DNAJC17 is localized in nuclear speckles and interacts with splicing machinery components.

DNAJC17 is a heat shock protein (HSP40) family member, identified in mouse as susceptibility gene for congenital hypothyroidism. DNAJC17 knockout mouse embryos die prior to implantation. In humans, germline homozygous mutations in DNAJC17 have been found in syndromic retinal dystrophy patients, while heterozygous mutations represent candidate pathogenic events for myeloproliferative disorders. Despite widespread expression and involvement in human diseases, DNAJC17 function is still poorly understood. Herein, we have investigated its function through high-throughput transcriptomic and proteomic approaches. DNAJC17-depleted cells transcriptome highlighted genes involved in general functional categories, mainly related to gene expression. Conversely, DNAJC17 interactome can be classified in very specific functional networks, with the most enriched one including proteins involved in splicing. Furthermore, several splicing-related interactors, were independently validated by co-immunoprecipitation and in vivo co-localization. Accordingly, co-localization of DNAJC17 with SC35, a marker of nuclear speckles, further supported its interaction with spliceosomal components. Lastly, DNAJC17 up-regulation enhanced splicing efficiency of minigene reporter in live cells, while its knockdown induced perturbations of splicing efficiency at whole genome level, as demonstrated by specific analysis of RNAseq data. In conclusion, our study strongly suggests a role of DNAJC17 in splicing-related processes and provides support to its recognized essential function in early development.

Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and incurable disease. Poor prognosis is due to multiple reasons, including acquisition of resistance to gemcitabine, the first-line chemotherapeutic approach. Thus, there is a strong need for novel therapies, targeting more directly the molecular aberrations of this disease. We found that chronic exposure of PDAC cells to gemcitabine selected a subpopulation of cells that are drug-resistant (DR-PDAC cells). Importantly, alternative splicing (AS) of the pyruvate kinase gene (PKM) was differentially modulated in DR-PDAC cells, resulting in promotion of the cancer-related PKM2 isoform, whose high expression also correlated with shorter recurrence-free survival in PDAC patients. Switching PKM splicing by antisense oligonucleotides to favor the alternative PKM1 variant rescued sensitivity of DR-PDAC cells to gemcitabine and cisplatin, suggesting that PKM2 expression is required to withstand drug-induced genotoxic stress. Mechanistically, upregulation of the polypyrimidine-tract binding protein (PTBP1), a key modulator of PKM splicing, correlated with PKM2 expression in DR-PDAC cell lines. PTBP1 was recruited more efficiently to PKM pre-mRNA in DR- than in parental PDAC cells. Accordingly, knockdown of PTBP1 in DR-PDAC cells reduced its recruitment to the PKM pre-mRNA, promoted splicing of the PKM1 variant and abolished drug resistance. Thus, chronic exposure to gemcitabine leads to upregulation of PTBP1 and modulation of PKM AS in PDAC cells, conferring resistance to the drug. These findings point to PKM2 and PTBP1 as new potential therapeutic targets to improve response of PDAC to chemotherapy.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells.

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.

ATM kinase enables the functional axis of YAP, PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

Gemcitabine triggers a pro-survival response in pancreatic cancer cells through activation of the MNK2/eIF4E pathway.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplastic disease. Gemcitabine, the currently used chemotherapeutic drug for PDAC, elicits only minor benefits, because of the development of escape pathways leading to chemoresistance. Herein, we aimed at investigating the involvement of the mitogen activating protein kinase interacting kinase (MNK)/eIF4E pathway in the acquired drug resistance of PDAC cells. Screening of a cohort of PDAC patients by immunohistochemistry showed that eIF4E phosphorylation correlated with disease grade, early onset of disease and worse prognosis. In PDAC cell lines, chemotherapeutic drugs induced MNK-dependent phosphorylation of eIF4E. Importantly, pharmacological inhibition of MNK activity synergistically enhanced the cytostatic effect of gemcitabine, by promoting apoptosis. RNA interference (RNAi) experiments indicated that MNK2 is mainly responsible for eIF4E phosphorylation and gemcitabine resistance in PDAC cells. Furthermore, we found that gemcitabine induced the expression of the oncogenic splicing factor SRSF1 and splicing of MNK2b, a splice variant that overrides upstream regulatory pathways and confers increased resistance to the drug. Silencing of SRSF1 by RNAi abolished this splicing event and recapitulated the effects of MNK pharmacological or genetic inhibition on eIF4E phosphorylation and apoptosis in gemcitabine-treated cells. Our results highlight a novel pro-survival pathway triggered by gemcitabine in PDAC cells, which leads to MNK2-dependent phosphorylation of eIF4E, suggesting that the MNK/eIF4E pathway represents an escape route utilized by PDAC cells to withstand chemotherapeutic treatments.

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

Alternative splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn.

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.

PATZ1 gene has a critical role in the spermatogenesis and testicular tumours.

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1(-/-) mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mis-localization could be associated to the development of TGCTs.

The RNA-binding protein Sam68 contributes to proliferation and survival of human prostate cancer cells.

The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68. Herein, we have investigated the expression and function of Sam68 in human prostate cancer cells. Analysis of specimens obtained from 20 patients revealed that Sam68 is upregulated at the protein level in 35% of the samples. Real-time polymerase chain reaction confirmed the results at the mRNA level in most patients. Downregulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression and reduced the proliferation rate. Moreover, depletion of Sam68 sensitized cells to apoptosis induced by DNA-damaging agents. Similarly, stable cell lines expressing a truncated GFP-Sam68(GSG) protein displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Our results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents.

Polycyclic aromatic hydrocarbon in inter-tidal mussel Perna perna: space-time observations, source investigation and genotoxicity.

The investigation aimed primarily at understanding the PAH record in inter-tidal mussel tissues and evaluating the bivalve performance as a bioindicator for oil contamination. The species Perna perna was used as test organism since it is abundantly distributed in coastal areas of the Americas, Africa and elsewhere. The study was carried out in Guanabara Bay and comprised two observation phases: phase one included seasonal sampling in 8 sites, whereas in phase two 4 sites were examined over 4 years. Among the 35 determined PAH (60-90 microg kg(-1) up to 4000-6000 microg kg(-1)) alkylated homologues predominated by more than 80%. The PAH profile in inter-tidal mussels is largely petrogenic with high contribution of dibenzothiophenes (DBT) and phenanthrenes (Ph). The prevailing petrogenic fingerprint, confirmed by diagnostic ratios, is linked to the properties of the inter-tidal habitat, which favors exposure to oil films. C2DBT/C2Ph and C3DBT/C3Ph ratios, however, show a wide range of values uncorrelated to specific oils. Micronucleus frequencies are significantly related to PAH concentrations, especially to those of alkylated homologues. Genotoxic expression appears at concentrations as low as 300 microg kg(-1) summation operator 35 PAH.

Molecular mechanisms utilized by alternative c-kit gene products in the control of spermatogonial proliferation and sperm-mediated egg activation.

The c-kit proto-oncogene plays a dual role in the control of male fertility in mice through two alternative gene products: (1). c-kit [the transmembrane tyrosine kinase receptor for stem cell factor (SCF)], which is expressed and functional in differentiating spermatogonia of the postnatal testis, in which c-kit is essential for pre-meiotic proliferation; and (2). tr-kit, an intracellular protein which is specifically accumulated during spermiogenesis through the use of an alternative intronic promoter, and which is able to trigger mouse egg activation when microinjected into the cytoplasm of metaphase II arrested oocytes. Here, we summarize the most recent findings about the molecular pathways through which c-kit regulates cell cycle progression in mitotic germ cells, and those through which sperm-derived tr-kit triggers parthenogenetic completion of meiosis II and pronuclear formation in microinjected mouse eggs.

Mutations in the YRB1 gene encoding yeast ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects.

