The secreted MSP domain of C. elegans VAPB homolog VPR-1 patterns the adult striated muscle mitochondrial reticulum via SMN-1.

The major sperm protein domain (MSPd) has an extracellular signaling function implicated in amyotrophic lateral sclerosis. Secreted MSPds derived from the C. elegans VAPB homolog VPR-1 promote mitochondrial localization to actin-rich I-bands in body wall muscle. Here we show that the nervous system and germ line are key MSPd secretion tissues. MSPd signals are transduced through the CLR-1 Lar-like tyrosine phosphatase receptor. We show that CLR-1 is expressed throughout the muscle plasma membrane, where it is accessible to MSPd within the pseudocoelomic fluid. MSPd signaling is sufficient to remodel the muscle mitochondrial reticulum during adulthood. An RNAi suppressor screen identified survival of motor neuron 1 (SMN-1) as a downstream effector. SMN-1 acts in muscle, where it colocalizes at myofilaments with ARX-2, a component of the Arp2/3 actin-nucleation complex. Genetic studies suggest that SMN-1 promotes Arp2/3 activity important for localizing mitochondria to I-bands. Our results support the model that VAPB homologs are circulating hormones that pattern the striated muscle mitochondrial reticulum. This function is crucial in adults and requires SMN-1 in muscle, likely independent of its role in pre-mRNA splicing.

The C. elegans VAPB homolog VPR-1 is a permissive signal for gonad development.

VAMP/synaptobrevin-associated proteins (VAPs) contain an N-terminal major sperm protein domain (MSPd) that is associated with amyotrophic lateral sclerosis. VAPs have an intracellular housekeeping function, as well as an extracellular signaling function mediated by the secreted MSPd. Here we show that the C. elegans VAP homolog VPR-1 is essential for gonad development. vpr-1 null mutants are maternal effect sterile due to arrested gonadogenesis following embryo hatching. Somatic gonadal precursor cells and germ cells fail to proliferate fully and complete their respective differentiation programs. Maternal or zygotic vpr-1 expression is sufficient to induce gonadogenesis and fertility. Genetic mosaic and cell type-specific expression studies indicate that vpr-1 activity is important in the nervous system, germ line and intestine. VPR-1 acts in parallel to Notch signaling, a key regulator of germline stem cell proliferation and differentiation. Neuronal vpr-1 expression is sufficient for gonadogenesis induction during a limited time period shortly after hatching. These results support the model that the secreted VPR-1 MSPd acts at least in part on gonadal sheath cell precursors in L1 to early L2 stage hermaphrodites to permit gonadogenesis.

Characterisation of novel microRNAs in the Black flying fox (Pteropus alecto) by deep sequencing.

BACKGROUND: Bats are a major source of new and emerging viral diseases. Despite the fact that bats carry and shed highly pathogenic viruses including Ebola, Nipah and SARS, they rarely display clinical symptoms of infection. Host factors influencing viral replication are poorly understood in bats and are likely to include both pre- and post-transcriptional regulatory mechanisms. MicroRNAs are a major mechanism of post-transcriptional gene regulation, however very little is known about them in bats. RESULTS: This study describes 399 microRNAs identified by deep sequencing of small RNA isolated from tissues of the Black flying fox, Pteropus alecto, a confirmed natural reservoir of the human pathogens Hendra virus and Australian bat lyssavirus. Of the microRNAs identified, more than 100 are unique amongst vertebrates, including a subset containing mutations in critical seed regions. Clusters of rapidly-evolving microRNAs were identified, as well as microRNAs predicted to target genes involved in antiviral immunity, the DNA damage response, apoptosis and autophagy. Closer inspection of the predicted targets for several highly supported novel miRNA candidates suggests putative roles in host-virus interaction. CONCLUSIONS: MicroRNAs are likely to play major roles in regulating virus-host interaction in bats, via dampening of inflammatory responses (limiting the effects of immunopathology), and directly limiting the extent of viral replication, either through restricting the availability of essential factors or by controlling apoptosis. Characterisation of the bat microRNA repertoire is an essential step towards understanding transcriptional regulation during viral infection, and will assist in the identification of mechanisms that enable bats to act as natural virus reservoirs. This in turn will facilitate the development of antiviral strategies for use in humans and other species.

