RBM25 regulates hypoxic cardiomyocyte apoptosis through CHOP-associated endoplasmic reticulum stress.

Ischemic heart failure (HF) is one of the leading causes of global morbidity and mortality; blocking the apoptotic cascade could help improve adverse outcomes of it. RNA-binding motif protein 25 (RBM25) is an RNA-binding protein related to apoptosis; however, its role remains unknown in ischemic HF. The main purpose of this study is to explore the mechanism of RBM25 in ischemic HF. Establishing an ischemic HF model and oxygen-glucose deprivation (OGD) model. ELISA was performed to evaluate the BNP level in the ischemic HF model. Echocardiography and histological analysis were performed to assess cardiac function and infarct size. Proteins were quantitatively and locationally analyzed by western blotting and immunofluorescence. The morphological changes of endoplasmic reticulum (ER) were observed with ER-tracker. Cardiac function and myocardial injury were observed in ischemic HF rats. RBM25 was elevated in cardiomyocytes of hypoxia injury hearts and localized in nucleus both in vitro and in vivo. In addition, cell apoptosis was significantly increased when overexpressed RBM25. Moreover, ER stress stimulated upregulation of RBM25 and promoted cell apoptosis through the CHOP related pathway. Finally, inhibiting the expression of RBM25 could ameliorate the apoptosis and improve cardiac function through blocking the activation of CHOP signaling pathway. RBM25 is significantly upregulated in ischemic HF rat heart and OGD model, which leads to apoptosis by modulating the ER stress through CHOP pathway. Knockdown of RBM25 could reverse apoptosis-mediated cardiac dysfunction. RBM25 may be a promising target for the treatment of ischemic HF.

Collective motions of fish originate from balanced local perceptual interactions and individual stochastics.

The movement of a group of biological individuals, such as fish schools, can evolve from disordered motions to synergistic movements or even ordered patterns. However, the physical origins behind such emergent phenomena of complex systems remain elusive. Here, we established a high-precision protocol for studying the collective behavior of biological groups in quasi-two-dimensional systems. Based on our video recording of ∼600h of fish movements, we extracted a force map of the interactions between fish from their trajectories using the convolution neural network. Presumably, this force implies the fish's perception of the surrounding individuals, the environment, and their response to social information. Interestingly, the fish in our experiments were predominantly in a seemingly disordered swarm state, but their local interactions were clearly specific. Combining such local interactions with the inherent stochasticity of the fish movements, we reproduced the collective motions of the fish through simulations. We demonstrated that a delicate balance between the specific local force and the intrinsic stochasticity is essential for ordered movements. This study presents implications for self-organized systems that use basic physical characterization to produce higher-level sophistication.

Circulating S-Glutathionylated cMyBP-C as a Biomarker for Cardiac Diastolic Dysfunction.

Background cMyBP-C (Cardiac myosin binding protein-C) regulates cardiac contraction and relaxation. Previously, we demonstrated that elevated myocardial S-glutathionylation of cMyBP-C correlates with diastolic dysfunction (DD) in animal models. In this study, we tested whether circulating S-glutathionylated cMyBP-C would be a biomarker for DD. Methods and Results Humans, African Green monkeys, and mice had DD determined by echocardiography. Blood samples were acquired and analyzed for S-glutathionylated cMyBP-C by immunoprecipitation. Circulating S-glutathionylated cMyBP-C in human participants with DD (n=24) was elevated (1.46±0.13-fold, P=0.014) when compared with the non-DD controls (n=13). Similarly, circulating S-glutathionylated cMyBP-C was upregulated by 2.13±0.47-fold (P=0.047) in DD monkeys (n=6), and by 1.49 (1.22-2.06)-fold (P=0.031) in DD mice (n=5) compared with the respective non-DD controls. Circulating S-glutathionylated cMyBP-C was positively correlated with DD in humans. Conclusions Circulating S-glutathionylated cMyBP-C was elevated in humans, monkeys, and mice with DD. S-glutathionylated cMyBP-C may represent a novel biomarker for the presence of DD.

Cardiac Resynchronization and Circulating Markers of Sarcoplasmic Reticulum Calcium Handling and Sudden Death Risk.

Cardiac resynchronization therapy (CRT) can improve heart function and decrease arrhythmic events. We tested whether CRT altered circulating markers of calcium handling and sudden death risk. Circulating cardiac sodium channel messenger RNA (mRNA) splicing variants indicate arrhythmic risk, and a reduction in sarco/endoplasmic reticulum calcium adenosine triphosphatase 2a (SERCA2a) is thought to diminish contractility in heart failure. CRT was associated with a decreased proportion of circulating, nonfunctional sodium channels and improved SERCA2a mRNA expression. Patients without CRT did not have improvement in the biomarkers. These changes might explain the lower arrhythmic risk and improved contractility associated with CRT.

Inhibition of the unfolded protein response reduces arrhythmia risk after myocardial infarction.

Ischemic cardiomyopathy is associated with an increased risk of sudden death, activation of the unfolded protein response (UPR), and reductions in multiple cardiac ion channels. When activated, the protein kinase-like ER kinase (PERK) branch of the UPR reduces protein translation and abundance. We hypothesized that PERK inhibition could prevent ion channel downregulation and reduce arrhythmia risk after myocardial infarct (MI). MI induced in mice by coronary artery ligation resulted in reduced ion channel levels, ventricular tachycardia (VT), and prolonged corrected intervals between the Q and T waves on the ECGs (QTc). Protein levels of major cardiac ion channels were decreased. MI cardiomyocytes showed significantly prolonged action potential duration and decreased maximum upstroke velocity. Cardiac-specific PERK KO reduced electrical remodeling in response to MI, with shortened QTc intervals, fewer VT episodes, and higher survival rates. Pharmacological PERK inhibition had similar effects. In conclusion, we found that activated PERK during MI contributed to arrhythmia risk by the downregulation of select cardiac ion channels. PERK inhibition prevented these changes and reduced arrhythmia risk. These results suggest that ion channel downregulation during MI is a fundamental arrhythmia mechanism and that maintenance of ion channel levels is antiarrhythmic.

Magnesium supplementation improves diabetic mitochondrial and cardiac diastolic function.

In heart failure and type 2 diabetes mellitus (DM), the majority of patients have hypomagnesemia, and magnesium (Mg) supplementation has improved cardiac function and insulin resistance. Recently, we have shown that DM can cause cardiac diastolic dysfunction (DD). Therefore, we hypothesized that Mg supplementation would improve diastolic function in DM. High-fat diet-induced diabetic mouse hearts showed increased cardiac DD and hypertrophy. Mice with DM showed a significantly increased E/e' ratio (the ratio of transmitral Doppler early filling velocity [E] to tissue Doppler early diastolic mitral annular velocity [e']) in the echocardiogram, left ventricular end diastolic volume (LVEDV), incidence of DD, left ventricular posterior wall thickness in diastole (PWTd), and ratio of heart weight to tibia length (HW/TL) when compared with controls. DM mice also had hypomagnesemia. Ventricular cardiomyocytes isolated from DM mice exhibited decreased mitochondrial ATP production, a 1.7- ± 0.2-fold increase of mitochondrial ROS, depolarization of the mitochondrial membrane potential, and mitochondrial Ca2+ overload. Dietary Mg administration (50 mg/ml in the drinking water) for 6 weeks increased plasma Mg concentration and improved cardiac function. At the cellular level, Mg improved mitochondrial function with increased ATP, decreased mitochondrial ROS and Ca2+ overload, and repolarized mitochondrial membrane potential. In conclusion, Mg supplementation improved mitochondrial function, reduced oxidative stress, and prevented DD in DM.

Mitochondrial Ca2+ flux modulates spontaneous electrical activity in ventricular cardiomyocytes.

INTRODUCTION: Ca2+ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na+-Ca2+ exchanger (NCX). Mitochondria participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in ventricular spontaneous electrical activity. METHODS: Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19-21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca2+ transients were determined by fluorescent imaging. RESULTS: In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca2+ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca2+ release from RYR2 or inositol trisphosphate receptors (IP3R), the pacemaker current (If) and mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca2+ flux influenced spontaneous beating independently of the SR Ca2+ release from RYR2, and the mitochondrial effect was dependent on IP3R SR Ca2+ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca2+ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels. CONCLUSIONS: Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca2+ uptake plays a modulatory role. Mitochondrial Ca2+ uptake through MCU is influenced by IP3R-dependent SR Ca2+ release.

RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA.

BACKGROUND: Although transcription is the initial process of gene expression, posttranscriptional gene expression regulation has also played a critical role for fine-tuning gene expression in a fast, precise, and cost-effective manner. Although the regulation of sodium channel α-subunit (SCN5A) mRNA expression has been studied at both transcriptional and pre-mRNA splicing levels, the molecular mechanisms governing SCN5A mRNA expression are far from clear. METHODS AND RESULTS: Herein, we show that, as evidenced by ribonucleoprotein immunoprecipitation assay, RNA binding protein Hu antigen R/ELAV like RNA binding protein 1 (HuR/ELAVL1) and myocyte enhancer factor-2C (MEF2C) transcription factor mRNA are associated. HuR positively regulated transcription factor MEF2C mRNA expression by protecting its mRNA from degradation. As demonstrated by both chromatin immunoprecipitation-quantitative polymerase chain reaction assay and an electrophoretic mobility shift assay, MEF2C enhanced SCN5A transcription by binding to a putative MEF2C binding site within SCN5A promoter region. Overexpression of HuR increased the expression of SCN5A mRNA, and this effect was attenuated by the presence of MEF2C small interfering RNA in cardiomyocytes. CONCLUSIONS: In conclusion, our results suggested that HuR participates in a combined network at the DNA and RNA levels that regulates SCN5A mRNA expression. HuR upregulates MEF2C mRNA expression by protecting MEF2C mRNA from degradation, and consequently, the elevated MEF2C enhances SCN5A mRNA transcription.

Mitochondrial Ca2+ Influx Contributes to Arrhythmic Risk in Nonischemic Cardiomyopathy.

BACKGROUND: Heart failure (HF) is associated with increased arrhythmia risk and triggered activity. Abnormal Ca2+ handling is thought to underlie triggered activity, and mitochondria participate in Ca2+ homeostasis. METHODS AND RESULTS: A model of nonischemic HF was induced in C57BL/6 mice by hypertension. Computer simulations were performed using a mouse ventricular myocyte model of HF. Isoproterenol-induced premature ventricular contractions and ventricular fibrillation were more prevalent in nonischemic HF mice than sham controls. Isolated myopathic myocytes showed decreased cytoplasmic Ca2+ transients, increased mitochondrial Ca2+ transients, and increased action potential duration at 90% repolarization. The alteration of action potential duration at 90% repolarization was consistent with in vivo corrected QT prolongation and could be explained by augmented L-type Ca2+ currents, increased Na+-Ca2+ exchange currents, and decreased total K+ currents. Of myopathic ventricular myocytes, 66% showed early afterdepolarizations (EADs) compared with 17% of sham myocytes (P<0.05). Intracellular application of 1 µmol/L Ru360, a mitochondrial Ca2+ uniporter-specific antagonist, could reduce mitochondrial Ca2+ transients, decrease action potential duration at 90% repolarization, and ameliorate EADs. Furthermore, genetic knockdown of mitochondrial Ca2+ uniporters inhibited mitochondrial Ca2+ uptake, reduced Na+-Ca2+ exchange currents, decreased action potential duration at 90% repolarization, suppressed EADs, and reduced ventricular fibrillation in nonischemic HF mice. Computer simulations showed that EADs promoted by HF remodeling could be abolished by blocking either the mitochondrial Ca2+ uniporter or the L-type Ca2+ current, consistent with the experimental observations. CONCLUSIONS: Mitochondrial Ca2+ handling plays an important role in EADs seen with nonischemic cardiomyopathy and may represent a therapeutic target to reduce arrhythmic risk in this condition.

Activation of the unfolded protein response downregulates cardiac ion channels in human induced pluripotent stem cell-derived cardiomyocytes.

RATIONALE: Heart failure is characterized by electrical remodeling that contributes to arrhythmic risk. The unfolded protein response (UPR) is active in heart failure and can decrease protein levels by increasing mRNA decay, accelerating protein degradation, and inhibiting protein translation. OBJECTIVE: Therefore, we investigated whether the UPR downregulated cardiac ion channels that may contribute to arrhythmogenic electrical remodeling. METHODS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to study cardiac ion channels. Action potentials (APs) and ion channel currents were measured by patch clamp recording. The mRNA and protein levels of channels and the UPR effectors were determined by quantitative RT-PCR and Western blotting. Tunicamycin (TM, 50 ng/mL and 5 µg/mL), GSK2606414 (GSK, 300 nmol/L), and 4µ8C (5 µmol/L) were utilized to activate the UPR, inhibit protein kinase-like ER kinase (PERK) and inositol-requiring protein-1 (IRE1), respectively. RESULTS: TM-induced activation of the UPR caused significant prolongation of the AP duration (APD) and a reduction of the maximum upstroke velocity (dV/dtmax) of the AP phase 0 in both acute (20-24 h) and chronic treatment (6 days). These changes were explained by reductions in the sodium, L-type calcium, the transient outward and rapidly/slowly activating delayed rectifier potassium currents. Nav1.5, Cav1.2, Kv4.3, and KvLQT1 channels showed concomitant reductions in mRNA and protein levels under activated UPR. Inhibition of PERK or IRE1 shortened the APD and reinstated dV/dtmax. The PERK branch regulated Nav1.5, Kv4.3, hERG, and KvLQT1. The IRE1 branch regulated Nav1.5, hERG, KvLQT1, and Cav1.2. CONCLUSIONS: Activated UPR downregulates all major cardiac ion currents and results in electrical remodeling in hiPSC-CMs. Both PERK and IRE1 branches downregulate Nav1.5, hERG, and KvLQT1. The PERK branch specifically downregulates Kv4.3, while the IRE1 branch downregulates Cav1.2. Therefore, the UPR contributed to electrical remodeling, and targeting the UPR might be anti-arrhythmic.

HuR-mediated SCN5A messenger RNA stability reduces arrhythmic risk in heart failure.

BACKGROUND: Downregulated sodium currents in heart failure (HF) have been linked to increased arrhythmic risk. Reduced expression of the messenger RNA (mRNA)-stabilizing protein HuR (also known as ELAVL1) may be responsible for the downregulation of sodium channel gene SCN5A mRNA. OBJECTIVE: The purpose of this article was to investigate whether HuR regulates SCN5A mRNA expression and whether manipulation of HuR benefits arrhythmia control in HF. METHODS: Quantitative real-time reverse-transcriptase polymerase chain reaction was used to investigate the expression of SCN5A. Optical mapping of the intact heart was adopted to study the effects of HuR on the conduction velocity and action potential upstroke in mice with myocardial infarct and HF after injection of AAV9 viral particles carrying HuR. RESULTS: HuR was associated with SCN5A mRNA in cardiomyocytes, and expression of HuR was downregulated in failing hearts. The association of HuR and SCN5A mRNA protected SCN5A mRNA from decay. Injection of AAV9 viral particles carrying HuR increased SCN5A expression in mouse heart tissues after MI. Optical mapping of the intact heart demonstrated that overexpression of HuR improved action potential upstroke and conduction velocity in the infarct border zone, which reduced the risk of reentrant arrhythmia after MI. CONCLUSION: Our data indicate that HuR is an important RNA-binding protein in maintaining SCN5A mRNA abundance in cardiomyocytes. Reduced expression of HuR may be at least partially responsible for the downregulation of SCN5A mRNA expression in ischemic HF. Overexpression of HuR may rescue decreased SCN5A expression and reduce arrhythmic risk in HF. Increasing mRNA stability to increase ion channel currents may correct a fundamental defect in HF and represent a new paradigm in antiarrhythmic therapy.

Abnormal sodium channel mRNA splicing in hypertrophic cardiomyopathy.

