Abstract
Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically ...disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.
Graphical Abstract
Graphical Abstract
SANS acts as a factor required for the release of tri-snRNPs from Cajal bodies following by recruitment of the tri-snRNP complex to nuclear speckles for spliceosome assembly. SANS may also participate in the snRNP recycling back to the Cajal bodies.
Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being ...understood, especially in the eye and retina. The
gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although
/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to
-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein
/harmonin with the stabilized acetylated
-catenin, especially in nuclei. In HEK293T cells, overexpression of
/harmonin significantly reduced cWnt signaling, but a
-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an
patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing
/harmonin as a suppressor of the cWnt/
-catenin pathway.
This is the first scholarly study of soldiers and guerrillas demobilized after the civil war in Mozambique (1979-1992). The book examines former soldiers' - from both sides - return to civilian life, ...and how their identity as veterans plays out in the political sphere.
Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, ...with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).
In a previous study, glyphosate-susceptible and -resistant giant ragweed biotypes grown in sterile field soil survived a higher rate of glyphosate than those grown in unsterile field soil, and the ...roots of the susceptible biotype were colonized by a larger number of soil microorganisms than those of the resistant biotype when treated with 1.6 kg ae ha−1 glyphosate. Thus, we concluded that soil-borne microbes play a role in glyphosate activity and now hypothesize that the ability of the resistant biotype to tolerate glyphosate may involve microbial interactions in the rhizosphere. The objective of this study was to evaluate differences in the rhizosphere microbial communities of glyphosate-susceptible and -resistant giant ragweed biotypes 3 d after a glyphosate treatment. Giant ragweed biotypes were grown in the greenhouse in unsterile field soil and glyphosate was applied at either 0 or 1.6 kg ha−1. Rhizosphere soil was sampled 3 d after the glyphosate treatment, and DNA was extracted, purified, and sequenced with the use of Illumina Genome Analyzer next-generation sequencing. The taxonomic distribution of the microbial community, diversity, genera abundance, and community structure within the rhizosphere of the two giant ragweed biotypes in response to a glyphosate application was evaluated by metagenomics analysis. Bacteria comprised approximately 96% of the total microbial community in both biotypes, and differences in the distribution of some microbes at the phyla level were observed. Select soil-borne plant pathogens (Verticillium and Xanthomonas) and plant-growth–promoting rhizobacteria (Burkholderia) present in the rhizosphere were influenced by either biotype or glyphosate application. We did not, however, observe large differences in the diversity or structure of soil microbial communities among our treatments. The results of this study indicate that challenging giant ragweed biotypes with glyphosate causes perturbations in rhizosphere microbial communities and that the perturbations differ between the susceptible and resistant biotypes. However, biological relevance of the rhizosphere microbial community data that we obtained by next-generation sequencing remains unclear. Nomenclature: Glyphosate; giant ragweed, Ambrosia trifida L.
Usher syndrome (USH) is the most common form of monogenic deaf‐blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular ...constituent of USH, so far. By introducing a human mutation into the harmonin‐encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas‐mediated gene repair or gene therapy in vitro. AAV‐based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.
Synopsis
The full phenotypic spectrum of Usher Syndrome is reflected in a pig model carrying a patient‐specific mutation after partial humanization of the porcine USH1C gene. Retinal function in USH1C pigs was improved when harmonin expression was reconstituted by AAV‐mediated gene therapy.
Partial humanization of the porcine USH1C gene was facilitated by the high degree of sequence conservation in the N‐terminal region of harmonin.
Vision loss within the first year of life in USH1C pigs was consistently indicated by behavior tests, clinical examination and morphological analysis.
Early onset of vision loss was correlated to disrupted photoreceptor cell architecture.
Ciliopathy mechanisms and their therapeutic correction were investigated in primary cells of USH1C individuals.
Local application and therapeutic efficacy of AAV‐mediated treatments was examined in USH1C pigs in vivo.
The full phenotypic spectrum of Usher Syndrome is reflected in a pig model carrying a patient‐specific mutation after partial humanization of the porcine USH1C gene. Retinal function in USH1C pigs was improved when harmonin expression was reconstituted by AAV‐mediated gene therapy.
This edited volume assembles contributions of leading scholars in the fields of statistical methods and applications in the social sciences. Multivariate scaling methods for categorical data, in ...particular correspondence analysis, are used to extract the most important dimensions from complex data tables and to visualize relationships in the data. The volume treats recent statistical developments, methodological considerations, and empirical applications. A special emphasis is placed on multiple aspects of space and their sociological significance: the reconstruction of "social spaces" with statistical methods, illustrations of spatial relations involving proximity, distance and inequality, and concrete interactions in urban neighbourhoods. The edited volume is meant to honour the lifetime achievements of Prof. Jörg Blasius (Chair of Sociology/ Empirical Research Methods, Bonn).
In previous research conducted on nonweed species, the efficacy of glyphosate was shown to be greater in unsterile soils compared to sterile soils and soil microorganisms were found to play an ...important role in glyphosate efficacy. Conducting greenhouse studies in microbe-free soil may therefore produce unreliable data, leading to erroneous conclusions. The objective of this study was to determine the effect of soil microorganisms on the response of glyphosate-resistant and -susceptible biotypes of three problematic weeds of the midwestern United States: giant ragweed, horseweed, and common lambsquarters. A greenhouse dose–response study was conducted on each of the three weed species grown in sterile and unsterile field soil, and the dry weight response of roots and shoots was measured. The three weed species responded differently to glyphosate when grown in the sterile and unsterile soil; that is, in the presence and absence of soil microbes. Soil microbes influenced the response of the susceptible and resistant giant ragweed biotypes and the susceptible common lambsquarters, but not the tolerant common lambsquarters or either horseweed biotype. The different responses of the three species to glyphosate in the presence and absence of soil microbes demonstrates that rhizosphere interactions are fundamental to the mode of action of glyphosate. These findings suggest that the range of tolerance to glyphosate observed in weeds and the evolution of resistance in weed biotypes may also be influenced by rhizosphere interactions. The soil media used in dose–response screenings to identify susceptible and resistant weed biotypes is very important. Unsterile field soil should be incorporated into growth media when conducting dose–response screenings to avoid false-positive results. In addition, researchers performing glyphosate dose–response assays should be aware of these findings. Nomenclature: Glyphosate; common lambsquarters, Chenopodium album L. CHEAL; giant ragweed, Ambrosia trifida L. AMBTR; horseweed, Conyza canadensis (L.) Cronq. ERICA.
Root colonization by soil microorganisms has been shown to increase the activity of glyphosate in resistant and susceptible biotypes of giant ragweed and a susceptible common lambsquarters biotype, ...but not in horseweed biotypes. The objective of this study was to investigate the colonization of roots in glyphosate-resistant and -susceptible giant ragweed and horseweed biotypes, and glyphosate-tolerant and -susceptible biotypes of common lambsquarters after a sublethal glyphosate application. The three weed species were grown separately in sterile and unsterile field soil and treated with glyphosate at two sublethal rates. Soil microbes were isolated from the roots onto sterile media 3 d after the glyphosate treatment. The susceptible biotypes of giant ragweed and horseweed grown in unsterile soil were colonized by more soil microbes at the higher rate of glyphosate, compared to the resistant biotype grown in unsterile soil. Oomycetes were isolated separately on a selective media and they were also more prevalent in the roots of the susceptible biotypes of each weed species grown in the unsterile soil when glyphosate was applied at the highest rate. Therefore, the ability of these three weed species to tolerate a glyphosate application may involve differences in the susceptibility to soil microbial colonization, especially oomycetes. Nomenclature: Glyphosate; common lambsquarters, Chenopodium album L. CHEAL; giant ragweed, Ambrosia trifida L. AMBTR; horseweed, Conyza canadensis (L.) Cronq. ERICA.
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