VAPB/ALS8 MSP ligands regulate striated muscle energy metabolism critical for adult survival in caenorhabditis elegans.

Mutations in VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), two motor neuron diseases that often include alterations in energy metabolism. We have shown that C. elegans and Drosophila neurons secrete a cleavage product of VAPB, the N-terminal major sperm protein domain (vMSP). Secreted vMSPs signal through Roundabout and Lar-like receptors expressed on striated muscle. The muscle signaling pathway localizes mitochondria to myofilaments, alters their fission/fusion balance, and promotes energy production. Here, we show that neuronal loss of the C. elegans VAPB homolog triggers metabolic alterations that appear to compensate for muscle mitochondrial dysfunction. When vMSP levels drop, cytoskeletal or mitochondrial abnormalities in muscle induce elevated DAF-16, the Forkhead Box O (FoxO) homolog, transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels in adults, and extends lifespan, despite reduced muscle mitochondria electron transport chain activity. Finally, Vapb knock-out mice exhibit abnormal muscular triacylglycerol levels and FoxO target gene transcriptional responses to fasting and refeeding. Our data indicate that impaired vMSP signaling to striated muscle alters FoxO activity, which affects energy metabolism. Abnormalities in energy metabolism of ALS patients may thus constitute a compensatory mechanism counterbalancing skeletal muscle mitochondrial dysfunction.

Secreted VAPB/ALS8 major sperm protein domains modulate mitochondrial localization and morphology via growth cone guidance receptors.

VIDEO ABSTRACT: The VAPB/ALS8 major sperm protein domain (vMSP) is implicated in amyotrophic lateral sclerosis and spinal muscular atrophy, yet its function in the nervous system is not well understood. In Caenorhabditis elegans and Drosophila, the vMSP is cleaved from its transmembrane anchor and secreted in a cell type-specific fashion. We show that vMSPs secreted by neurons act on Lar-like protein-tyrosine phosphatase and Roundabout growth cone guidance receptors expressed in striated muscle. This signaling pathway promotes Arp2/3-dependent actin remodeling and mitochondrial localization to actin-rich muscle I-bands. C. elegans VAPB mutants have mitochondrial localization, morphology, mobility, and fission/fusion defects that are suppressed by Lar-like receptor or Arp2/3 inactivation. Hence, growth cone guidance receptor pathways that remodel the actin cytoskeleton have unanticipated effects on mitochondrial dynamics. We propose that neurons secrete vMSPs to promote striated muscle energy production and metabolism, in part through the regulation of mitochondrial localization and function.

Sperm and oocyte communication mechanisms controlling C. elegans fertility.

During sexual reproduction in many species, sperm and oocyte secrete diffusible signaling molecules to help orchestrate the biological symphony of fertilization. In the Caenorhabditis elegans gonad, bidirectional signaling between sperm and oocyte is important for guiding sperm to the fertilization site and inducing oocyte maturation. The molecular mechanisms that regulate sperm guidance and oocyte maturation are being delineated. Unexpectedly, these mechanisms are providing insight into human diseases, such as amyotrophic lateral sclerosis, spinal muscular atrophy, and cancer. Here we review sperm and oocyte communication in C. elegans and discuss relationships to human disorders.

A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach.

MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small regulatory RNAs characterized in chickens we used a deep sequencing approach developed by Solexa (now Illumina Inc.). We sequenced three small RNA libraries prepared from different developmental stages of the chicken embryo (days five, seven, and nine) to produce over 9.5 million short sequence reads. We developed a bioinformatics pipeline to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected almost all of the previously known chicken miRNAs and their respective miRNA* sequences. In addition we discovered 449 new chicken miRNAs including 88 miRNA candidates. Of these, 430 miRNAs appear to be specific to the avian lineage. Another six new miRNAs had evidence of evolutionary conservation in at least one vertebrate species outside of the bird lineage. The remaining 13 putative miRNAs appear to represent chicken orthologs of known vertebrate miRNAs. We discovered 39 additional putative miRNA candidates originating from miRNA generating intronic sequences known as mirtrons.

Genomics of reproduction in nematodes: prospects for parasite intervention?

