Publications by authors named "Giuseppe Biamonti"

59 Publications

Post-Translational Modifications Modulate Proteinopathies of TDP-43, FUS and hnRNP-A/B in Amyotrophic Lateral Sclerosis.

Front Mol Biosci 2021 5;8:693325. Epub 2021 Jul 5.

Istituto di Genetica Molecolare "Luigi Luca Cavalli-Sforza" - Consiglio Nazionale delle Ricerce (CNR), Pavia, Italy.

It has been shown that protein low-sequence complexity domains (LCDs) induce liquid-liquid phase separation (LLPS), which is responsible for the formation of membrane-less organelles including P-granules, stress granules and Cajal bodies. Proteins harbouring LCDs are widely represented among RNA binding proteins often mutated in ALS. Indeed, LCDs predispose proteins to a prion-like behaviour due to their tendency to form amyloid-like structures typical of proteinopathies. Protein post-translational modifications (PTMs) can influence phase transition through two main events: i) destabilizing or augmenting multivalent interactions between phase-separating macromolecules; ii) recruiting or excluding other proteins and/or nucleic acids into/from the condensate. In this manuscript we summarize the existing evidence describing how PTM can modulate LLPS thus favouring or counteracting proteinopathies at the base of neurodegeneration in ALS.
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http://dx.doi.org/10.3389/fmolb.2021.693325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8287968PMC
July 2021

Reduced levels of prostaglandin I synthase: a distinctive feature of the cancer-free trichothiodystrophy.

Proc Natl Acad Sci U S A 2021 06;118(26)

Istituto di Genetica Molecolare L.L. Cavalli Sforza, Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy;

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely or Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.
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http://dx.doi.org/10.1073/pnas.2024502118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255998PMC
June 2021

HGF/c-Met Signalling in the Tumor Microenvironment.

Adv Exp Med Biol 2021 ;1270:31-44

Institute of Molecular Genetics (IGM); National Research Council (CNR), Pavia, Italy.

Recently, it has become clearer that tumor plasticity increases the chance that cancer cells could acquire new mechanisms to escape immune surveillance, become resistant to conventional drugs, and spread to distant sites.Effectively, tumor plasticity drives adaptive response of cancer cells to hypoxia and nutrient deprivation leading to stimulation of neoangionesis or tumor escape. Therefore, tumor plasticity is believed to be a great contributor in recurrence and metastatic dissemination of cancer cells. Importantly, it could be an Achilles' heel of cancer if we could identify molecular mechanisms dictating this phenotype.The reactivation of stem-like signalling pathways is considered a great determinant of tumor plasticity; in addition, a key role has been also attributed to tumor microenvironment (TME). Indeed, it has been proved that cancer cells interact with different cells in the surrounding extracellular matrix (ECM). Interestingly, well-established communication represents a potential allied in maintenance of a plastic phenotype in cancer cells supporting tumor growth and spread. An important signalling pathway mediating cancer cell-TME crosstalk is represented by the HGF/c-Met signalling.Here, we review the role of the HGF/c-Met signalling in tumor-stroma crosstalk focusing on novel findings underlying its role in tumor plasticity, immune escape, and development of adaptive mechanisms.
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http://dx.doi.org/10.1007/978-3-030-47189-7_2DOI Listing
January 2021

Brief Report: Neuroimaging Endophenotypes of Social Robotic Applications in Autism Spectrum Disorder.

J Autism Dev Disord 2021 Jul;51(7):2538-2542

Institute for Biomedical Research and Innovation (IRIB), Nationasssl Research Council, Messina, 98164, Italy.

A plethora of neuroimaging studies have focused on the discovery of potential neuroendophenotypes useful to understand the etiopathogenesis of autism and predict treatment response. Social robotics has recently been proposed as an effective tool to strengthen the current treatments in children with autism. However, the high clinical heterogeneity characterizing this disorder might interfere with behavioral effects. Neuroimaging is set to overcome these limitations by capturing the level of heterogeneity. Here, we provide a preliminary evaluation of the neural basis of social robotics and how extracting neural hallmarks useful to design more effective behavioral applications. Despite the endophenotype-oriented neuroimaging research approach is in its relative infancy, this preliminary evidence encourages innovation to address its current limitations.
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http://dx.doi.org/10.1007/s10803-020-04708-9DOI Listing
July 2021

Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs.

Int J Mol Sci 2020 Apr 17;21(8). Epub 2020 Apr 17.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche via Abbiategrasso 207, 27100 Pavia, Italy.

Heat shock activates the transcription of arrays of Satellite III (SatIII) DNA repeats in the pericentromeric heterochromatic domains of specific human chromosomes, the longest of which is on chromosome 9. Long non-coding SatIII RNAs remain associated with transcription sites where they form nuclear stress bodies or nSBs. The biology of SatIII RNAs is still poorly understood. Here, we show that SatIII RNAs and nSBs are detectable up to four days after thermal stress and are linked to defects in chromosome behavior during mitosis. Heat shock perturbs the execution of mitosis. Cells reaching mitosis during the first 3 h of recovery accumulate in pro-metaphase. During the ensuing 48 h, this block is no longer detectable; however, a significant fraction of mitoses shows chromosome segregation defects. Notably, most of lagging chromosomes and chromosomal bridges are bound to nSBs and contain arrays of SatIII DNA. Disappearance of mitotic defects at the end of day 2 coincides with the processing of long non-coding SatIII RNAs into a ladder of small RNAs associated with chromatin and ranging in size from 25 to 75 nt. The production of these molecules does not rely on DICER and Argonaute 2 components of the RNA interference apparatus. Thus, massive transcription of SatIII DNA may contribute to chromosomal instability.
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http://dx.doi.org/10.3390/ijms21082812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216065PMC
April 2020

An Intricate Connection between Alternative Splicing and Phenotypic Plasticity in Development and Cancer.

Cells 2019 Dec 21;9(1). Epub 2019 Dec 21.

Institute of Molecular Genetics (IGM); National Research Council (CNR), 27100 Pavia, Italy.

During tumor progression, hypoxia, nutrient deprivation or changes in the extracellular environment (i.e., induced by anti-cancer drugs) elicit adaptive responses in cancer cells. Cellular plasticity increases the chance that tumor cells may survive in a challenging microenvironment, acquire new mechanisms of resistance to conventional drugs, and spread to distant sites. Re-activation of stem pathways appears as a significant cause of cellular plasticity because it promotes the acquisition of stem-like properties through a profound phenotypic reprogramming of cancer cells. In addition, it is a major contributor to tumor heterogeneity, depending on the coexistence of phenotypically distinct subpopulations in the same tumor bulk. Several cellular mechanisms may drive this fundamental change, in particular, high-throughput sequencing technologies revealed a key role for alternative splicing (AS). Effectively, AS is one of the most important pre-mRNA processes that increases the diversity of transcriptome and proteome in a tissue- and development-dependent manner. Moreover, defective AS has been associated with several human diseases. However, its role in cancer cell plasticity and tumor heterogeneity remains unclear. Therefore, unravelling the intricate relationship between AS and the maintenance of a stem-like phenotype may explain molecular mechanisms underlying cancer cell plasticity and improve cancer diagnosis and treatment.
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http://dx.doi.org/10.3390/cells9010034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016785PMC
December 2019

Alternative splicing in Alzheimer's disease.

Aging Clin Exp Res 2021 Apr 3;33(4):747-758. Epub 2019 Oct 3.

Istituto di Genetica Molecolare "Luigi Luca Cavalli-Sforza", Consiglio Nazionale delle Ricerche (IGM-CNR), via Abbiategrasso, 207, 27100, Pavia, Italy.

Alzheimer's disease (AD) is the most frequent neurodegenerative disorder in the elderly, occurring in approximately 20% of people older than 80. The molecular causes of AD are still poorly understood. However, recent studies have shown that Alternative Splicing (AS) is involved in the gene expression reprogramming associated with the functional changes observed in AD patients. In particular, mutations in cis-acting regulatory sequences as well as alterations in the activity and sub-cellular localization of trans-acting splicing factors and components of the spliceosome machinery are associated with splicing abnormalities in AD tissues, which may influence the onset and progression of the disease. In this review, we discuss the current molecular understanding of how alterations in the AS process contribute to AD pathogenesis. Finally, recent therapeutic approaches targeting aberrant AS regulation in AD are also reviewed.
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http://dx.doi.org/10.1007/s40520-019-01360-xDOI Listing
April 2021

The Krebs Cycle Connection: Reciprocal Influence Between Alternative Splicing Programs and Cell Metabolism.

Front Oncol 2018 26;8:408. Epub 2018 Sep 26.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy.

Alternative splicing is a pervasive mechanism that molds the transcriptome to meet cell and organism needs. However, how this layer of gene expression regulation is coordinated with other aspects of the cell metabolism is still largely undefined. Glucose is the main energy and carbon source of the cell. Not surprisingly, its metabolism is finely tuned to satisfy growth requirements and in response to nutrient availability. A number of studies have begun to unveil the connections between glucose metabolism and splicing programs. Alternative splicing modulates the ratio between M1 and M2 isoforms of pyruvate kinase in this way determining the choice between aerobic glycolysis and complete glucose oxidation in the Krebs cycle. Reciprocally, intermediates in the Krebs cycle may impact splicing programs at different levels by modulating the activity of 2-oxoglutarate-dependent oxidases. In this review we discuss the molecular mechanisms that coordinate alternative splicing programs with glucose metabolism, two aspects with profound implications in human diseases.
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http://dx.doi.org/10.3389/fonc.2018.00408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168629PMC
September 2018

From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

Chem Rev 2018 04 30;118(8):4365-4403. Epub 2018 Mar 30.

IFOM - The FIRC Institute of Molecular Oncology , Milan , 20139 , Italy.

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.
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http://dx.doi.org/10.1021/acs.chemrev.7b00487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717669PMC
April 2018

A missense MT-ND5 mutation in differentiated Parkinson Disease cytoplasmic hybrid induces ROS-dependent DNA Damage Response amplified by DROSHA.

Sci Rep 2017 08 25;7(1):9528. Epub 2017 Aug 25.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (CNR), Pavia, 27100, Italy.

Genome integrity is continuously threatened by endogenous sources of DNA damage including reactive oxygen species (ROS) produced by cell metabolism. Factors of the RNA interference (RNAi) machinery have been recently involved in the cellular response to DNA damage (DDR) in proliferating cells. To investigate the impact of component of RNAi machinery on DDR activation in terminally differentiated cells, we exploited cytoplasmic hybrid (cybrid) cell lines in which mitochondria of sporadic Parkinson's disease patients repopulate neuroblastoma SH-SY5Y-Rho(0) cells. Upon differentiation into dopaminergic neuron-like cells, PD63 cybrid showed increased intracellular level of ROS and chronic DDR activation, compared to other cybrids with the same nuclear background. Importantly, DDR activation in these cells can be prevented by ROS scavenging treatment suggesting that ROS production is indeed causative of nuclear DNA damage. Sequence analysis of the mitogenomes identified a rare and heteroplasmic missense mutation affecting a highly conserved residue of the ND5-subunit of respiratory complex I, which accounts for ROS increase. We demonstrated that the assembly of nuclear DDR foci elicited by oxidative stress in these cells relies on DROSHA, providing the first evidence that components of RNAi machinery play a crucial role also in the mounting of ROS-induced DDR in non-replicating neuronal cells.
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http://dx.doi.org/10.1038/s41598-017-09910-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573376PMC
August 2017

First dual AK/GSK-3β inhibitors endowed with antioxidant properties as multifunctional, potential neuroprotective agents.

Eur J Med Chem 2017 Sep 9;138:438-457. Epub 2017 Jun 9.

European Research Centre for Drug Discovery and Development (NatSynDrugs), Department of Biotechnology, Chemistry, and Pharmacy, Università degli Studi di Siena via Aldo Moro 2, 53100 Siena, Italy.

The manuscript deals with the design, synthesis and biological evaluation of novel benzoxazinone-based and indole-based compounds as multifunctional neuroprotective agents. These compounds inhibit human adenosine kinase (hAK) and human glycogen synthase kinase 3 beta (hGSK-3β) enzymes. Computational analysis based on a molecular docking approach underlined the potential structural requirements for simultaneously targeting both proteins' allosteric sites. In silico hints drove the synthesis of appropriately decorated benzoxazinones and indoles (5a-s, and 6a-c) and biochemical analysis revealed their behavior as allosteric inhibitors of hGSK-3β. For both our hit 4 and the best compounds of the series (5c,l and 6b) the potential antioxidant profile was assessed in human neuroblastoma cell lines (IMR 32, undifferentiated and neuronal differentiated), by evaluating the protective effect of selected compounds against HO cytotoxicity and reactive oxygen species (ROS) production. Results showed a strong efficacy of the tested compounds, even at the lower doses, in counteracting the induced oxidative stress (50 μM of HO) and in preventing ROS formation. In addition, the tested compounds did not show any cytotoxic effect determined by the LDH release, at the concentration range analyzed (from 0.1 to 50 μM). This study allowed the identification of compound 5l, as the first dual hAK/hGSK-3β inhibitor reported to date. Compound 5l, which behaves as an effective antioxidant, holds promise for the development of new series of potential therapeutic agents for the treatment of neurodegenerative diseases characterized by an innovative pharmacological profile.
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http://dx.doi.org/10.1016/j.ejmech.2017.06.017DOI Listing
September 2017

