involving constitutive barriers and local and systemic defences, with a high metabolic cost for the plant. On the other hand Trichoderma harzianum T39, a beneficial microorganism, primes for defence against grapevine downy mildew. This mechanism presents advantages in terms of energy costs for the plant, because defence responses are expressed upon pathogen attack and, therefore, only when they are really needed. The present study aims to understand cellular processes involved in reaction to P. viticola inoculation in plants pre-treated with the biocontrol agent T. harzianum T39 in comparison to the water-treated and BTH-treated ones. By monitoring the kinetic of intercellular colonization of P. viticola, callose and lignin deposition in grapevine cell wall and the production of reactive oxygen species, we demonstrated a priming mechanism for the post-invasion reactions in T39- and BTH-treated plants with difference in timing and magnitude of responses. Moreover, to explore the processes involved in grapevine self-defence induced by T. harzianum T39 before and after pathogen inoculation, a proteomic approach was used following an 8-plex differential mass tags iTRAQ protocol. Among proteins identified and quantified by LC-MS-MS (more than 900), 89 were significantly modulated 1 day post P. viticola inoculation in water-treated plants. Eighty-three proteins were directly modulated by T39 prior pathogen infection and 104 in T39-treated plants at 1 day post P. viticola inoculation. A general re-direction of primary and secondary metabolism, together with a reaction involving early steps of recognition (receptors and downstream signalling molecules) were induced by P. viticola within the first day of infection in water-treated plants. On the contrary a general number of proteins including hormones signalling, pathogenesis related proteins and oxidative stress-related proteins, were induced or primed for induction upon pathogen inoculation in T39-treated plants. All these results offer a greater understand of the mechanisms underlying the grapevine T39-induced resistance and illustrate the priming effect of T39 on both defence-related proteins abundance and constitutive barriers formation. Further research will be focused on a detailed functional characterization of potential elicitors of grapevine defence mechanism

Dissecting the tripartite interaction between Vitis vinifera, Plasmopara viticola and the biocontrol agent Trichoderma harzianum T39

The biocontrol agent Trichoderma harzianum T39 reduce downy mildew severity on susceptible grapevines by inducing plant resistance. By cytological and molecular approaches, we aimed to identify mechanisms responsible of the grapevine self-defence. T39 resulted to induce a direct modulation of the receptor and recognition plant-machinery and activates a priming state for enhanced expression of stress-related proteins after pathogen inoculation

Phosphoproteomic analysis of induced resistance reveals activation of signal transduction processes by beneficial and pathogenic interaction in grapevine

Protein phosphorylation regulates several key processes of the plant immune system. Protein kinases and phosphatases are pivotal regulators of defense mechanisms elicited by resistance inducers. However, the phosphorylation cascades that trigger the induced resistance mechanisms in plants have not yet been deeply investigated. The beneficial fungus Trichoderma harzianum T39 (T39) induces resistance against grapevine downy mildew (Plasmopara viticola), but its efficacy could be further improved by a better understanding of the cellular regulations involved. We investigated quantitative changes in the grapevine phosphoproteome during T39-induced resistance to get an overview of regulatory mechanisms of downy mildew resistance. Immunodetection experiments revealed activation of the 45 and 49 kDa kinases by T39 treatment both before and after pathogen inoculation, and the phosphoproteomic analysis identified 103 phosphopeptides that were significantly affected by the phosphorylation cascades during T39-induced resistance. Peptides affected by T39 treatment showed comparable phosphorylation levels after P. viticola inoculation, indicating activation of the microbial recognition machinery before pathogen infection. Phosphorylation profiles of proteins related to photosynthetic processes and protein ubiquitination indicated a partial overlap of cellular responses in T39-treated and control plants. However, phosphorylation changes of proteins involved in response to stimuli, signal transduction, hormone signaling, gene expression regulation, and RNA metabolism were exclusively elicited by P. viticola inoculation in T39-treated plants. These results highlighted the relevance of phosphorylation changes during T39-induced resistance and identified key regulator candidates of the grapevine defense against downy mildew

Molecular characterisation of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp. TA144.

The Antarctic Gram-negative bacterium Psychrobacter sp. TA144 contains two small cryptic plasmids, called pTAUp and pTADw. pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway. Several attempts to produce the wild-type biologically active PsyRep in Escherichia coil failed, possibly due to auto-regulation of the protein population. However, the serendipitous occurrence of a Frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form. The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization. The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori(+) sequence. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved