We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.

Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABA A receptor ␥2 (GABA A R␥2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABA A R␥2 alternative splicing, we systematically screened minigenes derived from the GABA A R␥2 and human ␤-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABA A R␥2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Novadependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABA A R␥2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.The regulation of eukaryotic gene expression involves an ordered sequence of events within various subcellular compartments. RNA binding proteins (RBPs) play a critical role in this process, acting at multiple levels to regulate and diversify gene expression, and such functions are likely to be of particular importance to neurons. RBPs regulate processes such as alternative splicing, which is an important mechanism for generating complexity in specialized cells, particularly in neurons. For example, all of the major neurotransmitter receptors contain subunits that are alternatively spliced, and these variants can alter receptor localization, ligand binding, signal transduction, and electrophysiological properties (13,20,44,51). Moreover, RBPs and aberrant splicing of mRNAs encoding proteins critical for proper functioning of neurons have been associated with a number of neurological diseases (reviewed in reference 19). To understand the role RBPs play in regulating gene expression in neurons, it would be necessary to identify their complement of RNA targets and to understand how they are recognized amidst the complexity of the cellular environment.Regulation of alternative splicing occurs through recognition of cis-acting sequences in pre-mRNA by trans-acting factors. cis-acting repressor elements that mediate the exclusion of the neuron-specific exons in nonneuronal tissues have been identified through studies of neuron-specific splicing in transcripts such as c-src, the GABA A rece...

The Nova family of neuron-specific RNA-binding proteins were originally identified as targets in an autoimmune neurologic disease characterized by failure of motor inhibition. Nova-1 regulates alternative splicing of premRNAs encoding the inhibitory neurotransmitter receptor subunits GABA A Rc2 and GlyRa2 by directly binding intronic elements, resulting in enhancement of exon inclusion. Here we identify exon E4 in the Nova-1 pre-mRNA itself, encoding a phosphorylated protein domain, as an additional target of Nova-dependent splicing regulation in the mouse spinal cord. Nova binding to E4 is necessary and sufficient for Nova-dependent exon exclusion. E4 harbors five repeats of the known Nova-binding tetranucleotide YCAY and mutation of these elements destroys Nova-dependent regulation. Furthermore, swapping of the sites from Nova-1 and GABA A Rc2 indicates that the ability of Nova to enhance or repress alternative exon inclusion is dependent on the position of the Nova-binding element within the pre-mRNA. These studies demonstrate that in addition to its previously described role as a splicing activator, Nova autoregulates its own expression by acting as a splicing repressor.

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we know about the mechanisms regulating neuron-specific splicing and our understanding of neural function and disease.

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.

Anti-NeuN (Neuronal Nuclei) is a monoclonal antibody used extensively to specifically detect post-mitotic neurons. Anti-NeuN reactivity is predominantly nuclear; by western it detects multiple bands ranging in molecular weight from 45 kDa to >75 kDa. Expression screening putatively identified R3hdm2 as NeuN; however immunoprecipitation and mass spectrometry of the two major NeuN species at 45–50 kDa identified both as the RNA binding protein Rbfox3 (a member of the Fox family of alternative splicing factors), confirming and extending the identification of the 45 kDa band as Rbfox3 by Kim et al. Mapping of the anti-NeuN reactive epitopes in both R3hdm2 and Rbfox3 reveals a common proline- and glutamine-rich domain that lies at the N-terminus of the Rbfox3 protein. Our data suggests that alternative splicing of the Rbfox3 pre-mRNA itself leads to the production of four protein isoforms that migrate in the 45–50 kDa range, and that one of these splicing choices regulates Rbfox3/NeuN sub-cellular steady-state distribution, through the addition or removal of a short C-terminal extension containing the second half of a bipartite hydrophobic proline-tyrosine nuclear localization signal. Rbfox3 regulates alternative splicing of the Rbfox2 pre-mRNA, producing a message encoding a dominant negative form of the Rbfox2 protein. We show here that nuclear Rbfox3 isoforms can also enhance the inclusion of cryptic exons in the Rbfox2 mRNA, resulting in nonsense-mediated decay of the message, thereby contributing to the negative regulation of Rbfox2 by Rbfox3 through a novel mechanism.

The recent emergence of a pathogenic new non-01 serotype (0139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate .that 0139 strains have clear differences in the surface polysaccharides when compared with 01 strains: the lipopolysaccharide can be described as semirough. Southern hybridization with the 01 rjb region demonstrates that 0139 strains no longer contain any of the rjb genes required for the synthesis of the 01 0-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, 0139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the 01 rjb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcISI. Transposon insertion mutants defective in 0139 0-antigen (and capsule) biosynthesis map to the same fragment as VcISI. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyltransferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of 01 strains and the 0139 serotype strains may have evolved.