We identified two temperature-sensitive (ts) mutations in the essential gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single amino acid substitutions of evolutionarily conserved residues (A91D and R127K, respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.

The role of stem cell factor and of alternative c-kit gene products in the establishment, maintenance and function of germ cells.

The c-kit gene plays a fundamental role during the establishment, the maintenance and the function of germ cells. In the embryonal gonad the c-kit tyrosine kinase receptor and its ligand Stem Cell Factor (SCF) are required for the survival and proliferation of primordial germ cells. In the postnatal animal, c-kit/SCF are required for the production of the mature gametes in response to gonadotropic hormones, i.e. for the survival and/or proliferation of the only proliferating germ cells of the testis, the spermatogonia, and for the growth and maturation of the oocytes. Finally, a truncated c-kit product, tr-kit, specifically expressed in post-meiotic stages of spermatogenesis and present in mature spermatozoa, causes parthenogenetic activation when microinjected into mouse eggs, suggesting that it might play a role in the final function of the gametes, fertilization.

Role of c-kit in mammalian spermatogenesis.

The tyrosine-kinase receptor c-kit and its ligand, stem cell factor (SCF), are essential for the maintenance of primordial germ cells (PGCs) in both sexes. However, c-kit and a post-meiotic-specific alternative c-kit gene product play important roles also during post-natal stages of spermatogenesis. In the adult testis, the c-kit receptor is re-expressed in differentiating spermatogonia, but not in spermatogonial stem cells, whereas SCF is expressed by Sertoli cells under FSH stimulation. SCF stimulates DNA synthesis in type A spermatogonia cultured in vitro, and injection of anti-c-kit antibodies blocks their proliferation in vivo. A point mutation in the c-kit gene, which impairs SCF-mediated activation of phosphatidylinositol 3-kinase, does not cause any significant reduction in PGCs number during embryonic development, nor in spermatogonial stem cell populations. However males are completely sterile due to a block in the initial stages of spermatogenesis, associated to abolishment of DNA-synthesis in differentiating A1-A4 spermatogonia. With the onset of meiosis c-kit expression ceases, but a truncated c-kit product, tr-kit, is specifically expressed in post-meiotic stages of spermatogenesis, and is accumulated in mature spermatozoa. Microinjection of tr-kit into mouse eggs causes their parthenogenetic activation, suggesting that it might play a role in the final function of the gametes, fertilization.

Mutational analysis suggests that activation of the yeast pheromone response mitogen-activated protein kinase pathway involves conformational changes in the Ste5 scaffold protein.

Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases Gbetagamma (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with Gbetagamma. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5Delta mutation, yet supports mating even in ste4Delta ste5Delta cells when artificially dimerized by fusion to glutathione S-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5Delta cells, but does not allow mating of ste4Delta ste5Delta cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa ste4Delta ste5Delta cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when Gbetagamma is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of Gbetagamma to the RING-H2 domain may induce a conformational change that promotes association of the N- and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20.

The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding. Consequences for cAMP signaling pathway and characterization of a phosphatidic acid binding site.

Hormones and growth factors induce in many cell types the production of phosphatidic acid (PA), which has been proposed to play a role as a second messenger. We have previously shown in an acellular system that PA selectively stimulates certain isoforms of type 4 cAMP-phosphodiesterases (PDE4). Here we studied the effect of endogenous PA on PDE activity of transiently transfected MA10 cells overexpressing the PA-sensitive isoform PDE4D3. Cell treatment with inhibitors of PA degradation, including propranolol, induced an accumulation of endogenous PA accompanied by a stimulation of PDE activity and a significant decrease in both cAMP levels and protein kinase A activity. Furthermore, in FRTL5 cells, which natively express PDE4D3, pretreatment with compounds inducing PA accumulation prevented both cAMP increase and cAMP-responsive element-binding protein phosphorylation triggered by thyroid-stimulating hormone. To determine the mechanism of PDE stimulation by PA, endogenous phospholipids were labeled by preincubating MA10 cells overexpressing PDE4D3 with [(32)P]orthophosphate. Immuno- precipitation experiments showed that PA was specifically bound to PDE4D3, supporting the hypothesis that PDE4D3 activation occurs through direct binding of PA to the protein. PA binding site on PDE4D3 was characterized by engineering deletions of selected regions in the N-terminal regulatory domain of the enzyme. Deletion of amino acid residues 31-59 suppressed both PA-activating effect and PA binding, suggesting that this region rich in basic and hydrophobic residues contains the PA binding site. These observations strongly suggest that endogenous PA can modulate cAMP levels in intact cells, through a direct activation of PDE4D3.