Understanding reproductive processes in parasitic nematodes has the potential to lead to the informed design of new anthelmintics and control strategies. Little is known, however, about the molecular mechanisms underlying sex determination, gametogenesis and reproductive physiology for most parasitic nematodes. Together with comparative analyses of data for the free-living nematode Caenorhabditis elegans, molecular investigations are beginning to provide insights into the processes involved in reproduction and development in parasitic nematodes. Here, we review recent developments, focusing on technological aspects and on molecules associated with sex-specific differences in adult nematodes.

Native microRNA loop sequences can improve short hairpin RNA processing for virus gene silencing in animal cells.

Introduction of small interfering RNAs (siRNAs) into cells results in transitory silencing of target genes with complementary sequence. Incorporating siRNAs into short-hairpin RNAs (shRNAs) or microRNA-adapted shRNAs (shRNAmir) is a popular tool for targeted gene silencing. shRNAmirs mimicking endogenous pre-microRNAs (unprocessed hairpin microRNAs) are more difficult to design and result in longer RNA molecules. The use of microRNA (miRNA) loop sequences in shRNAs as an alternative to an entire pre-microRNA structure on silencing efficiency has not been studied extensively. This report shows that loop sequences derived from native miRNAs improves the efficiency of silencing due to the processing of the shRNAs into mature siRNAs.

Oesophagostomum dentatum: potential as a model for genomic studies of strongylid nematodes, with biotechnological prospects.

There are substantial gaps in the knowledge of the molecular processes of development and reproduction in parasitic nematodes, despite the fact that understanding such processes could lead to novel ways of treating and controlling parasitic diseases, through blocking or disrupting key biological pathways. Biotechnological advances through large-scale sequencing projects, approaches for the analysis of differential gene and protein expression and functional genomics (e.g., double-stranded RNA interference) now provide opportunities to investigate the molecular basis of developmental processes in some parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum (order Strongylida), may provide a platform for testing the function of genes from this and related nematodes, given that this species can be grown and maintained in culture in vitro for periods longer than other nematodes of the same order. In this article, we review relevant biological, biochemical and molecular biological and genomic information about O. dentatum and propose that the O. dentatum - pig system provides an attractive model for exploring molecular developmental and reproductive processes in strongylid nematodes, leading toward new intervention methods and biotechnological outcomes.

Ubiquitin-conjugating enzyme genes in Oesophagostomum dentatum.

Full-length genes representing different isoforms of the ubiquitin-conjugating enzyme UBC-2 were isolated from Oesophagostomum dentatum, cloned and sequenced. The alignment of their sequences (designated Od-ubc-2.1 to Od-ubc-2.3) revealed nucleotide variation at three positions within the predicted open reading frame of 444 bp. Substitutions were at positions 141 (A<-->G), 142 (A<-->G) and 296 (T<-->C). Both former substitutions resulted in amino acid changes from a glycine residue to an arginine residue, whereas the latter resulted in a change from isoleucine to threonine. Comparison of predicted OD-UBC-2 with UBC-2 (protein) homologues/orthologues from 12 other species representing nematodes, Drosophila melanogaster, Saccharomyces cerevisiae, mice and humans revealed identities between species varying from 77 to 100% at the amino acid level, and motifs associated with protein conformation and function were identified. While the function of a representative ubc-2 gene from O. dentatum could not be established in C. elegans, it is likely to play a key role in the catabolism of proteins and in the development of O. dentatum.

Molecular biology of reproduction and development in parasitic nematodes: progress and opportunities.

Molecular biological research on the development and reproduction of parasites is of major significance for many fundamental and applied areas of medical and veterinary parasitology. Together with knowledge of parasite biology and epidemiology, the application of molecular tools and technologies provides unique opportunities for elucidating developmental and reproductive processes in helminths. This article focuses specifically on recent progress in studying the molecular mechanisms of development, sexual differentiation and reproduction in parasitic nematodes of socio-economic importance and comparative analyses, where appropriate, with the free-living nematode Caenorhabditis elegans. It also describes the implications of such work for understanding reproduction, tissue migration, hypobiosis, signal transduction and host-parasite interactions at the molecular level, and for seeking new means of parasite intervention.

Haemonchus contortus: molecular characterisation of a small heat shock protein.

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development. Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein