RESUMEN

Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CCCTC-binding factor (CTCF) and its paralog BORIS. Additionally, we highlighted the role of CTCF and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition.

Asunto(s)

Empalme Alternativo , Autofagia , Neoplasias de la Mama , Factor de Unión a CCCTC , Metilación de ADN , Proteínas de la Membrana , Proteínas Proto-Oncogénicas , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Femenino , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Epigénesis Genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Células MCF-7 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo

RESUMEN

The therapeutic outcomes of ovarian cancer (OVCA) patients are majorly limited by the development of acquired chemo/radioresistance and the lack of targeted therapies. Accumulating studies demonstrate that microRNAs are involved in tumorigenesis and radioresistance. This study aims to illustrate the role of miR-588 in the radioresistance of OVCA cells. The levels of miR-588 and mRNAs were detected by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). OVCA cell viability, proliferative, migratory and invasive capacities were evaluated by the cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay and transwell assay, respectively. The luciferase activities of plasmids containing wild -type and mutant serine/arginine-rich splicing factor 6 (SRSF6) 3'-untranslated region in miR-588 silenced OVCA cells were detected by a luciferase reporter assay. We found that miR-588 was overexpressed in OVCA tissues and cells. Knockdown of miR-588 exerted an inhibitory effect on the proliferation, migration and invasion and strengthened the radiosensitivity of OVCA cells, whereas overexpression of miR-588 increased the radioresistance of OVCA cells. SRSF6 was verified to be targeted by miR-588 in OVCA cells. In addition, the expression level of miR-588 was negatively correlated with that of SRSF6 in OVCA clinical samples. Rescue assays indicated that SRSF6 knockdown reversed the effect of miR-588 inhibition of OVCA cells under radiation. Overall, miR-588 acts as an oncogene in OVCA and increases the radioresistance of OVCA cells by targeting SRSF6.

RESUMEN

Low level HIV transcription during modern antiretroviral therapy (ART) in persons with HIV is linked to residual inflammation and associated diseases, like cardiovascular disease and cancer. The "block and lock" approach to hold HIV in a state of deep latency may help decrease residual inflammation in a person with HIV on ART and thus improve health. A camptothecin analog topotecan (TPT) was previously implicated as an inhibitor of active HIV replication. Using an in vitro primary T cell model of HIV latency, we demonstrated that (i) TPT reduces HIV transcriptional activity in latently infected cells; (ii) downregulation of HIV RNA by TPT cannot be reversed by latency reversing agents; (iii) several primary and secondary mechanism of action of TPT may be involved in control of HIV replication; (iv) regulation of HIV RNA by TPT is dependent on splicing complexity; (v) increase in proportion of unspliced HIV transcripts was facilitated by intron retention and upregulation of splicing factors, specifically SRSF6, by TPT. Although high TPT dosing (10 µM) was needed to achieve the observed effects, viability of primary CD4+ T cells was not greatly affected. Because toxicity can be observed with TPT in persons with cancer, TPT is unlikely to be used as an anti-HIV agent in clinic, but our study provides proof that camptothetin has "block and lock" activity. Other camptothetin analogs, which are less toxic than TPT, should be designed and tested as HIV "block and lock" agents. IMPORTANCE HIV survives in a state of very low activity, called latency, for long periods in persons with HIV on antiretroviral therapy. This low activity of HIV is linked to residual inflammation and associated diseases, such as heart disease and cancer. New strategies are being explored to further silence the HIV provirus and suppress residual inflammation. This study provides strong evidence that the camptothetin analog, Topotecan, can reduce residual activity of HIV in an experimental model of HIV latency. While Topotecan itself is likely not suitable for use in the clinic due to its toxicity, other camptothetin analogs should be designed and investigated as "block and lock" agents.

Asunto(s)

Infecciones por VIH , Empalme del ARN , Topotecan , Latencia del Virus , Humanos , Infecciones por VIH/tratamiento farmacológico , Fosfoproteínas , Factores de Empalme Serina-Arginina , Topotecan/farmacología , Latencia del Virus/efectos de los fármacos

RESUMEN

Alternative splicing (AS) is a procedure during gene expression that allows the production of multiple mRNAs from a single gene, leading to a larger number of proteins with various functions. The alternative splicing (AS) of Fas (Apo-1/CD95) pre-mRNA can generate membrane-bound or soluble isoforms with pro-apoptotic and anti-apoptotic functions. SRSF6, a member of the Serine/Arginine-rich protein family, plays essential roles in both constitutive and alternative splicing. Here, we identified SRSF6 as an important regulatory protein in Fas AS. The cassette exon inclusion of Fas was decreased by SRSF6-targeting shRNA treatment, but increased by SRSF6 overexpression. The deletion and substitution mutagenesis of the Fas minigene demonstrated that the UGCCAA sequence in the cassette exon of the Fas gene causes the functional disruption of SRSF6, indicating that these sequences are essential for SRSF6 function in Fas splicing. In addition, biotin-labeled RNA-pulldown and immunoblotting analysis showed that SRSF6 interacted with these RNA sequences. Mutagenesis in the splice-site strength alteration demonstrated that the 5' splice-site, but not the 3' splice-site, was required for the SRSF6 regulation of Fas pre-mRNA. In addition, a large-scale RNA-seq analysis using GTEX and TCGA indicated that while SRSF6 expression was correlated with Fas expression in normal tissues, the correlation was disrupted in tumors. Furthermore, high SRSF6 expression was linked to the high expression of pro-apoptotic and immune activation genes. Therefore, we identified a novel RNA target with 5' splice-site dependence of SRSF6 in Fas pre-mRNA splicing, and a correlation between SRSF6 and Fas expression.

RESUMEN

Aberrant alternative splicing of pre-mRNA is an emerging cancer hallmark. Many cancer-associated genes undergo alternative splicing to produce multiple isoforms with diverse or even antagonistic functions. Oncogenic isoforms are often up-regulated, whereas tumor suppressive isoforms are down-regulated during tumorigenesis. Serine/arginine-rich splicing factor 6 (SRSF6) is an important splicing factor that regulates the alternative splicing of hundreds of target genes, including many cancer-associated genes. The potential roles of SRSF6 in cancers have attracted increasing attentions in the past decade. Accumulated pieces of evidence have shown that SRSF6 is a potential oncogenic gene that promotes oncogenic splicing when overexpressed. Targeting SRSF6 may suppress tumorigenesis. In this review, we describe the gene, mRNA, and protein structure of SRSF6; summarize the current understanding of the expression, functions, and regulatory mechanisms of SRSF6 during tumorigenesis; and discuss the potential application of targeting SRSF6 in cancer treatment.

RESUMEN

BACKGROUND: Long non-coding RNAs (lncRNA) are reported to influence colorectal cancer (CRC) progression. Currently, the functions of the lncRNA ZNF561 antisense RNA 1 (ZNF561-AS1) in CRC are unknown. METHODS: ZNF561-AS1 and SRSF6 expression in CRC patient samples and CRC cell lines was evaluated through TCGA database analysis, western blot along with real-time PCR. SRSF6 expression in CRC cells was also examined upon ZNF561-AS1 depletion or overexpression. Interaction between miR-26a-3p, miR-128-5p, ZNF561-AS1, and SRSF6 was examined by dual luciferase reporter assay, as well as RNA binding protein immunoprecipitation (RIP) assay. Small interfering RNA (siRNA) mediated knockdown experiments were performed to assess the role of ZNF561-AS1 and SRSF6 in the proliferative actives and apoptosis rate of CRC cells. A mouse xenograft model was employed to assess tumor growth upon ZNF561-AS1 knockdown and SRSF6 rescue. RESULTS: We find that ZNF561-AS1 and SRSF6 were upregulated in CRC patient tissues. ZNF561-AS1 expression was reduced in tissues from treated CRC patients but upregulated in CRC tissues from relapsed patients. SRSF6 expression was suppressed and enhanced by ZNF561-AS1 depletion and overexpression, respectively. Mechanistically, ZNF561-AS1 regulated SRSF6 expression by sponging miR-26a-3p and miR-128-5p. ZNF561-AS1-miR-26a-3p/miR-128-5p-SRSF6 axis was required for CRC proliferation and survival. ZNF561-AS1 knockdown suppressed CRC cell proliferation and triggered apoptosis. ZNF561-AS1 depletion suppressed the growth of tumors in a model of a nude mouse xenograft. Similar observations were made upon SRSF6 depletion. SRSF6 overexpression reversed the inhibitory activities of ZNF561-AS1 in vivo, as well as in vitro. CONCLUSION: In summary, we find that ZNF561-AS1 promotes CRC progression via the miR-26a-3p/miR-128-5p-SRSF6 axis. This study reveals new perspectives into the role of ZNF561-AS1 in CRC.

