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Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of a RBP to specific regions of its target RNAs in adherent human cell lines.

SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein-RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Global transcriptome reprogramming during spermatogenesis ensures timely expression of factors in each phase of male germ cell differentiation. Spermatocytes and spermatids require particularly extensive reprogramming of gene expression to switch from mitosis to meiosis and to support gamete morphogenesis. Here, we uncovered an extensive alternative splicing program during this transmeiotic differentiation. Notably, intron retention was largely the most enriched pattern, with spermatocytes showing generally higher levels of retention compared with spermatids. Retained introns are characterized by weak splice sites and are enriched in genes with strong relevance for gamete function. Meiotic intron-retaining transcripts (IRTs) were exclusively localized in the nucleus. However, differently from other developmentally regulated IRTs, they are stable RNAs, showing longer half-life than properly spliced transcripts. Strikingly, fate-mapping experiments revealed that IRTs are recruited onto polyribosomes days after synthesis. These studies reveal an unexpected function for regulated intron retention in modulation of the timely expression of select transcripts during spermatogenesis., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epithelial-to-mesenchymal transition (EMT) is associated with metastasis formation as well as with generation and maintenance of cancer stem cells. In this way, EMT contributes to tumor invasion, heterogeneity and chemoresistance. Morphological and functional changes involved in these processes require robust reprogramming of gene expression, which is only partially accomplished at the transcriptional level. Alternative splicing is another essential layer of gene expression regulation that expands the cell proteome. This step in post-transcriptional regulation of gene expression tightly controls cell identity between epithelial and mesenchymal states and during stem cell differentiation. Importantly, dysregulation of splicing factor function and cancer-specific splicing isoform expression frequently occurs in human tumors, suggesting the importance of alternative splicing regulation for cancer biology.In this review, we briefly discuss the role of EMT programs in development, stem cell differentiation and cancer progression. Next, we focus on selected examples of key factors involved in EMT and stem cell differentiation that are regulated post-transcriptionally through alternative splicing mechanisms. Lastly, we describe relevant oncogenic splice-variants that directly orchestrate cancer stem cell biology and tumor EMT, which may be envisioned as novel targets for therapeutic intervention.

The brain is made up of trillions of synaptic connections that together form neural networks needed for normal brain function and behavior. SLM2 is a member of a conserved family of RNA binding proteins, including Sam68 and SLM1, that control splicing of Neurexin1-3 pre-mRNAs. Whether SLM2 affects neural network activity is unknown. Here, we find that SLM2 levels are maintained by a homeostatic feedback control pathway that predates the divergence of SLM2 and Sam68. SLM2 also controls the splicing of Tomosyn2, LysoPLD/ATX, Dgkb, Kif21a, and Cask, each of which are important for synapse function. Cortical neural network activity dependent on synaptic connections between SLM2-expressing-pyramidal neurons and interneurons is decreased in Slm2-null mice. Additionally, these mice are anxious and have a decreased ability to recognize novel objects. Our data reveal a pathway of SLM2 homeostatic auto-regulation controlling brain network activity and behavior., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold., (© 2016 Nazio et al.)

We studied the thermal physiology of the Andean lizard Stenocercus guentheri in order to evaluate the possible effects of global warming on this species. We determined the preferred body temperature (T pref ), critical thermals (CTmin, CTmax), and hours of restriction and activity. T pref was 32.14±1.83°C; CTmin was 8.31°C in adults and 9.14°C in juveniles, whereas CTmax was 43.28°C in adults and 41.68°C in juveniles. To assess extinction risk, we used the model created by Sinervo et al. (2010) and predicted that 16.7% of populations will have a high risk of extinction by 2020, with an increase to 26.7% by 2050. These results suggest that this species, despite being able to maintain its T pref through behavioral thermoregulation and habitat selection, could be physiologically sensitive to climate warming; thus, the potential for local adaptation may be limited under a warmer climate. Further studies focusing on the ability of S. guentheri to evolve higher T pref and thermal tolerances are needed to understand the ability of this species to respond to climate change., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Most human genes encode multiple mRNA variants and protein products through alternative splicing of exons and introns during pre-mRNA processing. In this way, alternative splicing amplifies enormously the coding potential of the human genome and represents a powerful evolutionary resource. Nonetheless, the plasticity of its regulation is prone to errors and defective splicing underlies a large number of inherited and sporadic diseases, including cancer. One key cellular process affected by alternative splicing is the programmed cell death or apoptosis. Many apoptotic genes encode for splice variants having opposite roles in cell survival. This regulation modulates cell and tissue homeostasis and is implicated in both developmental and pathological processes. Furthermore, recent evidence has also unveiled splicing-mediated regulation of genes involved in autophagy, another essential process for tissue homeostasis. In this review, we highlight some of the best-known examples of alternative splicing events involved in cell survival. Emphasis is given to the role of this regulation in human cancer and in the response to chemotherapy, providing examples of how alternative splicing of apoptotic genes can be exploited therapeutically.