Editorial: DNA and RNA Metabolism Meet at Chromatin to Control Genome Stability.

Front Genet 2016 27;7:67. Epub 2016 Apr 27.

Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare Pavia, Italy.

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http://dx.doi.org/10.3389/fgene.2016.00067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846655PMC
May 2016

Molecular mechanisms of etoposide.

EXCLI J 2015 19;14:95-108. Epub 2015 Jan 19.

Istituto di Genetica Molecolare, CNR, via Abbiategrasso 207, Pavia.

Etoposide derives from podophyllotoxin, a toxin found in the American Mayapple. It was first synthesized in 1966 and approved for cancer therapy in 1983 by the U.S. Food and Drug Administration (Hande, 1998[25]). Starting from 1980s several studies demonstrated that etoposide targets DNA topoisomerase II activities thus leading to the production of DNA breaks and eliciting a response that affects several aspects of cell metabolisms. In this review we will focus on molecular mechanisms that account for the biological effect of etoposide.
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http://dx.doi.org/10.17179/excli2015-561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652635PMC
November 2015

The alternative splicing factor Nova2 regulates vascular development and lumen formation.

Nat Commun 2015 Oct 8;6:8479. Epub 2015 Oct 8.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, via Abbiategrasso 207, Pavia 27100, Italy.

Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the 'angioneurins' family.
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http://dx.doi.org/10.1038/ncomms9479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633719PMC
October 2015

Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells.

PLoS One 2015 7;10(7):e0130561. Epub 2015 Jul 7.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (CNR), Pavia, Italy.

Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130561PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495043PMC
April 2016

Studies on the ATP Binding Site of Fyn Kinase for the Identification of New Inhibitors and Their Evaluation as Potential Agents against Tauopathies and Tumors.

J Med Chem 2015 Jun 20;58(11):4590-609. Epub 2015 May 20.

†Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. De Gasperi 2, I-53100 Siena, Italy.

Fyn is a member of the Src-family of nonreceptor protein-tyrosine kinases. Its abnormal activity has been shown to be related to various human cancers as well as to severe pathologies, such as Alzheimer's and Parkinson's diseases. Herein, a structure-based drug design protocol was employed aimed at identifying novel Fyn inhibitors. Two hits from commercial sources (1, 2) were found active against Fyn with K(i) of about 2 μM, while derivative 4a, derived from our internal library, showed a K(i) of 0.9 μM. A hit-to-lead optimization effort was then initiated on derivative 4a to improve its potency. Slightly modifications rapidly determine an increase in the binding affinity, with the best inhibitors 4c and 4d having K(i)s of 70 and 95 nM, respectively. Both compounds were found able to inhibit the phosphorylation of the protein Tau in an Alzheimer's model cell line and showed antiproliferative activities against different cancer cell lines.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00140DOI Listing
June 2015

hnRNP L inhibits CD44 V10 exon splicing through interacting with its upstream intron.

Biochim Biophys Acta 2015 Jun 24;1849(6):743-50. Epub 2015 Jan 24.

School of life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea. Electronic address:

CD44 is a complex cell adhesion molecule that mediates communication and adhesion between adjacent cells as well as between cells and the extracellular matrix. CD44 pre-mRNA produces various mRNA isoforms through alternative splicing of 20 exons, among which exons 1-5 (C1-C5) and 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. CD44 V10 exon has important roles in breast tumor progression and Hodgkin lymphoma. Here we show that increased expression of hnRNP L inhibits V10 exon splicing of CD44 pre-mRNA, whereas reduced expression of hnRNP L promotes V10 exon splicing. In addition, hnRNP L also promotes V10 splicing of endogenous CD44 pre-mRNA. Through mutation analysis, we demonstrate that the effects of hnRNP L on V10 splicing are abolished when the CA-rich sequence on the upstream intron of V10 exon is disrupted. However, hnRNP L effects are stronger if more CA-repeats are provided. Furthermore, we show that hnRNP L directly contacts the CA-rich sequence. Importantly, we provide evidences that hnRNP L inhibits U2AF65 binding on the upstream Py tract of V10 exon. Our results reveal that hnRNP L is a new regulator for CD44 V10 exon splicing.
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http://dx.doi.org/10.1016/j.bbagrm.2015.01.004DOI Listing
June 2015

DNA-protein interaction dynamics at the Lamin B2 replication origin.

Cell Cycle 2015 ;14(1):64-73

a Molecular Biology Laboratory; Scuola Normale Superiore ; Pisa , Italy.

To date, a complete understanding of the molecular events leading to DNA replication origin activation in mammalian cells still remains elusive. In this work, we report the results of a high resolution chromatin immunoprecipitation study to detect proteins interacting with the human Lamin B2 replication origin. In addition to the pre-RC component ORC4 and to the transcription factors USF and HOXC13, we found that 2 components of the AP-1 transcription factor, c-Fos and c-Jun, are also associated with the origin DNA during the late G1 phase of the cell cycle and that these factors interact with ORC4. Both DNA replication and AP-1 factor binding to the origin region were perturbed by cell treatment with merbarone, a topoisomerase II inhibitor, suggesting that DNA topology is essential for determining origin function.
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http://dx.doi.org/10.4161/15384101.2014.973337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352957PMC
September 2015

SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene.

Biochim Biophys Acta 2014 Nov 8;1839(11):1132-40. Epub 2014 Sep 8.

School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea. Electronic address:

The product of proto-oncogene Ron is a human receptor for the macrophage-stimulating protein (MSP). Upon activation, Ron is able to induce cell dissociation, migration and matrix invasion. Exon 11 skipping of Ron pre-mRNA produces Ron△165 protein that is constitutively active even in the absence of its ligand. Here we show that knockdown of SRSF2 promotes the decrease of exon 11 inclusion, whereas overexpression of SRSF2 promotes exon 11 inclusion. We demonstrate that SRSF2 promotes exon 11 inclusion through splicing and transcription procedure. We also present evidence that reduced expression of SRSF2 induces a decrease in the splicing of both introns 10 and 11; by contrast, overexpression of SRSF2 induces an increase in the splicing of introns 10 and 11. Through mutation analysis, we show that SRSF2 functionally targets and physically interacts with CGAG sequence on exon 11. In addition, we reveal that the weak strength of splice sites of exon 11 is not required for the function of SRSF2 on the splicing of Ron exon 11. Our results indicate that SRSF2 promotes exon 11 inclusion of Ron proto-oncogene through targeting exon 11. Our study provides a novel mechanism by which Ron is expressed.
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http://dx.doi.org/10.1016/j.bbagrm.2014.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535357PMC
November 2014

The alternative splicing side of cancer.

Semin Cell Dev Biol 2014 Aug 19;32:30-6. Epub 2014 Mar 19.

Istituto di Genetica Molecolare - CNR, Via Abbiategrasso 207, 27011 Pavia, Italy.

Alternative splicing emerges as a potent and pervasive mechanism of gene expression regulation that expands the coding capacity of the genome and forms an intermediate layer of regulation between transcriptional and post-translational networks. Indeed, alternative splicing occupies a pivotal position in developmental programs and in the cell response to external and internal stimuli. Not surprisingly, therefore, its deregulation frequently leads to human disease. In this review we provide an updated overview of the impact of alternative splicing on tumorigenesis. Moreover, we discuss the intricacy of the reciprocal interactions between alternative splicing programs and signal transduction pathways, which appear to be crucially linked to cancer progression in response to the tumor microenvironment. Finally, we focus on the recently described interplay between splicing and chromatin organization which is expected to shed new lights into gene expression regulation in normal and cancer cells.
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http://dx.doi.org/10.1016/j.semcdb.2014.03.016DOI Listing
August 2014

CorrelaGenes: a new tool for the interpretation of the human transcriptome.

BMC Bioinformatics 2014 10;15 Suppl 1:S6. Epub 2014 Jan 10.

