Longman D et al. (2000) EMBO J "Functional characterization of SR and SR-related genes in Caenorhabditis elegans."

Longman D, Johnstone IL, Caceres JF

[EMBO J, 2000]

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.

T Kawano et al. (1999) International C. elegans Meeting "Analysis of SR protein genes in C. elegans"

H Sakamoto, T Kawano, M Fujita

[International C. elegans Meeting, 1999]

SR proteins contain one or two copies of RNA recognition motif (RRM) as RNA-binding domain and an arginine/serine-rich (RS) domain. Previous in vitro studies have revealed that mammalian SR proteins have essential roles in constitutive splicing of pre-mRNAs. In addition, some SR proteins have been shown to play an important role in splice site selection. To investigate the in vivo function of SR proteins, we first searched the C. elegans genome database and found five genes potentially encoding SR protein (ceSRp20, ceSC35, T28, W12.2 and W12.3). We next examined, using transgenic worms, the expression of these SR proteins fused with green fluorescent protein (GFP) under the control of their native promoters. All of these SR-GFP fusion proteins were localized in the nuclei of almost all somatic cells, consistent with that SR proteins are constitutive splicing factors as in mammalian cells. However, simultaneous suppression of two or more SR protein-coding genes by dsRNA-mediated interference resulted in drastic phenotypes, such as embryonic lethality, abnormal gonadal formation and decrease of fertility. These results raise the possibility that SR proteins also play important roles in the development and the germ cell differentiation in C. elegans . Disruption of genes encodong SR proteins and a search for target genes whose mRNA expression are affected in such mutants are currently in progress.

Lefebvre C et al. (2016) J Cell Sci "The ESCRT-II proteins are involved in shaping the sarcoplasmic reticulum."

Lefebvre C, Fourrage C, Legouis R, Matic I, Michelet X, Culetto E, Largeau C, Maniere X

[J Cell Sci, 2016]

The sarcoplasmic reticulum (SR) is a network of tubules and cisternae localized in close association with the contractile apparatus and regulating Ca(2+) dynamics within striated muscle cell. The SR maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here we report that ESCRT-II proteins play a novel function in the maintenance of SR integrity in C. elegans. ESCRT-II proteins colocalize with the SR marker ryanodine receptor UNC-68. The localization at the SR of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the SR network, associated with a calcium dynamics alteration. Our data provide evidence that ESCRT-II proteins are involved in SR shaping.

Tazir Y et al. (2009) Mol Biochem Parasitol "Molecular and functional characterisation of the heat shock protein 10 of Strongyloides ratti."

Younis AE, Soblik H, Beckmann S, Steisslinger V, Tazir Y, Erttmann KD, Steen H, Brattig NW, Grevelding CG

[Mol Biochem Parasitol, 2009]

Strongyloides stercoralis and S. ratti are intestinal parasitic nematodes infecting rats and humans, respectively. Both present extraordinary life cycles comprising a free-living generation in addition to parasitic stages. In search of molecules possibly involved in parasite-host interaction, we performed mass spectrometry to identify excretory/secretory products of S. ratti. Amongst others we detected homologs of the heat shock proteins HSP10 and HSP60 (Sr-HSP10 and Sr-HSP60). HSPs are well known as chaperones involved in stress responses of cells, but recent studies suggest additional roles of small HSPs for parasite biology including immune modulation. To characterise Sr-HSP10, we cloned its full-length cDNA, analysed the genomic organisation, tested its presumptive role as an interaction partner of Sr-HSP60, studied its transcription in the parasite, and expressed the protein to test its immune responses. The cDNA contains an open reading frame of 330bp encoding a polypeptide of 110 amino acids with an approximate molecular weight of 10kDa. The Sr-HSP10 protein is highly homologous to that of the human pathogen S. stercoralis with only eight amino acid substitutions. Analysis of the genomic organisation of the Sr-HSP10 locus revealed that the gene is linked head-to-head to the gene encoding Sr-HSP60, and both share a bidirectional promoter. RT-PCR experiments indicated potential independent expression of the Sr-HSPs genes. In situ hybridisation results demonstrate Sr-HSP10 transcription in the gut area. Mammalian and yeast two-hybrid assays show dimerisation of Sr-HSP10, but no binding to recombinant Sr-HSP60. Immunisation experiments finally revealed a strong immunogenicity of Sr-HSP10 and provided evidence for a role in regulating the host-parasite interaction.