The balance between self-renewal and differentiation of neural progenitor cells (NPCs) dictates neurogenesis and proper brain development. We found that the RNA- binding protein Sam68 (Khdrbs1) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs. Knockout of Khdrbs1 constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons. Sam68 function was linked to regulation of Aldh1a3 pre-mRNA 3'-end processing. Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination, favoring expression of a functional enzyme. The lower ALDH1A3 expression and activity in Khdrbs1 -/- NPCs results in reduced glycolysis and clonogenicity, thus depleting the embryonic NPC pool and limiting cortical expansion. Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex., Competing Interests: The authors declare that no competing interests exist.

Insulin-like growth factor (IGF) -1 is a pleiotropic hormone exerting mitogenic and anti-apoptotic effects. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. The biological significance of the IGF-1 splice variants and how the E-peptides affect the actions of mature IGF-1 are largely unknown. In this study we investigated the origin and conservation of the IGF-1 E-peptides and we compared the pattern of expression of the IGF-1 isoforms in vivo, in nine mammalian species, and in vitro using human and mouse IGF-1 minigenes. Our analysis showed that only IGF-1Ea is conserved among all vertebrates, whereas IGF-1Eb and IGF-1Ec are an evolutionary novelty originated from the exonization of a mammalian interspersed repetitive-b (MIR-b) element. Both IGF-1Eb and IGF-1Ec mRNAs were constitutively expressed in all mammalian species analyzed but their expression ratio varies greatly among species. Using IGF-1 minigenes we demonstrated that divergence in cis-acting regulatory elements between human and mouse conferred species-specific features to the exon 5 region. Finally, the protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains. In conclusion, exonization of a MIR-b element in the IGF-1 gene determined gain of exon 5 during mammalian evolution. Alternative splicing of this novel exon added new regulatory elements at the mRNA and protein level potentially able to regulate the mature IGF-1 across tissues and species., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Type 2 cannabinoid receptor (CB2) has been proposed to play a pivotal role in meiotic entry of male germ cells, similar to retinoic acid (RA). In this study, we showed that activation of CB2with the specific agonist JWH133 [3-(1',1'-dimethylbutyl)-1-deoxy-8-THC] (IC5010(-6)M) mimics epigenetic events induced by RA (IC5010(-7)M) in spermatogonia. Both JWH133 and RA treatments stimulate the expression of the meiotic genes c-KitandStra8, by up-regulating H3K4me3 and down-regulating H3K9me2 levels in genomic regions flanking the transcription start site. Moreover, both agents increase the expression ofPrdm9, the gene encoding a meiosis-specific histone, H3K4me3 methyltransferase, which marks hotspots of recombination in prophase I, thus resulting in a global increase in H3K4me3. Notably, prolonged administration of JWH133 to immature 7 dpp CD-1 mice induced an acceleration of the onset of spermatogenesis, whereas the specific CB2antagonist delayed germ cell differentiation. Thus, both hyper- and hypostimulation of CB2disrupted the temporal dynamics of the spermatogenic cycle. These findings highlight the importance of proper CB2signaling for the maintenance of a correct temporal progression of spermatogenesis and suggest a possible adverse effect of cannabis in deregulating this process.-Di Giacomo, D., De Domenico, E., Sette, C., Geremia, R., Grimaldi, P. Type 2 cannabinoid receptor contributes to the physiological regulation of spermatogenesis., (© FASEB.)

Immune cells sense foreign organisms through the evolutionarily conserved family of Toll-like receptors. Signaling from these receptors relies on oligomerization of adaptor molecules. In this issue of Structure, Vynke et al. (2016) shed light on the dynamical structure of the homo- and hetero-dimerization domain of MyD88, the main adaptor utilized by Toll-like receptors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre-messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3' splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model., (© 2015 Pagliarini et al.)

The p38 mitogen-activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) -mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4(+)  CD27(+)  CD45RA(+) naive T cells, both in healthy donors and in patients affected by the relapsing-remitting form of MS. Our data also indicate that p38 activation is essential for interleukin-17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4(+) T cells isolated from individuals with relapsing-remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17-mediated autoimmune diseases., (© 2015 John Wiley & Sons Ltd.)