Asunto(s)

Proteínas Portadoras/genética , Neoplasias Colorrectales/metabolismo , MicroARNs/metabolismo , Fosfoproteínas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Animales , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células HCT116 , Células HT29 , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Fosfoproteínas/genética , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Largo no Codificante/genética , Factores de Empalme Serina-Arginina/genética

RESUMEN

Alternative splicing (AS) occurs in nearly all human genes and abnormal AS has a close association with cancer. Serine and arginine­rich splicing factor 6 (SRSF6), a canonical member of the serine/arginine­rich protein family, has been characterized as an important regulator of AS. However, the role of SRSF6 in regulating AS in cancers has remained to be fully elucidated. In the present study, the median expression of SRSF6 in tumors was determined to be higher compared with that in matched normal tissues in 13 out of 16 cancer types from The Cancer Genome Atlas. To investigate the biological effects of SRSF6 overexpression, an SRSF6­overexpression model of HeLa cells was constructed and it was revealed that SRSF6 overexpression resulted in significantly higher apoptosis and lower proliferation compared to control cells. Transcriptome analysis indicated that overexpression of SRSF6 in cancer cells induced large­scale changes in transcriptional expression levels and AS. Two groups of cervical cancer tumor samples in which SRSF6 was differentially expressed were then selected to analyze potential SRSF6­regulated AS. It was determined that the pattern of SRSF6­regulated AS in clinical samples was similar to that in cancer cells and AS genes were enriched in DNA damage response (DDR) pathways, including DNA repair and double­strand break repair via homologous recombination. Furthermore, AS events regulated by SRSF6 were validated using reverse transcription­quantitative PCR. The present results highlighted that SRSF6 is able to trigger the activation of DDR pathways via regulation of AS to influence cancer progression. These results markedly expand the current understanding of the mechanisms underlying SRSF6­mediated gene regulation and suggest the potential use of SRSF6 as a therapeutic target in cancer.

Asunto(s)

Daño del ADN , Enzimas Reparadoras del ADN/genética , Reparación del ADN , Neoplasias/genética , Fosfoproteínas/genética , Factores de Empalme Serina-Arginina/genética , Empalme Alternativo , Apoptosis/fisiología , Proliferación Celular/fisiología , Biología Computacional/métodos , Enzimas Reparadoras del ADN/metabolismo , Bases de Datos Genéticas , Femenino , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Fosfoproteínas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo

RESUMEN

MicroRNAs (miRNAs) are short and non-coding RNAs binding to 3'UTR of target mRNAs to downregulate their expression. Recent studies have shown that miRNAs indirectly regulated alternative splicing (AS) by targeting splicing factors and caused shifts in splicing patterns of target genes. However, the roles of miRNA-regulating splicing factors in pancreatic cancer progression remain unknown. Herein, we reported that miR-193a-5p was markedly upregulated in pancreatic cancer tissues and cells and correlated with clinical outcomes of pancreatic cancer patients. Overexpression of miR-193a-5p contributed to the metastasis of pancreatic cancer cells both in vitro and in vivo. The mechanistic investigation suggested that miR-193a-5p modulated oxoglutarate dehydrogenase-like (OGDHL) and extracellular matrix protein 1 (ECM1) AS by targeting serine/arginine-rich splicing factor 6 (SRSF6), leading to the activation of the epithelial-to-mesenchymal transition (EMT) process. Together, our findings highlighted the role of miR-193a-5p-targeting SRSF6 in pancreatic cancer metastasis, which may serve as a novel target for pancreatic cancer diagnosis and therapy.

RESUMEN

Alzheimer's disease (AD) is the most common cause of dementia and cannot be cured. The etiology and pathogenesis of AD is still not fully understood, the genetics is considered to be one of the most important factors for AD onset, and the identified susceptible genes could provide clues to the AD mechanism and also be the potential targets. MicroRNA-146a-5p (miR-146a) is well known in the regulation of the inflammatory response, and the functional SNP of miR-146a was associated with AD risk. In this study, using a noninvasive nasal administration, we discovered that a miR-146a agomir (M146AG) rescued cognitive impairment in the APP/PS1 transgenic mouse and alleviated the overall pathological process in the AD mouse model, including neuroinflammation, glia activation, Aß deposit, and tau phosphorylation in hippocampi. Furthermore, the transcriptional analysis revealed that besides the effect of neuroinflammation, M146AG may serve as a multi-potency target for intervention in AD. In addition, Srsf6 was identified as a target of miR-146a, which may play a role in AD progression. In conclusion, our study supports that the nasal-to-brain pathway is efficient and operable for the brain administration of microRNAs (miRNAs), and that miR-146a may be a new potential target for AD treatment.

RESUMEN

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine-arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine-arginine-rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC-like kinases-1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.

Asunto(s)

Comunicación Autocrina/fisiología , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Bovinos , Femenino , Células de la Granulosa/citología

RESUMEN

OBJECTIVE: To investigate the molecular function of splicing factor SRSF6 in colorectal cancer (CRC) progression and discover candidate chemicals for cancer therapy through targeting SRSF6. DESIGN: We performed comprehensive analysis for the expression of SRSF6 in 311 CRC samples, The Cancer Genome Atlas and Gene Expression Omnibus (GEO) database. Functional analysis of SRSF6 in CRC was performed in vitro and in vivo. SRSF6-regulated alternative splicing (AS) and its binding motif were identified by next-generation RNA-sequencing and RNA immunoprecipitation sequencing (RIP-seq), which was validated by gel shift and minigene reporter assay. ZO-1 exon23 AS was investigated to mediate the function of SRSF6 in vitro and in vivo. Based on the analysis of domain-specific role, SRSF6-targeted inhibitor was discovered de novoby virtual screening in 4855 FDA-approved drugs and its antitumour effects were evaluated in vitroand in vivo. RESULTS: SRSF6 was frequently upregulated in CRC samples and associated with poor prognosis, which promoted proliferation and metastasis in vitro and in vivo. We identified SRSF6-regulated AS targets and discovered the SRSF6 binding motif. Particularly, SRSF6 regulates ZO-1 aberrant splicing to function as an oncogene by binding directly to its motif in the exon23. Based on the result that SRSF6 RRM2 domain plays key roles in regulating AS and biological function, indacaterol, a ß2-adrenergic receptor agonist approved for chronic obstructive pulmonary disease treatment, is identified as the inhibitor of SRSF6 to suppress CRC tumourigenicity. CONCLUSIONS: SRSF6 functions the important roles in mediating CRC progression through regulating AS, and indacaterol is repositioned as an antitumour drug through targeting SRSF6. ACCESSION NUMBERS: The accession numbers for sequencing data are SRP111763 and SRP111797.

Asunto(s)

Empalme Alternativo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Fosfoproteínas/genética , Factores de Empalme Serina-Arginina/genética , Animales , Antineoplásicos/farmacología , Proliferación Celular , Supervivencia Celular , Neoplasias Colorrectales/tratamiento farmacológico , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoprecipitación , Indanos/farmacología , Ratones , Isoformas de Proteínas , Quinolonas/farmacología , Análisis de Secuencia de ARN , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

Smooth muscle has a central role in bronchospasm-induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis-regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT-PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis-acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.

Asunto(s)

Empalme Alternativo , Asma/metabolismo , Enfermedades de los Caballos/metabolismo , Miocitos del Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/genética , Factores de Empalme Serina-Arginina/genética , Animales , Asma/genética , Asma/veterinaria , Células Cultivadas , Enfermedades de los Caballos/genética , Caballos , Pulmón/citología , Pulmón/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Regulación hacia Arriba

RESUMEN

Dendritic alteration of striatal medium spiny neurons is one of the earliest morphological abnormalities in Huntington's disease (HD). The main microtubule-associated protein in dendrites is MAP2. The low-molecular weight isoforms of MAP2 (LMW-MAP2) are the juvenile forms resulting from exclusion of the sequence encoded by exons E7-E9 and are downregulated after the early stages of neuronal development when E7-E9 exon-including high-molecular weight isoforms (HMW-MAP2) are favored. Splicing alteration has recently been proposed to contribute to HD in view of two pathogenic missplicing events resulting in a highly toxic N-terminal version of mutant huntingtin and in a detrimental imbalance in MAP Tau isoforms with three or four tubulin-binding repeats. Both splicing events are postulated targets of the SR splicing factor SRSF6 which has recently been reported to be dramatically altered in HD. SR proteins often regulate functionally related sets of genes and SRSF6 targets are enriched in genes involved in brain organogenesis including several actin-and tubulin-binding proteins. Here we hypothesized that MAP2 might be target of SRSF6 and altered in HD. By SRSF6 knockdown in neuroblastoma cells, we demonstrate that splicing of MAP2 E7-E9 exons is affected by SRSF6. We then show a disbalance in LMW and HMW MAP2 mRNA isoforms in HD striatum in favor of the juvenile LMW forms together with a decrease in total MAP2 mRNA. This is accompanied by a global decrease in total MAP2 protein due to almost total disappearance of HMW-MAP2 isoforms with preservation of LMW-MAP2 isoforms. Accordingly, the predominant dendritic MAP2 staining in striatal neuropil of control subjects is absent in HD cases. In these, MAP2-immunoreactivity is faint and restricted to neuronal cell bodies often showing a sharp boundary at the base of dendrites. Together, our results highlight the importance of splicing alteration in HD and suggest that MAP2 alteration contributes to dendritic atrophy.

Asunto(s)

Enfermedad de Huntington/genética , Proteínas Asociadas a Microtúbulos/genética , Empalme Alternativo/genética , Encéfalo/patología , Línea Celular Tumoral , Cuerpo Estriado/patología , Dendritas/patología , Exones , Técnicas de Silenciamiento del Gen/métodos , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuroblastoma/metabolismo , Neuronas/patología , Fosfoproteínas/metabolismo , ARN Mensajero/genética , Factores de Empalme Serina-Arginina/metabolismo