Ditgen D et al. (2018) Mol Biochem Parasitol "Comparative characterization of two galectins excreted-secreted from intestine-dwelling parasitic and free-living females of the soli-transmitted nematode Strongyloides."

Witt S, Paclik D, Brattig NW, Seeberger P, Reinhardt A, Jolodar A, Garcia-Hernandez M, Hansmann J, Anandarajah EM, Ditgen D, Lorenz E, Soblik H, Elshazly Younis A, Liebau E

[Mol Biochem Parasitol, 2018]

Helminths are complex pathogens that ensure their long-term survival by influencing the immune responses of their host. Excretory/secretory products (ESP) can exert immunoregulatory effects which foster parasite survival. Galectins represent a widespread group of -galactoside-binding proteins which are involved in a multitude of biological processes operative in parasite-host interaction. We had earlier identified seven galectins in Strongyloides ratti, four of them detected in the ESP of distinct developmental stages of the parasite. In the present report, we focused on the characterization of two of them, Sr-galectin-1 (Sr-Gal-1) and Sr-galectin-3 (Sr-Gal-3). While Sr-Gal-3 expression was strongest in parasitic females, Sr-Gal-1 was predominantly expressed in free-living females. Both proteins were cloned and recombinantly expressed in an E. coli expression system. Their glycan-binding activity was verified by haemagglutination and glycan array analysis. Furthermore, primary immunological activities of the Sr-galectins were initially investigated by the application of an in vitro mucosal 3D-culture model, comprising of mucosa-associated epithelial and dendritic cells. The Sr-galectins stimulated preferentially the release of the type 2 cytokines thymic stromal lymphopoietin and IL-22, a first indication for immunoregulatory activity. In addition, the Sr-galectins dose-dependently fostered cell migration. Our results confirm the importance of these carbohydrate-binding proteins in host-parasite-interaction by indicating possible interaction with the host mucosa-associated cells.

Kawano T et al. (2000) Mech Dev "Unique and redundant functions of SR proteins, a conserved family of splicing factors, in Caenorhabditis elegans development."

Fujita M, Sakamoto H, Kawano T

[Mech Dev, 2000]

Serine/arginine-rich proteins (SR proteins) constitute a family of RNA-binding proteins conserved throughout metazoans. The SR proteins are essential for constitutive pre-mRNA splicing and also affect regulated pre-mRNA splicing. We identified five putative genes encoding SR proteins (referred to as srp genes) in Caenorhabditis elegans, examined their expression using the gfp gene as a reporter, and suppressed their functions by double-stranded RNA-mediated interference (RNAi). The srp::gfp fusion genes were expressed in the nuclei of most somatic cells and showed no obvious tissue- or stage-specific expression. Simultaneous RNAi of the five srp genes resulted in embryonic lethality, whereas RNAi of individual srp genes caused no obvious morphological abnormality in the F1 progeny, indicating functional redundancy of the SR proteins. However, RNAi of several combinations of srp genes caused various developmental abnormalities, such as abnormal somatic gonad structures, delayed shift of the germ cell sexual differentiation, and abnormal spermatogenesis. Our results suggest that individual SR proteins have unique but somewhat redundant functions in C. elegans development.

Longman D et al. (2001) Curr Biol "Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans."