Genome integrity is constantly threatened by endogenous and exogenous factors. However, its preservation is ensured by a network of pathways that prevent and/or repair the lesion, which constitute the DNA damage response (DDR). Expression of the key proteins involved in the DDR is controlled by numerous post-transcriptional mechanisms, among which pre-mRNA splicing stands out. Intriguingly, several splicing factors (SFs) have been recently shown to play direct functions in DNA damage prevention and repair, which go beyond their expected splicing activity. At the same time, evidence is emerging that DNA repair proteins (DRPs) can actively sustain the DDR by acting as post-transcriptional regulator of gene expression, in addition to their well-known role in the mechanisms of signaling and repair of the lesion. Herein, we will review these non-canonical functions of both SFs and DRPs, which suggest the existence of a tight interplay between splicing regulation and canonical DNA safeguard mechanisms ensuring genome stability.

Alternative splicing (AS) represents a major resource for eukaryotic cells to expand the coding potential of their genomes and to finely regulate gene expression in response to both intra- and extracellular cues. Cancer cells exploit the flexible nature of the mechanisms controlling AS in order to increase the functional diversity of their proteome. By altering the balance of splice isoforms encoded by human genes or by promoting the expression of aberrant oncogenic splice variants, cancer cells enhance their ability to adapt to the adverse growth conditions of the tumoral microenvironment. Herein, we will review the most relevant cancer-related splicing events and the underlying regulatory mechanisms allowing tumour cells to rapidly adapt to the harsh conditions they may face during the occurrence and development of cancer.

Alternative splicing (AS) modulates many physiological and pathological processes. For instance, AS of the BCL-X gene balances cell survival and apoptosis in development and cancer. Herein, we identified the polypyrimidine tract binding protein (PTBP1) as a direct regulator of BCL-X AS. Overexpression of PTBP1 promotes selection of the distal 5' splice site in BCL-X exon 2, generating the pro-apoptotic BCL-Xs splice variant. Conversely, depletion of PTBP1 enhanced splicing of the anti-apoptotic BCL-XL variant. In vivo cross-linking experiments and site-directed mutagenesis restricted the PTBP1 binding site to a polypyrimidine tract located between the two alternative 5' splice sites. Binding of PTBP1 to this site was required for its effect on splicing. Notably, a similar function of PTBP1 in the selection of alternative 5' splice sites was confirmed using the USP5 gene as additional model. Mechanistically, PTBP1 displaces SRSF1 binding from the proximal 5' splice site, thus repressing its selection. Our study provides a novel mechanism of alternative 5' splice site selection by PTBP1 and indicates that the presence of a PTBP1 binding site between two alternative 5' splice sites promotes selection of the distal one, while repressing the proximal site by competing for binding of a positive regulator., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pancreatic endocrine tumors (PETs) are characterised by an indolent behaviour in terms of tumor growth. However, most patients display metastasis at diagnosis and no cure is currently available. Since the PI3K/AKT/mTOR axis is deregulated in PETs, the mTOR inhibitor RAD001 represents the first line treatment. Nevertheless, some patients do not respond to treatments and most acquire resistance. Inhibition of mTOR leads to feedback re-activation of PI3K activity, which may promote resistance to RAD001. Thus, PI3K represents a novel potential target for PETs. We tested the impact of three novel PI3K inhibitors (BEZ235, BKM120 and BYL719) on proliferation of PET cells that are responsive (BON-1) or unresponsive (QGP-1) to RAD001. BEZ235 was the most efficient in inhibiting proliferation in PET cells. Furthermore, combined treatment with BEZ235 and RAD001 exhibited synergic effects and was also effective in BON-1 that acquired resistance to RAD001 (BON-1 RR). Analysis of PI3K/AKT/mTOR pathway showed that RAD001 and BEZ235 only partially inhibited mTOR-dependent phosphorylation of 4EBP1. By contrast, combined therapy with the two inhibitors strongly inhibited phosphorylation of 4EBP1, assembly of the translational initiation complex and protein synthesis. Thus, combined treatment with BEZ235 may represent suitable therapy to counteract primary and acquired resistance to RAD001 in PETs.

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.

Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ∼150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2β, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2β and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5' splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein that transduces intracellular signaling pathways evoked by the Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 is composed of an N-terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain, separated by a short region. Upon ligand binding, TLR/IL-1Rs hetero- or homodimerize and recruit MyD88 through their respective TIR domains. Then, MyD88 oligomerizes via its DD and TIR domain and interacts with the interleukin-1 receptor-associated kinases (IRAKs) to form the Myddosome complex. We performed site-directed mutagenesis of conserved residues that are located in exposed regions of the MyD88-TIR domain and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of Glu(183), Ser(244), and Arg(288) impaired homodimerization of the MyD88-TIR domain, recruitment of IRAKs, and activation of NF-κB. Moreover, overexpression of two green fluorescent protein (GFP)-tagged MyD88 mini-proteins (GFP-MyD88151-189 and GFP-MyD88168-189), comprising the Glu(183) residue, recapitulated these effects. Importantly, expression of these dominant negative MyD88 mini-proteins competed with the function of endogenous MyD88 and interfered with TLR2/4-mediated responses in a human monocytic cell line (THP-1) and in human primary monocyte-derived dendritic cells. Thus, our studies identify novel residues of the TIR domain that are crucially involved in MyD88 homodimerization and TLR signaling in immune cells.

IL-1 is a master cytokine that plays a pivotal role in local and systemic inflammation and is involved in the host response to both physiological and pathological stimuli. The recent development of biological compounds targeting IL-1 activity in vivo has revealed the contribution of this cytokine to a large number of human inflammatory and autoimmune diseases. Nevertheless, these currently used drugs display some disadvantages that limit their use in several IL-1-driven diseases, urging for the identification of more powerful agents. The present article describes the identification of new small peptides displaying IL-1 inhibitory activity and covered by the patent WO2012122985. The experimental work in support of the patent's claims is also discussed. Specific attention is paid to the comparison with the original compound from which the peptides were originated: the IL-1 receptor antagonist (IL-1RA) protein. The potential advantages brought about by the present invention as well as the limits documented by the experimental results shown in the patent are also discussed.

Toll-like receptor (TLR)/interleukin (IL) 1 receptor (IL-1R) play a fundamental role in the immune response. These receptors are distributed in various cellular compartments and recognize different components of pathogens. All TLR/IL-1Rs, with the exception of TLR3, interact with MyD88, an intracellular adapter protein that triggers a signaling cascade that culminates in the expression of inflammatory genes. Because aberrant activation of TLR/IL-1Rs can promote the onset of inflammatory or autoimmune diseases and malignancies, this pathway has attracted considerable interest as a potential therapeutic target. Given the central role of MyD88 in TLR/IL-1R signaling, we set out different strategies to develop drugs that can block its function. Structural and functional analysis of the MyD88 domains allowed us to identify crucial residues required for MyD88 homodimerization. Moreover, we developed small cell-permeable peptides and peptidomimetic agents that inhibit MyD88 homodimerization and function. Our results pave the way for the development of new therapeutic drugs for the inhibition of MyD88-dependent signaling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The most frequent molecular abnormalities in pancreatic endocrine tumours (PETs) are mutations of the MEN1 gene, deregulation of the PI3K/AKT/mTOR signalling pathway and overactivation of growth factors and their receptors, such as the VEGF. On this basis, everolimus (Afinitor®; Novartis) and sunitinib (Sutent®; Pfizer) have both been approved by the FDA for the treatment of progressive, unresectable, locally advanced or metastatic PETs. However, molecular or surrogate markers able to predict the response of PET patients to treatment with these drugs are not available, and cancer cells treated with targeted therapies might develop escape pathways that evoke pro-survival feedback responses. The existence of cross-talk between different molecular pathways in PETs has been poorly investigated. In the present review, we present data supporting an important role for Src family kinases (SFKs) in PETs, together with the recent observation of a novel role for SFK in modulating the mTOR pathway activity. Of note, while treatment with everolimus triggered the activation of a survival response dependent on PI3K/AKT signalling in vitro, the simultaneous inhibition of SFKs blocked the activation of this unwanted escape signal. These studies might set the ground for the investigation of combined treatment of PETs with SFK and mTOR inhibitors., (Copyright © 2012 S. Karger AG, Basel.)

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB(1) and CB(2) in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB(1) is present at higher levels than CB(2) in NSCs. The eCB anandamide (AEA) or the CB(1)-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB(2)-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB(1), but not CB(2), antagonist, corroborating the specificity of the response. CB(1) activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB(1) stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB(1) activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB(1) activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB(1) in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.

Prostate cancer (PCa) remains one of the most frequent causes of death for cancer in the male population. Although the initial antiandrogenic therapies are efficacious, PCa often evolves into a hormone-resistant, incurable disease. The genetic and phenotypic heterogeneity of this type of cancer renders its diagnosis and cure particularly challenging. Mounting evidence indicates that alternative splicing, the process that allows production of multiple mRNA variants from each gene, contributes to the heterogeneity of the disease. Key genes for the biology of normal and neoplastic prostate cells, such as those encoding for the androgen receptor and cyclin D1, are alternatively spliced to yield protein isoforms with different or even opposing functions. This review illustrates some examples of genes whose alternative splicing regulation is relevant to PCa biology and discusses the possibility to exploit alternative splicing regulation as a novel tool for prognosis, diagnosis, and therapeutic approaches to PCa.