RESUMEN

Huntington's disease (HD) is caused by a CAG-repeat encoding a polyglutamine (polyQ) tract in the huntingtin protein. There is plenty of evidence of polyQ-driven toxicity. However, CAG repeat RNA-driven alteration of splicing has recently been proposed in analogy to CUG-repeat diseases. Here we review the reported alteration of the CAG-repeat associated splicing factor SRSF6 in brains of HD patients and mouse models and how this correlates with altered splicing of, at least, two microtubule-associated proteins in HD, namely MAPT (tau) and MAP2. Regarding tau, altered splicing of exon 10 has been reported, along with increased levels and 4R/3R-tau ratio and detection of tau in a new nuclear rod-shaped histopathological hallmark termed tau nuclear rod (TNR) or tau nuclear indentation (TNI). These findings, together with an attenuation of HD phenotype in R6/1 mice with tau deficiency and subsequent studies showing increased phosphorylation in mouse models and increased levels in CSF of patients, has led to proposing HD as a tauopathy. Regarding MAP2, an increase in its juvenile form and a decrease in total MAP2 together with redistribution from dendrites to soma is observed in HD patients, which may contribute to the dendritic atrophy in HD. Furthermore, MAP2 positive structures filling nuclear indentations have occasionally been found and co-localized with tau. Therefore, altered MAP function with imbalance in tau/MAP2 content could contribute to HD striatal atrophy and dysfunction. Besides, TNIs might be indicative of such MAP abnormalities. TNIs are also found in early pathology Alzheimer's disease and in tauopathy mice over-expressing mutant 4R-tau. This indicates that tau alteration is sufficient for TNI detection, which becomes a marker of increased total tau and/or altered 4R/3R-tau ratio and reporter of pathology-associated nuclear indentations. Altogether, these recent studies suggest that correcting the SRSF6-driven missplicing and/or microtubule-associated imbalance might be of therapeutic value in HD.

Asunto(s)

Encéfalo/patología , Citoesqueleto/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Fosfoproteínas/genética , Factores de Empalme Serina-Arginina/genética , Empalme Alternativo/fisiología , Animales , Citoesqueleto/genética , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/patología , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Expansión de Repetición de Trinucleótido , Proteínas tau/genética , Proteínas tau/metabolismo

RESUMEN

Alternative splicing (AS) is a pivotal mechanism for the expansion of gene diversity, which determines the cellular fate or specification. However, the effect of AS networks on brown adipogenesis has not been comprehensively investigated. In this study, we identified the discriminative splicing profiles of RNA-binding motif protein 4a-knockout (RBM4a-/-) brown adipocytes (BAs) and compared them with those of their wild-type counterparts through deep RNA-sequencing. Among these candidates, RBM4a ablation enhanced the relative level of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1-4) transcripts, which were predominantly generated in embryonic BAs. By contrast, most of the Nova1 transcripts were exon 4-included (Nova1+4) in mature BAs. The Nova1 isoforms exhibited differential effects on repressing the development of BAs. Moreover, overexpression of Nova1 proteins reduced the serine/arginine splicing factor 6 (SRSF6) level by enhancing the generation of intron 2-included (SRSF6+intron 2) transcripts, which are a putative candidate of the AS-coupled nonsense-mediated decay mechanism. Furthermore, we observed the positive effect of SRSF6 on BA development. These results highlight the hierarchical role of RBM4a in an AS cascade that manipulates brown adipogenesis.

Asunto(s)

Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Empalme Alternativo , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/genética , Animales , Línea Celular , Masculino , Ratones , Ratones Noqueados , Antígeno Ventral Neuro-Oncológico

RESUMEN

Long non-coding RNAs (lncRNAs) play crucial roles in many biological and pathological processes, including tumor metastasis. Here we reported a novel lncRNA, LINC01133 that was downregulated by TGF-ß, which could inhibit epithelial-mesenchymal transition (EMT) and metastasis in colorectal cancer (CRC) cells. An alternative splicing factor SRSF6 was identified to directly interact with LINC01133, and SRSF6 promoted EMT and metastasis in CRC cells independent of LINC01133 And we confirmed that the EMT process was regulated by LINC01133 in CRC cells dependent on the presence of SRSF6. The observation for LINC01133 to inhibit metastasis was also verified in vivo. Moreover clinical data showed that the LINC01133 expression was positively correlated with E-cadherin, and negatively correlated with Vimentin, and there was a robust association of low LIINC01133 expression in tumors with poor survival in CRC samples. These data suggest that LINC01133 inhibits the EMT and metastasis by directly binding to SRSF6 as a target mimic, and may serve as a prognostic biomarker and an effective target for anti-metastasis therapies for CRC.