Involvement of type 4 cAMP-phosphodiesterase in the myogenic differentiation of L6 cells.

Myogenic cell differentiation is induced by Arg(8)-vasopressin, whereas high cAMP levels and protein kinase A (PKA) activity inhibit myogenesis. We investigated the role of type 4 phosphodiesterase (PDE4) during L6-C5 myoblast differentiation. Selective PDE4 inhibition resulted in suppression of differentiation induced by vasopressin. PDE4 inhibition prevented vasopressin-induced nuclear translocation of the muscle-specific transcription factor myogenin without affecting its overall expression level. The effects of PDE4 inhibition could be attributed to an increase of cAMP levels and PKA activity. RNase protection, reverse transcriptase PCR, immunoprecipitation, Western blot, and enzyme activity assays demonstrated that the PDE4D3 isoform is the major PDE4 expressed in L6-C5 myoblasts and myotubes, accounting for 75% of total cAMP-hydrolyzing activity. Vasopressin cell stimulation caused a biphasic increase of PDE4 activity, which peaked at 2 and 15 min and remained elevated for 48 h. In the continuous presence of vasopressin, cAMP levels and PKA activity were lowered. PDE4D3 overexpression increased spontaneous and vasopressin-dependent differentiation of L6-C5 cells. These results show that PDE4D3 plays a key role in the control of cAMP levels and differentiation of L6-C5 cells. Through the modulation of PDE4 activity, vasopressin inhibits the cAMP signal transduction pathway, which regulates myogenesis possibly by controlling the subcellular localization of myogenin.

Activation of the mitogen-activated protein kinase ERK1 during meiotic progression of mouse pachytene spermatocytes.

Okadaic acid (OA) causes meiotic progression and chromosome condensation in cultured pachytene spermatocytes and an increase in maturation promoting factor (cyclin B1/cdc2 kinase) activity, as evaluated by H1 phosphorylative activity in anti-cyclin B1 immunoprecipitates. OA also induces a strong increase of phosphorylative activity toward the mitogen-activated protein kinase substrate myelin basic protein (MBP). Immunoprecipitation experiments with anti-extracellular signal-regulated kinase 1 (ERK1) or anti-ERK2 antibodies followed by MBP kinase assays, and direct in-gel kinase assays for MBP, show that p44/ERK1 but not p42/ERK2 is stimulated in OA-treated spermatocytes. OA treatment stimulates phosphorylation of ERK1, but not of ERK2, on a tyrosine residue involved in activation of the enzyme. ERK1 immunoprecipitated from extracts of OA-stimulated spermatocytes induces a stimulation of H1 kinase activity in extracts from control pachytene spermatocytes, whereas immunoprecipitated ERK2 is uneffective. We also show that natural G(2)/M transition in spermatocytes is associated to intracellular redistribution of ERKs, and their association with microtubules of the metaphase spindle. Preincubation of cultured pachytene spermatocytes with PD98059 (a selective inhibitor of ERK-activating kinases MEK1/2) completely blocks the ability of OA to induce chromosome condensation and progression to meiotic metaphases. These results suggest that ERK1 is specifically activated during G(2)/M transition in mouse spermatocytes, that it contributes to the mechanisms of maturation promoting factor activation, and that it is essential for chromosome condensation associated with progression to meiotic metaphases.