Reduced levels of prostaglandin I2 synthase: a distinctive feature of the cancer-free trichothiodystrophy.

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 ERCC2/XPD or ERCC3/XPB 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 ERCC2/XPD 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 I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 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 PTGIS 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.

HGF/c-Met Signalling in the Tumor Microenvironment.

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.

Alternative splicing in Alzheimer's disease.

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.

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

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.

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

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.

The alternative splicing side of cancer.

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.

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

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.

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

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.

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

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.

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

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.

Phosphorylation of SRSF1 is modulated by replicational stress.

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.

Cellular stress and RNA splicing.

In response to physical and chemical stresses that affect protein folding and, thus, the execution of normal metabolic processes, cells activate gene-expression strategies aimed at increasing their chance of survival. One target of several stressing agents is pre-mRNA splicing, which is inhibited upon heat shock. Recently, the molecular basis of this splicing inhibition has begun to emerge. Interestingly, different mechanisms seem to be in place to block constitutive pre-mRNA splicing and to affect alternative splicing regulation. This could be important to modulate gene expression during recovery from stress. Thus, pre-mRNA splicing emerges as a central mechanism to integrate cellular and metabolic stresses into gene-expression profiles.

Alternative splicing of tumor suppressors and oncogenes.

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.

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

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.

DNA replication, development and cancer: a homeotic connection?

The homeotic proteins are transcription factors, highly conserved in metazoan organisms, exerting a pivotal role in development and differentiation. They individually display a loose specificity for the DNA sequence they can bind, but operate mainly in multi-molecular associations that assure their target and function specificity. Homeotic proteins are known to play a role in the positive or negative regulation of cell proliferation. Furthermore, many homeotic proteins are actually proto-oncogenes, since different translocations involving their genes cause tumors, particularly in the hematopoietic system. A one-hybrid screen to detect proteins with affinity for the lamin B2 replication origin identified three homeotic proteins, namely HoxA13, HoxC10 and HoxC13. Recent data demonstrate that the HoxC13 oncoprotein specifically associates with replication foci and binds in vitro and in vivo to several human DNA replication origins. Moreover, Hox proteins interact with geminin, a regulator of cell cycle progression, and control the interaction of this protein with the DNA replication licensing factor Ctd1. Thus, the homeotic proteins, by participating directly in the function of DNA replication origins, may provide a direct link between the accurate regulation of DNA replication required by the morphogenetic program and the deregulation of this process typical of cancer.

Homeotic proteins participate in the function of human-DNA replication origins.

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13-origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.

Transcription of Satellite DNAs in Mammals.

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.

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

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.

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

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.

Constitutive heterochromatin: a surprising variety of expressed sequences.

The organization of chromosomes into euchromatin and heterochromatin is amongst the most important and enigmatic aspects of genome evolution. Constitutive heterochromatin is a basic yet still poorly understood component of eukaryotic chromosomes, and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Although recent evidence indicates that the presence of transcribed genes in constitutive heterochromatin is a conserved trait that accompanies the evolution of eukaryotic genomes, the term heterochromatin is still considered by many as synonymous of gene silencing. In this paper, we comprehensively review data that provide a clearer picture of transcribed sequences within constitutive heterochromatin, with a special emphasis on Drosophila and humans.