BACKGROUND: Our previous studies showed that in ischemic and nonischemic heart failure (HF), the voltage-gated cardiac Na+ channel α subunit (SCN5A) mRNA is abnormally spliced to produce two truncated transcript variants (E28C and D) that activate the unfolded protein response (UPR). We tested whether SCN5A post-transcriptional regulation was abnormal in hypertrophic cardiomyopathy (HCM). MATERIAL AND METHODS: Human heart tissue was obtained from HCM patients. The changes in relative abundances of SCN5A, its variants, splicing factors RBM25 and LUC7A, and PERK, a major effector of the UPR, were analyzed by real time RT-PCR and the expression changes were confirmed by Western Blot. RESULTS: We found reduced full-length transcript, increased SCN5A truncation variants and activation of UPR in HCM when compared to control hearts. In these patients, real time RT-PCR revealed that HCM patients had decreased SCN5A mRNA to 27.8±4.07% of control (P<0.01) and an increased abundance of E28C and E28D (3.4±0.3 and 2.8±0.3-fold, respectively, P<0.05). PERK mRNA increased 8.2±3.1 fold (P<0.01) in HCM patients. Western blot confirmed a significant increase of PERK. CONCLUSIONS: These data suggested that the full-length SCN5A was reduced in patients with HCM. This reduction was accompanied by abnormal SCN5A pre-mRNA splicing and UPR activation. These changes may contribute to the arrhythmic risk in HCM.

Protein 4.1R Influences Myogenin Protein Stability and Skeletal Muscle Differentiation.

Protein 4.1R (4.1R) isoforms are expressed in both cardiac and skeletal muscle. 4.1R is a component of the contractile apparatus. It is also associated with dystrophin at the sarcolemma in skeletal myofibers. However, the expression and function of 4.1R during myogenesis have not been characterized. We now report that 4.1R expression increases during C2C12 myoblast differentiation into myotubes. Depletion of 4.1R impairs skeletal muscle differentiation and is accompanied by a decrease in the levels of myosin heavy and light chains and caveolin-3. Furthermore, the expression of myogenin at the protein, but not mRNA, level is drastically decreased in 4.1R knockdown myocytes. Similar results were obtained using MyoD-induced differentiation of 4.1R-/- mouse embryonic fibroblast cells. von Hippel-Lindau (VHL) protein is known to destabilize myogenin via the ubiquitin-proteasome pathway. We show that 4.1R associates with VHL and, when overexpressed, reverses myogenin ubiquitination and stability. This suggests that 4.1R may influence myogenesis by preventing VHL-mediated myogenin degradation. Together, our results define a novel biological function for 4.1R in muscle differentiation and provide a molecular mechanism by which 4.1R promotes myogenic differentiation.

Obstructive Sleep Apnea and Circulating Potassium Channel Levels.

BACKGROUND: Cardiac arrhythmias and sudden cardiac death are more frequent in patients with obstructive sleep apnea (OSA). OSA is associated with QT prolongation, and QT prolongation is an independent risk factor for sudden cardiac death. Because QT prolongation can be mediated by potassium channel loss of function, we tested whether OSA or continuous positive airway pressure therapy altered mRNA expression of circulating white blood cell potassium channels. METHODS AND RESULTS: In total, 28 patients with OSA newly diagnosed by polysomnogram and 6 participants without OSA were enrolled. Potassium channel levels in white blood cells at baseline and at a 4-week follow-up visit were compared. There was a significant inverse correlation between the severity of the OSA stratified by apnea-hypopnea index and mRNA expression of the main potassium channels assessed: KCNQ1 (r=-0.486, P=0.007), KCNH2 (r=-0.437, P=0.016), KCNE1 (r=-0.567, P=0.001), KCNJ2 (r=-0.442, P=0.015), and KCNA5 (r=-0.468, P=0.009). In addition, KCNQ1, KCNH2, and KCNE1 inversely correlated with the oxygen desaturation index 4. After 4 weeks of continuous positive airway pressure therapy, circulating KCNQ1 and KCNJ2 were increased 1.4±0.4-fold (P=0.040) and 2.1±1.4-fold (P=0.046) in the moderate OSA group. Compared with patients with mild or moderate OSA, patients with severe OSA had a persistently higher apnea-hypopnea index (mild 2.0±1.8, moderate 1.0±0.9, severe 5.8±5.6; P=0.015), perhaps explaining why the potassium channel changes were not seen in the severe OSA group. CONCLUSIONS: The mRNA expression of most potassium channels inversely correlates with the severity of OSA and hypoxemia. Continuous positive airway pressure therapy improves circulating KCNQ1 and KCNJ2 in patients with moderate OSA.

