• A platform independent RNA-Seq protocol for the detection of transcriptome complexity.Calabrese C, Mangiulli M, Manzari C, Paluscio AM, Caratozzolo MF, Marzano F, Kurelac I, D’Erchia AM, D’Elia D, Licciulli F, Liuni S, Picardi E, Attimonelli M, Gasparre G, Porcelli AM, Pesole G, Sbisà E, Tullo A.

    BMC Genomics. 2013 Dec 5;14:855. doi: 10.1186/1471-2164-14-855.

    (PMID: 24308330)


    Background: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform.

    Results: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol.

    Conclusion: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.

  • Respiratory complex I is essential to induce a Warburg profile in mitochondria-defective tumor cells.Calabrese C, Iommarini L, Kurelac I, Calvaruso MA, Capristo M, Lollini PL, Nanni P, Bergamini C, Nicoletti G, Giovanni CD, Ghelli A, Giorgio V, Caratozzolo MF, Marzano F, Manzari C, Betts CM, Carelli V, Ceccarelli C, Attimonelli M, Romeo G, Fato R, Rugolo M, Tullo A, Gasparre G, Porcelli AM.

    Cancer Metab. 2013 Mar 18;1(1):11. doi: 10.1186/2049-3002-1-11.

    (PMID: 24280190)


    Background: Aerobic glycolysis, namely the Warburg effect, is the main hallmark of cancer cells. Mitochondrial respiratory dysfunction has been proposed to be one of the major causes for such glycolytic shift. This hypothesis has been revisited as tumors appear to undergo waves of gene regulation during progression, some of which rely on functional mitochondria. In this framework, the role of mitochondrial complex I is still debated, in particular with respect to the effect of mitochondrial DNA mutations in cancer metabolism. The aim of this work is to provide the proof of concept that functional complex I is necessary to sustain tumor progression.

    METHODS: Complex I-null osteosarcoma cells were complemented with allotopically expressed complex I subunit 1 (MT-ND1). Complex I re-assembly and function recovery, also in terms of NADH consumption, were assessed. Clones were tested for their ability to grow in soft agar and to generate tumor masses in nude mice. Hypoxia levels were evaluated via pimonidazole staining and hypoxia-inducible factor-1α (HIF-1α) immunoblotting and histochemical staining. 454-pyrosequencing was implemented to obtain global transcriptomic profiling of allotopic and non-allotopic xenografts.

    RESULTS: Complementation of a truncative mutation in the gene encoding MT-ND1, showed that a functional enzyme was required to perform the glycolytic shift during the hypoxia response and to induce a Warburg profile in vitro and in vivo, fostering cancer progression. Such trigger was mediated by HIF-1α, whose stabilization was regulated after recovery of the balance between α-ketoglutarate and succinate due to a recuperation of NADH consumption that followed complex I rescue.

    CONCLUSION: Respiratory complex I is essential for the induction of Warburg effect and adaptation to hypoxia of cancer cells, allowing them to sustain tumor growth. Differently from other mitochondrial tumor suppressor genes, therefore, a complex I severe mutation such as the one here reported may confer anti-tumorigenic properties, highlighting the prognostic values of such genetic markers in cancer.

  • Genome-wide analysis of differentially expressed genes and splicing isoforms in clear cell renal cell carcinoma.Valletti A, Gigante M, Palumbo O, Carella M, Divella C, Sbisà E, Tullo A, Picardi E, D’Erchia AM, Battaglia M, Gesualdo L, Pesole G, Ranieri E.

    PLoS One. 2013 Oct 23;8(10):e78452. doi: 10.1371/journal.pone.0078452. eCollection 2013.

    (PMID: 24194935)


    Clear cell renal cell carcinoma (ccRCC) is the most common malignant renal epithelial tumor and also the most deadly. To identify molecular changes occurring in ccRCC, in the present study we performed a genome wide analysis of its entire complement of mRNAs. Gene and exon-level analyses were carried out by means of the Affymetrix Exon Array platform. To achieve a reliable detection of differentially expressed cassette exons we implemented a novel methodology that considered contiguous combinations of exon triplets and candidate differentially expressed cassette exons were identified when the expression level was significantly different only in the central exon of the triplet. More detailed analyses were performed for selected genes using quantitative RT-PCR and confocal laser scanning microscopy. Our analysis detected over 2,000 differentially expressed genes, and about 250 genes alternatively spliced and showed differential inclusion of specific cassette exons comparing tumor and non-tumoral tissues. We demonstrated the presence in ccRCC of an altered expression of the PTP4A3, LAMA4, KCNJ1 and TCF21 genes (at both transcript and protein level). Furthermore, we confirmed, at the mRNA level, the involvement of CAV2 and SFRP genes that have previously been identified. At exon level, among potential candidates we validated a differentially included cassette exon in DAB2 gene with a significant increase of DAB2 p96 splice variant as compared to the p67 isoform. Based on the results obtained, and their robustness according to both statistical analysis and literature surveys, we believe that a combination of gene/isoform expression signature may remarkably contribute, after suitable validation, to a more effective and reliable definition of molecular biomarkers for ccRCC early diagnosis, prognosis and prediction of therapeutic response.

  • Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α.Bartoletti-Stella A, Mariani E, Kurelac I, Maresca A, Caratozzolo MF, Iommarini L, Carelli V, Eusebi LH, Guido A, Cenacchi G, Fuccio L, Rugolo M, Tullo A, Porcelli AM, Gasparre G.

    Cell Death Dis. 2013 Jun 13;4:e663. doi: 10.1038/cddis.2013.187.

    (PMID: 23764844)


    Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1β inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

  • Regulation of the expression of CLU isoforms in endometrial proliferative diseases.Fuzio P, Valletti A, Napoli A, Napoli G, Cormio G, Selvaggi L, Liuni S, Pesole G, Maiorano E, Perlino E.

    Int J Oncol. 2013 Jun;42(6):1929-44. doi: 10.3892/ijo.2013.1894. Epub 2013 Apr 11.

    (PMID: 23589125)


    Clusterin (CLU) is a nearly ubiquitous multifunctional protein synthesized in different functionally divergent isoforms, sCLU and nCLU, playing a crucial role by keeping a balance between cell proliferation and death. Studying in vivo CLU expression we found a higher mRNA expression both in neoplastic and hyperplastic tissues in comparison to normal endometria; in particular, by RT-qPCR we demonstrated an increase of the specific sCLU isoform in the neoplastic and hyperplastic endometrial diseases. On the contrary, no CLU increase was detected at the protein level. The CLU gene transcriptional activity was upregulated in the hyperplastic and neoplastic tissues, indicating the existence of a fine post-trans-criptional regulation of CLU expression possibly responsible for the protein decrease in the malignant disease. A specific CLU immunoreactivity was present in all the endometrial glandular cells in comparison to the other cellular compartments where CLU immunoreactivity was lower or absent. In conclusion, our results suggest the existence of a complex regulatory mechanism of CLU gene expression during the progression from normal to malignant cells, possibly contributing to endometrial carcinogenesis. Moreover, the specific alteration of the sCLU:nCLU ratio associated with the pathological stage, suggests a possible usage of CLU as molecular biomarker for the diagnosis/prognosis of endometrial proliferative diseases.

  • BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.Vieux-Rochas M, Bouhali K, Mantero S, Garaffo G, Provero P, Astigiano S, Barbieri O, Caratozzolo MF, Tullo A, Guerrini L, Lallemand Y, Robert B, Levi G, Merlo GR.

    PLoS One. 2013;8(1):e51700. doi: 10.1371/journal.pone.0051700. Epub 2013 Jan 29.

    (PMID: 23382810)


    The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

  • A novel biclustering algorithm for the discovery of meaningful biological correlations between miRNAs and mRNAs
    Pio G, Ceci M, D’Elia D, Loglisci C, Malerba D
    BMC Bioinformatics 2013, 14(Suppl 7):S8 doi:10.1186/1471-2105-14-S7-S8. Epub 2013 Apr 22.

    Background: microRNAs (miRNAs) are a class of small non-coding RNAs which have been recognized as ubiquitous post-transcriptional regulators. The analysis of interactions between different miRNAs and their target genes is necessary for the understanding of miRNAs’ role in the control of cell life and death. In this paper we propose a novel data mining algorithm, called HOCCLUS2, specifically designed to bicluster miRNAs and target messenger RNAs (mRNAs) on the basis of their experimentally-verified and/or predicted interactions. Indeed, existing biclustering approaches, typically used to analyze gene expression data, fail when applied to miRNA:mRNA interactions since they usually do not extract possibly overlapping biclusters (miRNAs and their target genes may have multiple roles), extract a huge amount of biclusters (difficult to browse and rank on the basis of their importance) and work on similarities of feature values (do not limit the analysis to reliable interactions).

    ResultsTo overcome these limitations, HOCCLUS2 i) extracts possibly overlapping biclusters, to catch multiple roles of both miRNAs and their target genes; ii) extracts hierarchically organized biclusters, to facilitate bicluster browsing and to distinguish between universe and pathway-specific miRNAs; iii) extracts highly cohesive biclusters, to consider only reliable interactions; iv) ranks biclusters according to the functional similarities, computed on the basis of Gene Ontology, to facilitate bicluster analysis.

    ConclusionsOur results show that HOCCLUS2 is a valid tool to support biologists in the identification of context-specific miRNAs regulatory modules and in the detection of possibly unknown miRNAs target genes. Indeed, results prove that HOCCLUS2 is able to extract cohesiveness-preserving biclusters, when compared with competitive approaches, and statistically confirm (at a confidence level of 99%) that mRNAs which belong to the same biclusters are, on average, more functionally similar than mRNAs which belong to different biclusters. Finally, the hierarchy of biclusters provides useful insights to understand the intrinsic hierarchical organization of miRNAs and their potential multiple interactions on target genes.

  • Semi- supervised ensemble learning to boost miRNA target predictions.
    Gianvito Pio, Domenica D’Elia, Donato Malerba, Michelangelo Ceci
    EMBnet.journal 2013, 19(A):74-75. DOI: 10.14806/ej.19.A.669

    The huge amount of data produced by the advent of Next Generation Sequencing (NGS) technologies is providing scientists with an unprecedented potential to investigate and shed light on remote secrets of genomes. We have developed a new tool based on biclustering techniques, i.e. HOCCLUS2 which is able to significantly correlate multiple miRNAs and their predicted targets to detect potential miRNA:mRNA regulatory modules. However, experiments performed on predicted interactions led to observe that the noise (i.e., false positives) introduced by prediction algorithms can substantially affect the significance of the discovered modules. In order to overcome this issue, we have developed a probabilistic method which is able to build a more reliable dataset, combining data produced by several well-known prediction algorithms. The main goal of this work is to combine the prediction score of several prediction algorithms in a single stronger classifier, in order to improve the reliability of the obtained predictions. This tool could greatly help in the interpretation of NGS miRNAs profile analysis with respect to their effects by using genome-wide predictions of their targets.

  • 2013 Annual General Meeting: Publicity & Public Relations Committee Report.
    Domenica D’Elia
    EMBnet.journal 2013, 19(1):28-29.

    On May 2013 the Publicity & Public Relation Project Committee (P&PR PC), elected during the EMBnet AGM 2010, brings to conclusion its three-year mandate. This article reports the committee’s activity during the last year (May 2012-2013) and also makes a summary of the overall committee’s achievements as compared with the objectives set out in the business plan drafted at the beginning of its mandate in 2010.