Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.     

SRrp37, a novel splicing regulator located in the nuclear speckles and nucleoli,interacts with SC35 and modulates alternative pre‐mRNA splicing in vivo

We report here the identification and characterization of a novel SR‐related protein, referred to as SRrp37, based on its apparent molecular weight and subcellular location. SRrp37 was identified through a yeast two‐hybrid screen during the course of searching for proteins interacting with pNO40, a ribosomal 60S core subunit. SRrp37 exhibited two alternative spliced isoforms generated by differential usage of the translation start site with the longer one, SRrp37, initiating at first exon and the shorter, SRrp37‐2, starting from exon 2. Three distinct motifs can be discerned in the SRrp37 protein: (1) a serine–arginine (SR) dipeptide enriched domain, (2) a polyserine stretch, and (3) a potential nucleolar localization signal comprising a long array of basic amino acids. SRrp37's message was translated in tissue‐specific patterns with both isoforms expressed at comparable levels in tissues showing expression. Indirect immunofluorescence analysis with an anti‐SRrp37 antibody, as well as an experiment using myc‐tagged proteins, demonstrated that SRrp37 was localized in nucleoli and nuclear speckles. GST pull‐down assay showed that SRrp37 interacted physically with SC35. Using adenovirus E1A and chimeric calcitonin/dhfr constructs as splicing reporter minigenes, we found that SRrp37 modulated alternative 5′ and 3′ splicing in vivo. Together, SRrp37 may participate directly in splicing regulation or indirectly through interaction with SC35. Studies on this novel splicing regulator may provide new information on the intricate splicing machinery as related to the RNA metabolism involving processing of mRNA and rRNA. J. Cell. Biochem. 108: 304–314, 2009. © 2009 Wiley‐Liss, Inc.     

NFBD1/MDC1 stabilizes oncogenic MDM2 to contribute to cell fate determination in response to DNA damage

In response to DNA damage, NFBD1/MDC1 induces the accumulation of DNA repair machinery such as MRN complex at the sites of damaged DNA to form nuclear foci. In this study, we found that NFBD1 directly interacts with MDM2 and increases its stability. During adriamycin (ADR)-mediated apoptosis, expression levels of NFBD1 reduced in association with the down-regulation of MDM2. Enforced expression of NFBD1 resulted in a significant stabilization of MDM2. Consistent with these observations, siRNA-mediated knockdown of the endogenous NFBD1 decreased the amounts of the endogenous MDM2. Immunoprecipitation and in vitro pull-down assays demonstrated that NFBD1 interacts with MDM2 through its COOH-terminal BRCT domains. In accordance with our recent results, enforced expression of NFBD1 rendered cells resistant to DNA damage. Similar results were also obtained in cells expressing exogenous MDM2. Taken together, our present findings suggest that NFBD1-mediated stabilization contributes to cell survival in response to DNA damage.     

Uhrf2 is important for DNA damage response in vascular smooth muscle cells

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.     

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to γ-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer γ-hexa-chloro-cyclohexane (γ-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon γ-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of γ-H2AX in γ-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.     

Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo

Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein.     

PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint

Phosphorylation of histone H2AX on Ser 139 (γH2AX) is one of the earliest events in the response to DNA double-strand breaks; however, the subsequent removal of γH2AX from chromatin is less understood, despite being a process tightly coordinated with DNA repair. Previous studies in yeast have identified the Pph3 phosphatase (the PP4C orthologue) as important for the dephosphorylation of γH2AX. By contrast, work in human cells attributed this activity to PP2A. Here, we report that PP4 contributes to the dephosphorylation of γH2AX, both at the sites of DNA damage and in undamaged chromatin in human cells, independently of a role in DNA repair. Furthermore, depletion of PP4C results in a prolonged checkpoint arrest, most likely owing to the persistence of mediator of DNA damage checkpoint 1 (MDC1) at the sites of DNA lesions. Taken together, these results indicate that PP4 is an evolutionarily conserved γH2AX phosphatase.     

泛素E3连接酶接头蛋白SPOP和MDM2在DNA损伤修复中的作用

DNA损伤修复是维持细胞基因组稳定性和完整性的基础,越来越多的研究发现,E3泛素连接酶在DNA损伤修复中起着重要的作用.该文将介绍DNA损伤修复的机制、DNA损伤修复与疾病的关系、及E3泛素连接酶接头蛋白MDM2和SPOP在DNA损伤修复中的作用.重点围绕DNA损伤修复的两条通路:E3泛素连接酶接头蛋白SPOP与ATM...     

Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage

The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G₁, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.     

The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)-rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK(3/6)-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.     

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C‐to‐T transition at position +6 in exon‐7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C‐to‐T transition in SMN2 creates a putative binding site for the RNA‐binding protein Sam68. RNA pull‐down assays and UV‐crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon‐7 skipping. Moreover, mutations in the Sam68‐binding site of SMN2 or in the RNA‐binding domain of Sam68 completely abrogated its effect on exon‐7 skipping. Retroviral infection of dominant‐negative mutants of Sam68 that interfere with its RNA‐binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon‐7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.     

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

RBFOX1 and RBFOX2 are alternative splicing factors that are predominantly expressed in the brain and skeletal muscle. They specifically bind the RNA element UGCAUG, and regulate alternative splicing positively or negatively in a position-dependent manner. The molecular basis for the position dependence of these and other splicing factors on alternative splicing of their targets is not known. We explored the mechanisms of RBFOX splicing activation and repression using an MS2-tethering assay. We found that the Ala/Tyr/Gly-rich C-terminal domain is sufficient for exon activation when tethered to the downstream intron, whereas both the C-terminal domain and the central RRM are required for exon repression when tethered to the upstream intron. Using immunoprecipitation and mass spectrometry, we identified hnRNP H1, RALY, and TFG as proteins that specifically interact with the C-terminal domain of RBFOX1 and RBFOX2. RNA interference experiments showed that hnRNP H1 and TFG modulate the splicing activity of RBFOX1/2, whereas RALY had no effect. However, TFG is localized in the cytoplasm, and likely modulates alternative splicing indirectly.     

Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.     

A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.     

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.     

S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and γH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.     

Spatially distinct functions of Clb2 in the DNA damage response

In budding yeast four mitotic cyclins (Clb1–4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G₂/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.     

Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria

In temperate lakes, asynchronous cycles in surface water temperatures and incident ultraviolet (UV) radiation expose aquatic organisms to damaging UV radiation at different temperatures. The enzyme systems that repair UV‐induced DNA damage are temperature dependent, and thus potentially less effective at repairing DNA damage at lower temperatures. This hypothesis was tested by examining the levels of UV‐induced DNA damage in the freshwater crustacean Daphnia pulicaria in the presence and absence of longer‐wavelength photoreactivating radiation (PRR) that induces photoenzymatic repair (PER) of DNA damage. By exposing both live and dead (freeze‐killed) Daphnia as well as raw DNA to UV‐B in the presence and absence of PRR, we were able to estimate the relative importance and temperature dependence of PER (light repair), nucleotide excision repair (NER, dark repair), and photoprotection (PP). Total DNA damage increased with increasing temperature. However, the even greater increase in DNA repair rates at higher temperatures led net DNA damage (total DNA damage minus repair) to be greater at lower temperatures. Photoprotection accounted for a much greater proportion of the reduction in DNA damage than did repair. Experiments that looked at survival rates following UV exposure demonstrated that PER increased survival rates. The important implication is that aquatic organisms that depend heavily on DNA repair processes may be less able to survive high UV exposure in low temperature environments. Photoprotection may be more effective under the low temperature, high UV conditions such as are found in early spring or at high elevations.