Background: The amount of gene expression data available in public repositories has grown exponentially in the last years, now requiring new data mining tools to transform them in information easily accessible to biologists.

Results: By exploiting expression data publicly available in the Gene Expression Omnibus (GEO) database, we developed a new bioinformatics tool aimed at the identification of genes whose expression appeared simultaneously altered in different experimental conditions, thus suggesting co-regulation or coordinated action in the same biological process. To accomplish this task, we used the 978 human GEO Curated DataSets and we manually performed the selection of 2,109 pair-wise comparisons based on their biological rationale. The lists of differentially expressed genes, obtained from the selected comparisons, were stored in a PostgreSQL database and used as data source for the CorrelaGenes tool. Our application uses a customized Association Rule Mining (ARM) algorithm to identify sets of genes showing expression profiles correlated with a gene of interest. The significance of the correlation is measured coupling the Lift, a well-known standard ARM index, and the χ(2) p value. The manually curated selection of the comparisons and the developed algorithm constitute a new approach in the field of gene expression profiling studies. Simulation performed on 100 randomly selected target genes allowed us to evaluate the efficiency of the procedure and to obtain preliminary data demonstrating the consistency of the results.

Conclusions: The preliminary results of the simulation showed how CorrelaGenes could contribute to the characterization of molecular pathways and biological processes integrating data obtained from other applications and available in public repositories.
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http://dx.doi.org/10.1186/1471-2105-15-S1-S6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016313PMC
May 2014

Oncogenic alternative splicing switches: role in cancer progression and prospects for therapy.

Int J Cell Biol 2013 27;2013:962038. Epub 2013 Oct 27.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Via Abbiategrasso 207, 27100 Pavia, Italy.

Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments.
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http://dx.doi.org/10.1155/2013/962038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826442PMC
November 2013

Alternative splicing of tumor suppressors and oncogenes.

Cancer Treat Res 2013 ;158:95-117

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, 27100, Italy.

Alternative splicing is a fundamental mechanism to modulate gene expression programs in response to different growth and environmental stimuli. There is now ample evidence that alternative splicing errors, caused by mutations in cis-acting elements and defects and/or imbalances in trans-acting factors, may be causatively associated to cancer progression. Recent work indicates the existence of an intricate network of interactions between alternative splicing events and signal transduction pathways. In this network, splicing factors occupy a central position and appear to function both as targets and effectors of regulatory circuits. Thus, a change in their activity deeply affects alternative splicing profiles and hence the cell behavior. Here, we discuss a number of cases that exemplify the involvement of deregulated alternative splicing in tumor progression.
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http://dx.doi.org/10.1007/978-3-642-31659-3_4DOI Listing
April 2016

A 2-nt RNA enhancer on exon 11 promotes exon 11 inclusion of the Ron proto-oncogene.

Oncol Rep 2014 Jan 5;31(1):450-5. Epub 2013 Nov 5.

School of life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.

Ron is a human receptor for the macrophage-stimulating protein (MSP). Exon 11 skipping of Ron pre-mRNA produces the Ron∆165 protein that has a deletion of a 49 amino acid region in the β-chain extracellular domain. Ron∆165 is constitutively active even in the absence of its ligand. Through stepwise deletion analysis, we identified a 2-nt RNA enhancer, which is located 74 nt upstream from the 5' splice site of exon 11, for exon 11 inclusion. Through double-base and single-base substitution analysis of the 2-nt RNA, we demonstrated that the GA, CC, UG and AC dinucleotides on exon 11, in addition to the wild-type AG sequence, function as enhancers for exon 11 inclusion of the Ron pre-mRNA.
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http://dx.doi.org/10.3892/or.2013.2835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528306PMC
January 2014

HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition.

Nucleic Acids Res 2013 Oct 17;41(18):8665-79. Epub 2013 Jul 17.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), 27100 Pavia, Italy, International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy and Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Dulbecco Telethon Institute at San Raffaele Scientific Institute, 20132 Milan, Italy.

Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. ΔRon, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of ΔRon and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of ΔRon, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote ΔRon production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.
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http://dx.doi.org/10.1093/nar/gkt579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794575PMC
October 2013

Pre-mRNA processing factors meet the DNA damage response.

Front Genet 2013 6;4:102. Epub 2013 Jun 6.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche Pavia, Italy.