Asunto(s)

Movimiento Celular , Neoplasias Colorrectales/metabolismo , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , ARN Largo no Codificante/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Animales , Antígenos CD , Cadherinas/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células HCT116 , Células HT29 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Metástasis Linfática , Ratones Endogámicos NOD , Ratones SCID , Fosfoproteínas/genética , Interferencia de ARN , ARN Largo no Codificante/genética , Factores de Empalme Serina-Arginina/genética , Transducción de Señal , Factores de Tiempo , Transfección , Carga Tumoral , Vimentina/metabolismo

RESUMEN

Huntington's disease (HD) is an adult-onset neurodegenerative disorder caused by a mutated CAG repeat in the huntingtin gene that is translated into an expanded polyglutamine tract. The clinical manifestation of HD is a progressive physical, cognitive, and psychiatric deterioration that is eventually fatal. The mutant huntingtin protein is processed into several smaller fragments, which have been implicated as critical factors in HD pathogenesis. The search for proteases responsible for their production has led to the identification of several cleavage sites on the huntingtin protein. However, the origin of the small N-terminal fragments that are found in HD postmortem brains has remained elusive. Recent mapping of huntingtin fragments in a mouse model demonstrated that the smallest N-terminal fragment is an exon 1 protein. This discovery spurred our hypothesis that mis-splicing as opposed to proteolysis could be generating the smallest huntingtin fragment. We demonstrated that mis-splicing of mutant huntingtin intron 1 does indeed occur and results in a short polyadenylated mRNA, which is translated into an exon 1 protein. The exon 1 protein fragment is highly pathogenic. Transgenic mouse models containing just human huntingtin exon 1 develop a rapid onset of HD-like symptoms. Our finding that a small, mis-spliced HTT transcript and corresponding exon 1 protein are produced in the context of an expanded CAG repeat has unraveled a new molecular mechanism in HD pathogenesis. Here we present detailed models of how mis-splicing could be facilitated, what challenges remain in this model, and implications for therapeutic studies.

Asunto(s)

Enfermedad de Huntington/genética , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Empalme del ARN , Animales , Humanos

RESUMEN

Bim is a member of the pro-apoptotic BH3-only Bcl-2 family of proteins. Bim gene undergoes alternative splicing to produce three predominant splicing variants (BimEL, BimL and BimS). The smallest variant BimS is the most potent inducer of apoptosis. Zinc (Zn(2+)) has been reported to stimulate apoptosis in various cell types. In this study, we examined whether Zn(2+) affects the expression of Bim in human neuroblastoma SH-SY5Y cells. Zn(2+) triggered alterations in Bim splicing and induced preferential generation of BimS, but not BimEL and BimL, in a dose- and time-dependent manner. Other metals (cadmium, cobalt and copper) and stresses (oxidative, endoplasmic reticulum and genotoxic stresses) had little or no effect on the expression of BimS. To address the mechanism of Zn(2+)-induced preferential generation of BimS, which lacks exon 4, we developed a Bim mini-gene construct. Deletion analysis using the Bim mini-gene revealed that predicted binding sites of the SR protein SRSF6, also known as SRp55, are located in the intronic region adjacent to exon 4. We also found that mutations in the predicted SRSF6-binding sites abolished generation of BimS mRNA from the mutated Bim mini-gene. In addition, a UV cross-linking assay followed by Western blotting showed that SRSF6 directly bound to the predicted binding site and Zn(2+) suppressed this binding. Moreover, Zn(2+) stimulated SRSF6 hyper-phosphorylation. TG003, a cdc2-like kinase inhibitor, partially prevented Zn(2+)-induced generation of BimS and SRSF6 hyper-phosphorylation. Taken together, our findings suggest that Zn(2+) inhibits the activity of SRSF6 and promotes elimination of exon 4, leading to preferential generation of BimS.

Asunto(s)

Empalme Alternativo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Zinc/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Secuencia de Bases , Proteína 11 Similar a Bcl2 , Sitios de Unión , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme Serina-Arginina , Transcripción Genética