Alternative splicing (AS) is one of the key processes involved in the regulation of gene expression in eukaryotic cells. AS catalyzes the removal of intronic sequences and the joining of selected exons, thus ensuring the correct processing of the primary transcript into the mature mRNA. The combinatorial nature of AS allows a great expansion of the genome coding potential, as multiple splice-variants encoding for different proteins may arise from a single gene. Splicing is mediated by a large macromolecular complex, the spliceosome, whose activity needs a fine regulation exerted by cis-acting RNA sequence elements and trans-acting RNA binding proteins (RBP). The activity of both core spliceosomal components and accessory splicing factors is modulated by their reversible phosphorylation. The kinases and phosphatases involved in these posttranslational modifications significantly contribute to AS regulation and to its integration in the complex regulative network that controls gene expression in eukaryotic cells. Herein, we will review the major canonical and noncanonical splicing factor kinases and phosphatases, focusing on those whose activity has been implicated in the aberrant splicing events that characterize neoplastic transformation.

Mammalian tissues display a remarkable complexity of splicing patterns. Nevertheless, only few examples of tissue-specific splicing regulators are known. Herein, we characterize a novel splicing regulator named RBM11, which contains an RNA Recognition Motif (RRM) at the amino terminus and a region lacking known homology at the carboxyl terminus. RBM11 is selectively expressed in brain, cerebellum and testis, and to a lower extent in kidney. RBM11 mRNA levels fluctuate in a developmentally regulated manner, peaking perinatally in brain and cerebellum, and at puberty in testis, in concomitance with differentiation events occurring in neurons and germ cells. Deletion analysis indicated that the RRM of RBM11 is required for RNA binding, whereas the carboxyl terminal region permits nuclear localization and homodimerization. RBM11 is localized in the nucleoplasm and enriched in SRSF2-containing splicing speckles. Transcription inhibition/release experiments and exposure of cells to stress revealed a dynamic movement of RBM11 between nucleoplasm and speckles, suggesting that its localization is affected by the transcriptional status of the cell. Splicing assays revealed a role for RBM11 in the modulation of alternative splicing. In particular, RBM11 affected the choice of alternative 5' splice sites in BCL-X by binding to specific sequences in exon 2 and antagonizing the SR protein SRSF1. Thus, our findings identify RBM11 as a novel tissue-specific splicing factor with potential implication in the regulation of alternative splicing during neuron and germ cell differentiation.

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68(-/-) germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis.

Pancreatic endocrine tumours (PETs) are rare and heterogeneous neoplasms, often diagnosed at metastatic stage, for which no cure is currently available. Recently, activation of two pathways that support proliferation and invasiveness of cancer cells, the Src family kinase (SFK) and mammalian target of rapamycin (mTOR) pathways, was demonstrated in PETs. Since both pathways represent suitable targets for therapeutic intervention, we investigated their possible interaction in PETs. Western blot and immunofluorescence analyses indicated that SFK and mTOR activity correlate in PET cell lines. We also found that SFKs coordinate cell adhesion and spreading with activation of the mTOR pathway in PET cells. Live cell metabolic labelling and biochemical studies demonstrated that SFK activity enhance mTOR-dependent translation initiation. Furthermore, microarray analysis of the mRNAs associated with polyribosomes revealed that SFKs regulate mTOR-dependent translation of specific transcripts, with an enrichment in mRNAs encoding cell cycle proteins. Importantly, a synergic inhibition of proliferation was observed in PET cells concomitantly treated with SFK and mTOR inhibitors, without activation of the phosphatidylinositol 3-kinase/AKT pro-survival pathway. Tissue microarray analysis revealed activation of Src and mTOR in some PET samples, and identified phosphorylation of 4E-BP1 as an independent marker of poor prognosis in PETs. Thus, our work highlights a novel link between the SFK and mTOR pathways, which regulate the translation of mRNAs for cell cycle regulators, and suggest that crosstalk between these pathways promotes PET cell proliferation.

Sam68 plays an essential role in mouse spermatogenesis and male fertility. Herein, we report an interaction between Sam68 and the phosphorylated forms of the RNA polymerase II (RNAPII) in meiotic spermatocytes. RNase treatment decreased but did not abolish the interaction, consistently with in vitro binding of RNAPII to the Sam68 carboxyl-terminal region. Sam68 retention in the spermatocyte nucleus was dependent on the integrity of cellular RNAs, suggesting that the protein is recruited to transcriptionally active chromatin. Mouse knockout models characterized by stage-specific arrest of spermatogenesis and staining with the phosphorylated form of RNAPII documented that Sam68 expression is confined to the transcriptionally active stages of spermatogenesis. Furthermore, Sam68 associates with splicing regulators in germ cells and we report that alternative splicing of Sgce exon 8 is regulated in a Sam68-dependent manner during spermatogenesis. RNA and chromatin crosslink immunoprecipitation experiments showed that Sam68 binds in vivo to sequences surrounding the intron 7/exon 8 boundary, thereby affecting the recruitment of the phosphorylated RNAPII and of the general splicing factor U2AF65. These results suggest that Sam68 regulates alternative splicing at transcriptionally active sites in differentiating germ cells and provide new insights into the regulation of Sam68 expression during spermatogenesis.