It is well-known that DNA-damaging agents induce genome instability, but only recently have we begun to appreciate that chromosomes are fragile per se and frequently subject to DNA breakage. DNA replication further magnifies such fragility, because it leads to accumulation of single-stranded DNA. Recent findings suggest that chromosome fragility is similarly increased during transcription. Transcripts produced by RNA polymerase II (RNAPII) are subject to multiple processing steps, including maturation of 5' and 3' ends and splicing, followed by transport to the cytoplasm. RNA maturation starts on nascent transcripts and is mediated by a number of diverse proteins and ribonucleoprotein particles some of which are recruited cotranscriptionally through interactions with the carboxy-terminal domain of RNAPII. This coupling is thought to maximize efficiency of pre-mRNA maturation and directly impacts the choice of alternative splice sites. Mounting evidence suggests that lack of coordination among different RNA maturation steps, by perturbing the interaction of nascent transcripts with the DNA template, has deleterious effects on genome stability. Thus, in the absence of proper surveillance mechanisms, transcription could be a major source of DNA damage in cancer. Recent high-throughput screenings in human cells and budding yeast have identified several factors implicated in RNA metabolism that are targets of DNA damage checkpoint kinases: ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3 related) (Tel1 and Mec1 in budding yeast, respectively). Moreover, inactivation of various RNA processing factors induces accumulation of γH2AX foci, an early sign of DNA damage. Thus, a complex network is emerging that links DNA repair and RNA metabolism. In this review we provide a comprehensive overview of the role played by pre-mRNA processing factors in the cell response to DNA damage and in the maintenance of genome stability.
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http://dx.doi.org/10.3389/fgene.2013.00102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674313PMC
June 2013

Alternative splicing and cancer.

J Nucleic Acids 2012 20;2012:363809. Epub 2012 May 20.

Inserm U1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, 69008 Lyon, France.

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http://dx.doi.org/10.1155/2012/363809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364008PMC
August 2012

Making alternative splicing decisions during epithelial-to-mesenchymal transition (EMT).

Cell Mol Life Sci 2012 Aug 19;69(15):2515-26. Epub 2012 Feb 19.

Istituto di Genetica Molecolare - Consiglio Nazionale delle Ricerche, Pavia, Italy.

Alternative splicing generates multiple mRNAs from a single transcript and is a major contributor to proteomic diversity and to the control of gene expression in complex organisms. Not surprisingly, this post-transcriptional event is tightly regulated in different tissues and developmental stages. An increasing body of evidences supports a causative role of aberrant alternative splicing in cancer. However, very little is known about its impact on cellular processes crucially involved in tumor progression. The aim of this review is to discuss the link between alternative splicing and the epithelial-to-mesenchymal transition (EMT), one of the major routes by which cancer cells acquire invasive capabilities and become metastatic. We begin with a brief overview of alternative splicing. Next, we discuss alternative splicing factors that regulate EMT. Finally, we provide examples of target genes presenting alternative splicing changes that contribute to the morphological conversions in the EMT process.
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http://dx.doi.org/10.1007/s00018-012-0931-7DOI Listing
August 2012

Phosphorylation of SRSF1 is modulated by replicational stress.

Nucleic Acids Res 2012 Feb 7;40(3):1106-17. Epub 2011 Oct 7.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy.

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.
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http://dx.doi.org/10.1093/nar/gkr837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3273819PMC
February 2012

Transcription of Satellite DNAs in Mammals.

Prog Mol Subcell Biol 2011 ;51:95-118

INSERM U823; Institut Albert Bonniot, Université Joseph Fourier-Grenoble, La Tronche BP170, 38042, Grenoble cedex 9, France,

Centromeric and pericentric regions have long been regarded as transcriptionally inert portions of chromosomes. A number of studies in the past 10 years disproved this dogma and provided convincing evidence that centromeric and pericentric sequences are transcriptionally active in several biological contexts.In this chapter, we provide a comprehensive picture of the various contexts (cell growth and differentiation, stress, effect of chromatin organization) in which these sequences are expressed in mouse and human cells and discuss the possible functional implications of centromeric and pericentric sequences activation and/or of the resulting noncoding RNAs. Moreover, we provide an overview of the molecular mechanisms underlying the activation of centromeric and pericentromeric sequences as well as the structural features of encoded RNAs.
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http://dx.doi.org/10.1007/978-3-642-16502-3_5DOI Listing
April 2016
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