RBFOX2 promotes protein 4.1R exon 16 selection via U1 snRNP recruitment.

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5' splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5' splice site, but not the engineered strong 5' splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5' splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5' splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5' splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.

Role of RBM25/LUC7L3 in abnormal cardiac sodium channel splicing regulation in human heart failure.

BACKGROUND: Human heart failure is associated with decreased cardiac voltage-gated Na+ channel current (encoded by SCN5A), and the changes have been implicated in the increased risk of sudden death in heart failure. Nevertheless, the mechanism of SCN5A downregulation is unclear. A number of human diseases are associated with alternative mRNA splicing, which has received comparatively little attention in the study of cardiac disease. Splicing factor expression profiles during human heart failure and a specific splicing pathway for SCN5A regulation were explored in this study. METHODS AND RESULTS: Gene array comparisons between normal human and heart failure tissues demonstrated that 17 splicing factors, associated with all major spliceosome components, were upregulated. Two of these splicing factors, RBM25 and LUC7L3, were elevated in human heart failure tissue and mediated truncation of SCN5A mRNA in both Jurkat cells and human embryonic stem cell-derived cardiomyocytes. RBM25/LUC7L3-mediated abnormal SCN5A mRNA splicing reduced Na+ channel current 91.1±9.3% to a range known to cause sudden death. Overexpression of either splicing factor resulted in an increase in truncated mRNA and a concomitant decrease in the full-length SCN5A transcript. CONCLUSIONS: Of the 17 mRNA splicing factors upregulated in heart failure, RBM25 and LUC7L3 were sufficient to explain the increase in truncated forms and the reduction in full-length Na+ channel transcript. Because the reduction in channels was in the range known to be associated with sudden death, interruption of this abnormal mRNA processing may reduce arrhythmic risk in heart failure.

Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5' exons of protein 4.1R gene.

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3' splice sites (ss) of exon 2'/2. Here, we demonstrate that exon 1 and 2'/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3' ss and exon 1B to the proximal 3' ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter-driven splicing patterns but differed from 1A-promoter-driven splicing patterns, suggesting that promoter identity affects exon 2'/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2'/2 junction CAGAGAA, a sequence that overlaps the distal U2AF(35)-binding 3' ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3' ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3' ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.

Inhibition of PAX3 by TGF-beta modulates melanocyte viability.

The protein encoded by paired-box homeotic gene 3 (PAX3) is a key regulator of the microphthalmia-associated transcription factor (Mitf) in the melanocyte lineage. Here, we show that PAX3 expression in skin is directly inhibited by TGF-beta/Smads. UV irradiation represses TGF-beta in keratinocytes, and the repression of TGF-beta/Smads upregulates PAX3 in melanocytes, which is associated with a UV-induced melanogenic response and consequent pigmentation. Furthermore, the TGF-beta-PAX3 signaling pathway interacts with the p53-POMC/MSH-MC1R signaling pathway, and both are crucial in melanogenesis. The activation of p53-POMC/MSH-MC1R signaling is required for the UV-induced melanogenic response because PAX3 functions in synergy with SOX10 in a cAMP-response element (CRE)-dependent manner to regulate the transcription of Mitf. This study will provide a rich foundation for further research on skin cancer prevention by enabling us to identify targeted small molecules in the signaling pathways of the UV-induced melanogenic response that are highly likely to induce naturally protective pigmentation.

Novel splicing factor RBM25 modulates Bcl-x pre-mRNA 5' splice site selection.

RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-x(S) 5' splice site (5' ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-x(L). Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-x(S) 5' ss selection, leading to decreased Bcl-x(S) isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5' ss; however, it was not required when a strong consensus 5' ss was present. In support of a role for RBM25 in modulating the selection of a 5' ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-x(S) 5' ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.