Src associated in mitosis, of 68 kDa (Sam68) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family. Although ubiquitously expressed, Sam68 plays very specialized roles in different cellular environments. In most cells, Sam68 resides in the nucleus and is involved in several steps of mRNA processing, from transcription, to alternative splicing, to nuclear export. In addition, Sam68 translocates to the cytoplasm upon cell stimulation, cell cycle transitions or viral infections, where it takes part to signaling complexes and associates with the mRNA translation machinery. Recent evidence has linked Sam68 function to the onset and progression of endocrine tumors, such as prostate and breast carcinomas. Notably, all the biochemical activities reported for Sam68 seem to be implicated in carcinogenesis. Herein, we review the recent advancement in the knowledge of Sam68 function and regulation and discuss it in the frame of its participation to neoplastic transformation and tumor progression.

Fertilization is a very complex biological process that requires the perfect cooperation between two highly specialized cells: the male and female gametes. The oocyte provides the physical space where this process takes place, most of the energetic need, and half of the genetic contribution. The spermatozoon mostly contributes the other half of the chromosomes and it is specialized to reach and to penetrate the oocyte. Notably, the mouse oocyte and early embryo are transcriptionally inactive. Hence, they fully depend on the maternal mRNAs and proteins stored during oocyte maturation to drive the onset of development. The new embryo develops autonomously around the four-cell stage, when maternal supplies are exhausted and the zygotic genome is activated in mice. This oocyte-to-embryo transition needs an efficient and tightly regulated translation of the maternally-inherited mRNAs, which likely contributes to embryonic genome activation. Full understanding of post-transcriptional regulation of gene expression in early embryos is crucial to understand the reprogramming of the embryonic genome, it might help driving reprogramming of stem cells in vitro and will likely improve in vitro culturing of mammalian embryos for assisted reproduction. Nevertheless, the knowledge of the mechanism(s) underlying this fundamental step in embryogenesis is still scarce, especially if compared to other model organisms. We will review here the current knowledge on the post-transcriptional control of gene expression in mouse early embryos and discuss some of the unanswered questions concerning this fascinating field of biology.

Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here we used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA-binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Using this strategy, we identified highly specific patterns of mRNA recruitment to the polysomes that are synchronized with the cell cycle. A large number of the mRNAs recovered with translating ribosomes contain motifs for the RNA-binding proteins DAZL (deleted in azoospermia-like) and CPEB (cytoplasmic polyadenylation element-binding protein). Although a Dazl role in early germ cell development is well established, no function has been described during oocyte-to-embryo transition. We demonstrate that CPEB1 regulates Dazl post-transcriptionally, and that DAZL is essential for meiotic maturation and embryonic cleavage. In the absence of DAZL synthesis, the meiotic spindle fails to form due to disorganization of meiotic microtubules. Therefore, Cpeb1 and Dazl function in a progressive, self-reinforcing pathway to promote oocyte maturation and early embryonic development.

Background: Previous studies demonstrated that ST1959, a triazole derivative endowed with immunomodulatory activities, also exerts inhibitory effects on proliferation and survival of a panel of tumor cells. In this study, we sought to ascertain the effects of ST1959 on the growth of androgen-dependent and androgen-independent prostate cancer (PCa) cells., Methods: The growth of androgen-dependent (LNCaP) and androgen-independent (PC3, DU-145) cells was analyzed in vitro both in the presence and absence of ST1959. Modulation of cyclin and androgen receptor (AR) expression following treatment with ST1959 was analyzed by Western blot and cytofluorimetric analysis., Results: We observed that ST1959 causes a significant growth inhibition of LNCaP cells without affecting proliferation of androgen-insensitive DU-145 and PC3 cell lines. These effects were associated with G0/G1 cell cycle arrest and down-regulation of cyclin D1, A and B and AR expression., Conclusions: Our present findings indicate that the anti-proliferative activity of ST1959 on cell growth of androgen-dependent LNCaP PCa cells may be brought about by decreasing expression of functional AR and selected cyclins, ultimately leading to cell growth inhibition., (© 2010 Wiley-Liss, Inc.)

