Ravi K. Singh

NF-κB–YY1–miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.

Pre-mRNA splicing in disease and therapeutics

In metazoans, alternative splicing of genes is essential for regulating gene expression and contributing to functional complexity. Computational predictions, comparative genomics, and transcriptome profiling of normal and diseased tissues indicate that an unexpectedly high fraction of diseases are caused by mutations that alter splicing. Mutations in cis elements cause missplicing of genes that alter gene function and contribute to disease pathology. Mutations of core spliceosomal factors are associated with hematolymphoid neoplasias, retinitis pigmentosa, and microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Mutations in the trans regulatory factors that control alternative splicing are associated with autism spectrum disorder, amyotrophic lateral sclerosis (ALS), and various cancers. In addition to discussing the disorders caused by these mutations, this review summarizes therapeutic approaches that have emerged to correct splicing of individual genes or target the splicing machinery.

Genotoxic Stress Induces Coordinately Regulated Alternative Splicing of the p53 Modulators MDM2 and MDM4

The tumor suppressor protein p53 is a transcription factor that induces G(1) arrest of the cell cycle and/or apoptosis. The murine double-minute protein MDM2 and its homologue MDM4 (also known as MDMX) are critical regulators of p53. Altered transcripts of the human homologue of mdm2, MDM2, have been identified in human tumors, such as invasive carcinoma of the breast, lung carcinoma, and liposarcoma. MDM2 alternate forms act to negatively regulate the normal MDM2 gene product, thus activating p53. Although many reports have documented a plethora of tumor types characterized by MDM2 alternative transcripts, few have investigated the signals that might initiate alternative splicing. We have identified a novel role of these alternative MDM2 transcripts in the normal surveillance mechanism of the cell and in DNA damage response. We report that alternate forms of MDM2 are detected after UV irradiation. Furthermore, we show that mouse cells treated with UV are also characterized by alternative transcripts of mdm2, suggesting that this is an important and evolutionarily conserved mechanism for regulating the expression of MDM2/mdm2. An additional p53 regulator and mdm2 family member, MDM4, is likewise alternatively spliced following UV irradiation. By activating alternative splicing of both MDM2 and MDM4, yet another layer of p53 regulation is initiated by the cells in response to damage. A stepwise model for malignant conversion by which alternate forms of MDM2 and MDM4 place selective pressure on the cells to acquire additional alterations in the p53 pathway is herein proposed.

The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression

Cardiac dysfunction is the second leading cause of death in myotonic dystrophy type 1 (DM1), primarily because of arrhythmias and cardiac conduction defects. A screen of more than 500 microRNAs (miRNAs) in a DM1 mouse model identified 54 miRNAs that were differentially expressed in heart. More than 80% exhibited downregulation toward the embryonic expression pattern and showed a DM1-specific response. A total of 20 of 22 miRNAs tested were also significantly downregulated in human DM1 heart tissue. We demonstrate that many of these miRNAs are direct MEF2 transcriptional targets, including miRNAs for which depletion is associated with arrhythmias or fibrosis. MEF2 protein is significantly reduced in both DM1 and mouse model heart samples, and exogenous MEF2C restores normal levels of MEF2 target miRNAs and mRNAs in a DM1 cardiac cell culture model. We conclude that loss of MEF2 in DM1 heart causes pathogenic features through aberrant expression of both miRNA and mRNA targets.

Rbfox2-Coordinated Alternative Splicing of Mef2d and Rock2 Controls Myoblast Fusion during Myogenesis

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition.

Rotavirus nonstructural protein NSP4 induces heterotypic antibody responses during natural infection in children.

Seroconversion of immunoglobulin A (IgA) and immunoglobulin G (IgG) (> or =4-fold rise) to rotavirus nonstructural protein 4 (NSP4) was determined, by use of enzyme-linked immunosorbent assay with fusion proteins glutathione S-transferase (GST)-NSP4 from strains SA11 (A), 116E (B), and RRV (C), in 40 children with acute rotavirus gastroenteritis and in 30 with the same disease due to other pathogens. The IgG seroconversion rates in the rotavirus group were 67.5%, 70%, and 60% when recombinant (r) NSP4A, -B, and -C, respectively, were used as antigen in the assay, and, for rotavirus-uninfected children, rates were 10%, 13%, and 7%. IgA seroconversion occurred in 57%, 70%, and 50%, respectively, of children with rotavirus gastroenteritis; in rotavirus-uninfected children, 1 child each seroconverted to the different rNSP4s. Among 9 children infected with strain NSP4A, 7, 6, and 5 children showed IgG seroconversion, and, among 18 infected with NSP4A, -B, and -C, 16, 17, and 15, respectively, showed IgG seroconversion. Between NSP4A-infected and NSP4B-infected children, IgA responses were similar to IgG responses. In conclusion, significant NSP4-specific antibody response occurs in natural rotavirus infection, and the antibody response appears to be broad and heterotypic in nature.

Pumilio1 Haploinsufficiency Leads to SCA1-like Neurodegeneration by Increasing Wild-Type Ataxin1 Levels

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

The RNA-binding protein Rbfox1 regulates splicing required for skeletal muscle structure and function

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

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.

The Splicing Factor FUBP1 Is Required for the Efficient Splicing of Oncogene MDM2 Pre-mRNA

Background: MDM2 is alternatively spliced into shorter isoforms under DNA damage and in several cancers through unknown mechanisms. Results: FUBP1 inactivation decreases splicing efficiency of an MDM2 minigene and causes exon skipping of endogenous MDM2 under normal conditions. Its overexpression attenuates damage-induced skipping of MDM2 exons. Conclusion: FUBP1 positively regulates efficient MDM2 splicing. Significance: This work uncovers an important mechanism regulating splicing efficiency of the oncogene MDM2. Alternative splicing of the oncogene MDM2 is a phenomenon that occurs in cells in response to genotoxic stress and is also a hallmark of several cancer types with important implications in carcinogenesis. However, the mechanisms regulating this splicing event remain unclear. Previously, we uncovered the importance of intron 11 in MDM2 that affects the splicing of a damage-responsive MDM2 minigene. Here, we have identified discrete cis regulatory elements within intron 11 and report the binding of FUBP1 (Far Upstream element-Binding Protein 1) to these elements and the role it plays in MDM2 splicing. Best known for its oncogenic role as a transcription factor in the context of c-MYC, FUBP1 was recently described as a splicing regulator with splicing repressive functions. In the case of MDM2, we describe FUBP1 as a positive splicing regulatory factor. We observed that blocking the function of FUBP1 in in vitro splicing reactions caused a decrease in splicing efficiency of the introns of the MDM2 minigene. Moreover, knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions. These results indicate that FUBP1 is also a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1.