Control of the heat stress‐induced alternative splicing of a subset of genes by hnRNP K

Pre‐mRNA splicing is widely repressed upon heat shock in eukaryotic cells. However, it has been shown that HSP105 pre‐mRNA is alternatively spliced in response to heat stress. Using RNAi screening in HeLa cells, we found that RNA‐binding proteins hnRNP K and PSF/SFPQ are necessary for the exon 12 exclusion of HSP105 during heat stress. Moreover, exon array analyses showed that a group of genes is alternatively spliced during heat stress in an hnRNP K‐dependent manner, whereas hnRNP K is not necessary for the stress‐induced alternative splicing of the remaining genes. Among the latter group, we found that SRp38/SRSF10 and SC35/SRSF2 are necessary for the inclusion of exon 13 of TNRC6A during heat stress. Thus, our study clearly showed that several RNA‐binding proteins are involved in the splicing regulation in response to heat stress in mammalian cells.     

FilaminB is required for the directed localization of cell–cell adhesion molecules in embryonic epithelial development

Filamin proteins cross‐link F‐actin and form a scaffold for numerous signal transduction systems. In this study, we show that filaminB is apically enriched in avian embryonic epithelium, and colocalizes with cell adhesion molecules and circumferential F‐actin. FilaminB knockdown in the neural tube and somites decreases the accumulation of N‐cadherin and ZO‐1 protein at cell junctions, and promotes disruption of these tissues and the presence of neuronal aggregates within the lumen of the neural tube. This phenotype resembles that of human congenital condition, periventricular heterotopia (PH). FilaminB knockdown in MDCK cells suggests that filaminB is required for the apical accumulation of adhesion molecules in the junctional complex and subsequent epithelium formation. We further suggest that the reduction of structural integrity of the neural epithelium caused by the loss of Filamin function may also result in formation of the neuronal nodules found in PH patients. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Lectins as a Tool for Detecting Neural Stem/Progenitor Cells in the Adult Mouse Brain

Glycoconjugates are biopolymers that are broadly distributed in the central nervous system, including the cell surface of neural stem cells or neural precursor cells (NSCs/NPCs). Glycoconjugates can be recognized by carbohydrate‐binding proteins, lectins. Two lectins, Phaseolus vulgaris lectin agglutinin E‐form (PHA‐E4) and wheat germ agglutinin (WGA) have been reported to be useful in isolating NSCs/NPCs by fluorescence‐activated cell sorting (FACS) or immunopanning methods. In this study, we analyzed the lectin‐binding properties of NSCs/NPCs in two neurogenic regions of the adult mouse brain to determine whether PHA‐E4 and WGA exhibit specific binding patterns on sections and whether there are other lectins presenting the binding pattern similar to those of PHA‐E4 and WGA in lectin histochemistry. Among nine types of lectins, peanut agglutinin was localized to the white matter and four lectins bound to cells within the subventricular zone (SVZ) of the lateral ventricle. Lectin histochemistry combined with immunohistochemistry demonstrated that one lectin, Ricinus communis agglutinin, specifically detected type A neuronal precursors and that the remaining three lectins, Agaricus bisporus agglutinin (ABA), PHA‐E4, and WGA, recognized type B NSCs and type C transient amplifying cells in the SVZ. These three lectins also recognized type 1 quiescent neural progenitors and type 2a amplifying neural progenitors in the subgranular layer of the dentate gyrus. Lectin histochemistry of the neurosphere culture also yielded similar results. These observations suggest that, in addition to PHA‐E4 and WGA, ABA lectin may also be applicable in FACS or immunopanning for the isolation of NSCs/NPCs. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Loss of dystrophin and the microtubule‐binding protein ELP‐1 causes progressive paralysis and death of adult C. elegans

EMAP‐like proteins (ELPs) are conserved microtubule‐binding proteins that function during cell division and in the behavior of post‐mitotic cells. In Caenorhabditis elegans, ELP‐1 is broadly expressed in many cells and tissues including the touch receptor neurons and body wall muscle. Within muscle, ELP‐1 is associated with a microtubule network that is closely opposed to the integrin‐based adhesion sites called dense bodies. To examine ELP‐1 function, we utilized an elp‐1 RNA interference assay and screened for synthetic interactions with mutated adhesion site proteins. We reveal a synthetic lethal relationship between ELP‐1 and the dystrophin‐like protein, DYS‐1. Reduction of ELP‐1 in a dystrophin [dys‐1(cx18)] mutant results in adult animals with motility defects, splayed and hypercontracted muscle with altered cholinergic signaling. Worms fill with vesicles, become flaccid, and die. We conclude that ELP‐1 is a genetic modifier of a C. elegans model of muscular dystrophy. Developmental Dynamics, 2009. © 2009 Wiley‐Liss, Inc.     

Matrin3 binds directly to intronic pyrimidine‐rich sequences and controls alternative splicing

Matrin3 is an RNA‐binding protein that is localized in the nuclear matrix. Although various roles in RNA metabolism have been reported for Matrin3, in vivo target RNAs to which Matrin3 binds directly have not been investigated comprehensively so far. Here, we show that Matrin3 binds predominantly to intronic regions of pre‐mRNAs. Photoactivatable Ribonucleoside‐Enhanced Cross‐linking and Immunoprecipitation (PAR‐CLIP) analysis using human neuronal cells showed that Matrin3 recognized pyrimidine‐rich sequences as binding motifs, including the polypyrimidine tract, a splicing regulatory element. Splicing‐sensitive microarray analysis showed that depletion of Matrin3 preferentially increased the inclusion of cassette exons that were adjacent to introns that contained Matrin3‐binding sites. We further found that although most of the genes targeted by polypyrimidine tract binding protein 1 (PTBP1) were also bound by Matrin3, Matrin3 could control alternative splicing in a PTBP1‐independent manner, at least in part. These findings suggest that Matrin3 is a splicing regulator that targets intronic pyrimidine‐rich sequences.     

Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis

FUS, EWS, and TAF15 belong to the TET family of structurally similar DNA/RNA‐binding proteins. Mutations in the FUS gene have recently been discovered as a cause of familial amyotrophic lateral sclerosis (FALS). Given the structural and functional similarities between the three genes, we screened TAF15 and EWS in 263 and 94 index FALS cases, respectively. No coding variants were found in EWS, while we identified six novel changes in TAF15. Of these, two 24 bp deletions and a R388H missense variant were also found in healthy controls. A D386N substitution was shown not to segregate with the disease in the affected pedigree. A single A31T and two R395Q changes were identified in FALS cases but not in over 1,100 controls. Interestingly, one of the R395Q FALS cases also harbors a TARDBP mutation (G384R). Altogether, these results suggest that additional studies are needed to determine whether mutations in the TAF15 gene represent a cause of FALS. © 2011 Wiley‐Liss, Inc.     

miR‐29a inhibition normalizes HuR over‐expression and aberrant AU‐rich mRNA stability in invasive cancer

The activities of RNA‐binding proteins are perturbed in several pathological conditions, including cancer. These proteins include tristetraprolin (TTP, ZFP36) and HuR (ELAVL1), which respectively promote the decay or stability of adenylate‐uridylate‐rich (AU‐rich) mRNAs. Here, we demonstrated that increased stabilization and subsequent over‐expression of HuR mRNA were coupled to TTP deficiency. These findings were observed in breast cancer cell lines with an invasive phenotype and were further confirmed in ZFP36‐knockout mouse fibroblasts. We show that TTP–HuR imbalance correlated with increased expression of AU‐rich element (ARE) mRNAs that code for cancer invasion genes. The microRNA miR‐29a was abundant in invasive breast cancer cells when compared to non‐tumourigenic cell types. When normal breast cells were treated with miR‐29a, HuR mRNA and protein expression were up‐regulated. MiR‐29a recognized a seed target in the TTP 3′ UTR and a cell‐permeable miR‐29a inhibitor increased TTP activity towards HuR 3′ UTR. This led to HuR mRNA destabilization and restoration of the aberrant TTP–HuR axis. Subsequently, the cancer invasion factors uPA, MMP‐1 and MMP‐13, and cell invasiveness, were decreased. The TTP:HuR mRNA ratios were also perturbed in samples from invasive breast cancer patients when compared with normal tissues, and were associated with invasion gene expression. This study demonstrates that an aberrant ARE‐mediated pathway in invasive cancer can be normalized by targeting the aberrant and functionally coupled TTP–HuR axis, indicating a potential therapeutic approach. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Same gene, surprising difference: adult neuronal ceroid lipofuscinosis linked to CLN6, mutated in variant late-infantile form

References 1.Jalanko A, Braulke T. Neuronal ceroid lipofuscinoses. Biochim Biophys Acta 2009 : 1793 : 697–709. 2.Abecasis GR, Cherny SS, Cookson WO, Cardon LR. Merlin‐rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 2002 : 30 : 97–101. 3.Getty AL, Pearce DA. Interactions of the proteins of neuronal ceroid lipofuscinosis: clues to function. Cell Mol Life Sci 2011 : 68 : 453–447. Kufs disease, the major adult form of neuronal ceroid lipofuscinosis, caused by mutations in CLN6 Arsov et al. (2011) American Journal of Human Genetics 88(5): 566–573.     

Labelling cell structures and tracking cell lineage in zebrafish using SNAP‐tag

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP‐tag technology and O⁶‐benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long‐term imaging and allows, unlike most of the autofluorescent proteins, the use of acid fixatives such as trichloroacetic acid. Furthermore, the use of O⁶‐benzylguanine derivatives bearing caged fluorescein allows cell lineage tracing through photo‐deprotection of the fluorophore and its detection either through fluorescence microscopy or through immunohistochemistry after fixation using anti‐fluorescein antibodies. Developmental Dynamics 240:820–827, 2011. © 2011 Wiley‐Liss, Inc.     

Different aggregation states of a nuclear localization signal‐tagged 25‐kDa C‐terminal fragment of TAR RNA/DNA‐binding protein 43 kDa

The mechanism and cause of motor neuronal cell death in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder, are unknown; gain of function of oligomers and aggregation of misfolded proteins, including carboxyl‐terminal fragments (CTFs) of TAR RNA/DNA‐binding protein 43 kDa (TDP‐43), have been proposed as important causative factors in the onset of ALS. We recently reported that a nuclear localization signal (NLS)‐tagged 25‐kDa CTF of TDP‐43 (TDP25) could decrease the cell‐death proportion compared with that promoted by TDP25. Here, we show oligomeric states of NLS‐TDP25 and its detailed localization property using super‐resolution fluorescence microscopy, FRET, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy analysis. NLS‐TDP25 efficiently formed a nucleolar cap structure via RNA binding in the presence of actinomycin D, but TDP25 did not. Although cytoplasmic inclusion bodies including TDP25 had a disordered and immobile structure, NLS‐TDP25 in the nucleolus was ordered and dynamic. In the diffuse state, TDP25 formed fewer oligomers and interacted with the molecular chaperone, HSP70; however, NLS‐TDP25 formed oligomers. These results suggested that NLS‐tagged TDP25 can change its structure to use ordered oligomeric but nontoxic state. Moreover, the structure of ordered oligomers as well as nuclear sequestration may be important in mediating cytotoxicity in ALS pathology.     

The ETFDH c.158A>G Variation Disrupts the Balanced Interplay of ESE‐ and ESS‐Binding Proteins thereby Causing Missplicing and Multiple Acyl‐CoA Dehydrogenation Deficiency

Multiple acyl‐CoA dehydrogenation deficiency is a disorder of fatty acid and amino acid oxidation caused by defects of electron transfer flavoprotein (ETF) or its dehydrogenase (ETFDH). A clear relationship between genotype and phenotype makes genotyping of patients important not only diagnostically but also for prognosis and for assessment of treatment. In the present study, we show that a predicted benign ETFDH missense variation (c.158A>G/p.Lys53Arg) in exon 2 causes exon skipping and degradation of ETFDH protein in patient samples. Using splicing reporter minigenes and RNA pull‐down of nuclear proteins, we show that the c.158A>G variation increases the strength of a preexisting exonic splicing silencer (ESS) motif UAGGGA. This ESS motif binds splice inhibitory hnRNP A1, hnRNP A2/B1, and hnRNP H proteins. Binding of these inhibitory proteins prevents binding of the positive splicing regulatory SRSF1 and SRSF5 proteins to nearby and overlapping exonic splicing enhancer elements and this causes exon skipping. We further suggest that binding of hnRNP proteins to UAGGGA is increased by triggering synergistic hnRNP H binding to GGG triplets located upstream and downsteam of the UAGGGA motif. A number of disease‐causing exonic elements that induce exon skipping in other genes have a similar architecture as the one in ETFDH exon 2.     

ABLIM1 splicing is abnormal in skeletal muscle of patients with DM1 and regulated by MBNL,CELF and PTBP1

Myotonic dystrophy type 1 (DM1) is an RNA‐mediated disorder characterized by muscle weakness, cardiac defects and multiple symptoms and is caused by expanded CTG repeats within the 3′ untranslated region of the DMPK gene. In this study, we found abnormal splicing of actin‐binding LIM protein 1 (ABLIM1) in skeletal muscles of patients with DM1 and a DM1 mouse model (HSA^LR). An exon 11 inclusion isoform is expressed in skeletal muscle and heart of non‐DM1 individuals, but not in skeletal muscle of patients with DM1 or other adult human tissues. Moreover, we determined that ABLIM1 splicing is regulated by several splice factors, including MBNL family proteins, CELF1, 2 and 6, and PTBP1, using a cellular splicing assay. MBNL proteins promoted the inclusion of ABLIM1 exon 11, but other proteins and expanded CUG repeats repressed exon 11 of ABLIM1. This result is consistent with the hypothesis that MBNL proteins are trapped by expanded CUG repeats and inactivated in DM1 and that CELF1 is activated in DM1. However, activation of PTBP1 has not been reported in DM1. Our results suggest that the exon 11 inclusion isoform of ABLIM1 may have a muscle‐specific function, and its abnormal splicing could be related to muscle symptoms of DM1.     

Autoantibody Specificities and Type I Interferon Pathway Activation in Idiopathic Inflammatory Myopathies

Myositis is a heterogeneous group of autoimmune diseases, with different pathogenic mechanisms contributing to the different subsets of disease. The aim of this study was to test whether the autoantibody profile in patients with myositis is associated with a type I interferon (IFN) signature, as in patients with systemic lupus erythematous (SLE). Patients with myositis were prospectively enrolled in the study and compared to healthy controls and to patients with SLE. Autoantibody status was analysed using an immunoassay system and immunoprecipitation. Type I IFN activity in whole blood was determined using direct gene expression analysis. Serum IFN‐inducing activity was tested using peripheral blood cells from healthy donors. Blocking experiments were performed by neutralizing anti‐IFNAR or anti‐IFN‐α antibodies. Patients were categorized into IFN high and IFN low based on an IFN score. Patients with autoantibodies against RNA‐binding proteins had a higher IFN score compared to patients without these antibodies, and the IFN score was related to autoantibody multispecificity. Patients with dermatomyositis (DM) and inclusion body myositis (IBM) had a higher IFN score compared to the other subgroups. Serum type I IFN bioactivity was blocked by neutralizing anti‐IFNAR or anti‐IFN‐α antibodies. To conclude, a high IFN score was not only associated with DM, as previously reported, and IBM, but also with autoantibody monospecificity against several RNA‐binding proteins and with autoantibody multispecificity. These studies identify IFN‐α in sera as a trigger for activation of the type I IFN pathway in peripheral blood and support IFN‐α as a possible target for therapy in these patients.