Sam68 is a multifunctional RNA-binding protein highly expressed in the gonads, whose ablation causes male infertility. Herein, we have investigated Sam68 expression in the adult ovary and its function in female fertility. Immunohistochemistry showed that Sam68 was localized in the nucleus of oocytes and follicular cells at all stages of folliculogenesis. Sam68(-/-) females were severely subfertile, and they showed a delay in the age of first pregnancy, increased breeding time for successful pregnancy and yielded smaller litters. Morphological analyses indicated a significant reduction in the number of secondary and pre-antral follicles in the ovary. These defects were associated with alteration of oestrous cycles and a reduced number of ovulated oocytes, which were only partially restored by the administration of exogenous gonadotropins. Crosslinking/immunoprecipitation experiments showed that Sam68 directly binds the mRNAs for the follicle-stimulating hormone (FSH) and the luteinizing hormone receptors (Fshr and Lhcgr), which were downregulated in ovaries of adult knockout females. Stimulation of immature females with FSH-like pregnant mare serum gonadotropin (PMSG), or of follicular cells with the FSH second messenger analogue 8Br-cAMP, caused the upregulation of Sam68. The increase in Sam68 levels paralleled that of the Fshr and Lhcgr mRNAs in the pre-ovulatory follicle and was required to allow accumulation of these transcripts in follicular cells. These studies identify a new crucial function for Sam68 in the regulation of female fertility and indicate that this protein is required to insure proper expression of the gonadotropin receptor transcripts in pre-ovulatory follicles in adult ovary.

Objectives: Toll-like receptor 9 (TLR9), which recognizes hypomethylated DNA [cytosine-phosphate-guanine (CpG)], plays a role in the maintenance of serological memory and has been recently implicated in the pathogenesis of SLE. We previously reported that in vitro TLR9 triggers memory B-cell differentiation into antibody-producing cells, and that the MyD88-inhibitor ST2825 blocks TLR9-induced plasma cell (PC) generation. Here, we investigated whether memory B cells produce autoantibodies in SLE patients with active disease or in clinical remission, and whether ST2825 could inhibit PC generation in SLE patients., Methods: Peripheral blood mononuclear cells from 10 SLE patients in clinical remission and 2 with active SLE were cultured in the presence of CpG with or without ST2825. Phenotypical analysis of CpG-stimulated cells was performed by flow cytometry. Supernatants were collected to measure antibody production by ELISA and to detect autoantibodies by IF., Results: CpG-induced TLR9 stimulation caused autoantibody secretion in patients with active disease and in the majority of patients in clinical remission. Inhibition of MyD88 completely blocked the de novo generation of PCs and the secretion of autoantibodies., Conclusions: Autoreactive B cells persist in SLE patients during disease remission in the circulating B-cell memory pool. TLR9-dependent activation of memory B cells by pathogens could be one of the mechanisms triggering relapses in SLE. Compounds targeting the TLR/MyD88 pathway may be used as novel therapeutic tools to treat acute disease and to prevent relapses in SLE patients.

Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell-derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli.

Spinal Muscular Atrophy (SMA) is a neurodegenerative disease with high impact in the human population, being the leading genetic cause of death in infancy. No cure is currently available for SMA, raising interest in the development of novel therapeutic strategies for this disease. Much of the effort in this sense has been aimed at increasing the SMN2-derived transcript levels, either by improving transcription rate or by reprogramming exon 7 splicing. Herein, we discuss recent findings on the regulation of SMN2 gene expression, focusing on splicing modulation as a therapeutic target. We review the literature regarding splicing factors involved in the regulation of exon 7 splicing in SMN2, and discuss the role played in this process by the RNA binding protein Sam68, a novel crucial regulator of SMN2 splicing.

Background: TR-KIT, a truncated form of KIT (the KITL receptor), corresponding to the c-terminal half of the intracellular split tyrosine kinase domain, is expressed during the haploid stages of mouse spermatogenesis, and is one of the candidate sperm factors possibly involved in egg activation at fertilization., Methods: Immunocytochemistry of adult human testis, and studies of human semen samples from volunteer donors through immunofluorescence, confocal microscopy, flow cytometry, western blot and RT-PCR analyses were performed., Results: We show that the TR-KIT is expressed during spermiogenesis in the human testis, and that it is maintained in human ejaculated spermatozoa. TR-KIT is localized both in the equatorial segment and in the sub-acrosomal region of the human sperm head. The equatorial localization of the TR-KIT persists after the spontaneous acrosome reaction. Cytometric analysis of several sperm samples from volunteer donors, showed variable degrees of the TR-KIT-specific immunolabeling, and a significant inverse correlation (Pearson's coefficient, r = -0.76, P < 0.0001, n = 23) of the TR-KIT positivity with markers of sperm damage, i.e. DNA fragmentation, as revealed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) analysis and the intense clusterin positivity. We also found less significant inverse correlation with altered head morphology (r = -0.47, P < 0.05, n = 23) and direct correlation with sperm forward motility parameters (r = 0.59, P < 0.01, n = 23)., Conclusions: The TR-KIT is present in the equatorial region of human spermatozoa, which is the first sperm component entering into the oocyte cytoplasm after fusion with the egg. This localization is consistent with the function previously proposed for this protein in mice. In addition, the TR-KIT represents a potential predictive parameter of human sperm quality.

