Cross-Regulation between an Alternative Splicing Activator and a Transcription Repressor Controls Neurogenesis

Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Molecular cell (Impact Factor: 14.02). 09/2011; 43(5):843-50. DOI: 10.1016/j.molcel.2011.08.014
Source: PubMed

ABSTRACT Neurogenesis requires the concerted action of numerous genes that are regulated at multiple levels. However, how different layers of gene regulation are coordinated to promote neurogenesis is not well understood. We show that the neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) negatively regulates REST (NRSF), a transcriptional repressor of genes required for neurogenesis. nSR100 directly promotes alternative splicing of REST transcripts to produce a REST isoform (REST4) with greatly reduced repressive activity, thereby activating expression of REST targets in neural cells. Conversely, REST directly represses nSR100 in nonneural cells to prevent the activation of neural-specific splicing events. Consistent with a critical role for nSR100 in the inhibition of REST activity, blocking nSR100 expression in the developing mouse brain impairs neurogenesis. Our results thus reveal a fundamental role for direct regulatory interactions between a splicing activator and transcription repressor in the control of the multilayered regulatory programs required for neurogenesis.

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    • "Although these observations highlight the importance of combinatorial regulation in splicing, nothing is known about how multiple splicing factors act together to shape splicing networks at single neuron resolution. Characterization of individual target transcripts within splicing networks have identified important splicing events that contribute to specific neuronal phenotypes (Aoto et al., 2013; Raj et al., 2011; Yano et al., 2010). However, the difficulty of systematic network interrogation in vertebrate models has made it challenging to perform functional analyses of substantial numbers of targets within splicing regulatory networks in vivo. "
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    ABSTRACT: Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single-cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA-binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems.
    Molecular Cell 06/2014; DOI:10.1016/j.molcel.2014.05.004 · 14.02 Impact Factor
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    • "Interestingly, in several cancer types REST transcripts undergo frequent and complex AS,105 and in lung cancer this is regulated by the neuronal splice factor SRRM4.106 In neuronal cells, REST and SRRM4 form a regulatory feedback loop107 and it will be interesting to explore the interplay between these proteins in PCa. Since NEPC is extremely aggressive with survival of <1 year,101 it will be important to explore the functional role of AS in NEPC, the mechanisms of its regulation and the role of REST transcriptional complex in NEPC biology (Figure 3). "
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    ABSTRACT: Alternative splicing (AS) is a crucial step in gene expression. It is subject to intricate regulation, and its deregulation in cancer can lead to a wide array of neoplastic phenotypes. A large body of evidence implicates splice isoforms in most if not all hallmarks of cancer, including growth, apoptosis, invasion and metastasis, angiogenesis, and metabolism. AS has important clinical implications since it can be manipulated therapeutically to treat cancer and represents a mechanism of resistance to therapy. In prostate cancer (PCa) AS also plays a prominent role and this review will summarize the current knowledge of alternatively spliced genes with important functional consequences. We will highlight accumulating evidence on AS of the components of the two critical pathways in PCa: androgen receptor (AR) and phosphoinositide 3-kinase (PI3K). These observations together with data on dysregulation of splice factors in PCa suggest that AR and PI3K pathways may be interconnected with previously unappreciated splicing regulatory networks. In addition, we will discuss several lines of evidence implicating splicing regulation in the development of the castration resistance.
    Asian Journal of Andrology 05/2014; 16(4). DOI:10.4103/1008-682X.127825 · 2.60 Impact Factor
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    • "CLIP assay was performed as described previously [48,49]. Briefly, HeLa cells were UV cross-linked at 254 nm (UV-B) with 600 J/cm2 in a UV Stratalinker 1800 crosslinker (Stratagene, La Jolla, CA); lysed in wash buffer containing 1× PBS, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, and 0.5% NP-40; supplemented with 0.015 U/μl RNasein Plus (N261, Promega) and RQ RNase-Free DNase (M610A, Promega); and immunoprecipitated for 2 h at 4°C with 5 μg polyclonal anti-TDP-43, anti-FUS/TLS, and rabbit IgG control (AB-105-C, R&D Systems, Minneapolis, MN) antibodies bound in advance to Dynabeads Protein G (100.04 D, Invitrogen). "
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    ABSTRACT: A long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1_2 (NEAT1_2), constitutes nuclear bodies known as "paraspeckles". Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons. In situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1_2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1_2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1_2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1_2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1_2 lncRNA. The observation indicating specific assembly of NEAT1_2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation. NEAT1_2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS.
    Molecular Brain 07/2013; 6(1):31. DOI:10.1186/1756-6606-6-31 · 4.90 Impact Factor
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