McGarvey T, Johnstone IL, Blencowe BJ, Longman D, McCracken S, Caceres JF

[Curr Biol, 2001]

Background: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. Results: In the present study, we show that SRm 160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleoticle sequence. This suggests that it functions as a general coactivator by interacting with different SIR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm 160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm 160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy, In the present study, we demonstrate that RNAi of CeSRm 160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. Conclusions: The observation that simultaneous suppression of CeSRm 160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm 160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.

Anandarajah EM et al. (2016) Mol Biochem Parasitol "SPARC (secreted protein acidic and rich in cysteine) of the intestinal nematode Strongyloides ratti is involved in mucosa-associated parasite-host interaction."

Erttmann KD, Brattig NW, Ditgen D, Hansmann J, Anandarajah EM, Liebau E

[Mol Biochem Parasitol, 2016]

The secreted protein acidic and rich in cysteine (SPARC), found in the excretory/secretory products of Strongyloides ratti, is most strongly expressed in parasitic females. Since SPARC proteins are involved in the modulation of cell-matrix interactions, a role of the secreted S. ratti SPARC (Sr-SPARC) in the manifestation of the parasite in the host's intestine is postulated. The full-length cDNA of Sr-SPARC was identified and the protein was recombinantly expressed. The purified protein was biologically active, able to bind calcium, and to attach to mucosa-associated human cells. Addition of Sr-SPARC to an in vitro mucosal three-dimensional-cell culture model led to a time-dependent release of the cytokines TNF-, IL-22, IL-10 and TSLP. Of importance, exposure with Sr-SPARC fostered wound closure in an intestinal epithelial cell model. Here, we demonstrate for the first time that SPARC released from the nematode is a multifunctional protein affecting the mucosal immune system.

Gao L et al. (2024) Proc Natl Acad Sci U S A "Voltage-induced calcium release in Caenorhabditis elegans body muscles."

Gao L, Ardiel E, Kaplan JM, Nurrish S

[Proc Natl Acad Sci U S A, 2024]

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in <i>Caenorhabditis elegans</i> body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Chen, Ron et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "E01A2.4/NKAP, an evolutionarily conserved SR-related protein involved in mRNA splicing"

Chen, Ron, Ahringer, Julie

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

RNA processing is an essential mechanism to regulate gene expression. A group of serine/arginine (SR)-rich proteins are crucial factors in the processing machineries involved in RNA splicing and metabolism. We previously identified E01A2.4 in a screen for new synthetic multivulval genes, a group of genetically interacting genes, many of which regulate gene expression and are required to prevent inappropriate vulval development. E01A2.4 is an essential gene and encodes a conserved protein with an N-terminal SR-rich region. We found that E01A2.4 is a nuclear protein and RNAi of E01A2.4 causes embryonic lethality, with an arrest phenotype similar to many RNA processing and splicing factors (1). Furthermore, E01A2.4 was found to genetically interact with F11A10.2, the ortholog of vertebrate splicing factor 3a (2). This gen etic analysis suggests that E01A2.4 may have a role in mRNA splicing, similar to other SR-repeat containing proteins. To further characterise the molecular mechanism of E01A2.4, we studied its human ortholog, NKAP (3). In human tissue culture cells, transiently expressed NKAP was recognised by the pan-SR antibody mAb104, indicating that NKAP is a SR-related protein. In addition, NKAP co-localised with a SR protein SC-35 in nuclear speckles, sites enriched with other SR proteins and RNA splicing factors, implying a role in mRNA splicing. To test for a possible role in splicing we conducted over-expression and loss of function studies of NKAP in HeLa cells and found that these treatments altered the splicing pattern of both an E1A mini-gene reporter and endogenous mRNA transcripts. We are currently conducting RNAseq analyses in C. elegans to study the consequences on RNA processing of E01A2.4 knockdown. Taken together, E01A2.4/NKAP is a new SR-related protein involved in mRNA splicing. Reference: 1. EMBO Journal, 2001 20(15): 3984; 2. 2003 International Worm Meeting 684; 3. Biochem Biophys Res Commun. 2003 Oct 24;310(3):720; Immunity. 2009 May;30(5):696.