The importance of obtaining stem cells through alternative methods has increased progressively in the recent years due to the potential role that embryonic stem (ES) cells play in the field of regenerative medicine. In this regard, generation of parthenogenetic blastocysts allows the production of ethic-free ES cells without the need to manipulate normal embryos. Our work was aimed at clarifying whether variations in the method adopted to generate diploid parthenogenetic blastocysts could determine differences in the quality of blastocysts produced. In vitro development of mouse oocytes activated with three protocols, using Sr2+ and cytochalasin for different time, was compared with that of in vivo fertilized embryos. We have evaluated the efficiency of blastocyst formation and analysed the expression pattern of the stemness markers OCT4, CDX2, and NANOG. Our results indicate that the yield of diploid parthenogenotes and the segregation of the stemness marker OCT4 in the developing blastocyst are influenced by the parthenogenetic protocol adopted. Particularly, even if all methods tested allowed the production of blastocysts in vitro, the correct segregation of OCT4 occurred only in blastocysts developed from oocytes concomitantly treated for 4 h with Sr2+ and cytochalasin D. Our results indicate that the protocol employed to develop parthenogenetic blastocysts in vitro affects the quality of cells in the inner cell mass.

Defects in the regulation of alternative splicing have strong relevance in the onset and progression of several types of human cancer. Modulation of alternative splicing allows cancer cells to adapt to hostile environments through production of specific mRNA variants. In particular, genotoxic stress exerted by chemotherapeutic drugs or irradiation strongly affects splicing of many genes. A key role in this aberrant regulation is played by the unbalanced expression of several splicing factors in cancer cells. Among them, the RNA-binding protein Sam68, which is overexpressed in various tumors, was shown to accumulate in nuclear foci of active transcription, together with other splicing regulators, and to affect splicing of target mRNAs in response to genotoxic stress. We suggest that subcellular redistribution of splicing factors is guided by changes in chromatin conformation elicited by DNA-damaging drugs. This event might represent an escape mechanism used by cancer cells to survive to genotoxic insults through expression of pro-survival, cancer-specific gene products.

TLRs boost antimicrobial response mechanisms by epithelial cells and represent the first line of defense at mucosal sites. In view of these immunomodulatory properties, TLR stimulation may represent a novel means to activate anticancer immune responses. In the present study, the ability of TLR ligands to affect the recruitment of different immune cell populations by human prostate cancer cell lines and the underlying mechanisms were investigated. We showed that LNCaP and DU-145 cells express functionally active TLR3 and TLR5. Treatment with their respective agonists, polyinosinic:polycytidylic acid and flagellin, rapidly triggered NF-kappaB-dependent upregulation of different inflammatory molecules, as assayed by microarray and ELISA. Furthermore, we demonstrated that conditioned media from polyinosinic:polycytidylic acid- and flagellin-treated LNCaP and DU-145 cells induced the recruitment of different leukocyte subpopulations, suggesting that TLR stimulation is able to activate the earliest step of immune response mediated by soluble factors. Interestingly, the more aggressive cancer cell line PC3 expressed TLR3 and TLR5 but failed to respond to TLR agonists in terms of NF-kappaB activation and the ability to attract immune effectors. Overall, these data show for the first time that TLR3 and TLR5 stimulation of human prostate cancer cells triggers the production of chemokines, which, in turn, favor the attraction of immune effectors, thereby representing a tool to enhance the efficacy of conventional therapies by stimulating anticancer immune responses.

The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b induction remain unsolved. Here, we identify the RNA-binding protein ASF/SF2 as a critical, allele-specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2 associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous transcript in prostate cancer (PCa) cells. SF2 association was altered by the CCND1 G/A870 polymorphism, which resides in the splice donor site controlling transcript-b production. This finding was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation of G870-derived transcript-b. Finally, the relevance of SF2 function was established, as SF2 strongly correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production. Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies designed to suppress the oncogenic alternative splicing event., ((c)2010 AACR.)

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.