Mapping targets for small nucleolar RNAs in yeast

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions

Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

Cyclic oscillations in the level of phosphatidylinositol 3-phosphate in phagosomes, regulated by two phosphoinositide kinases and one phosphatase, are critical for phagosome maturation and degradation of apoptotic cells

Mrp1 is involved in lipid presentation and iNKT cell activation by Streptococcus pneumoniae

The CD1d pathway present lipid antigens resulting in the activation of iNKT cells but the complete pathway remains to be fully elucidated. Here, Chandra et al. use an siRNA screen and identify Mrp1 as crucial for CD1d lipid presentation and activation of iNKT in the context of Streptococcus pneumoniae infection

DNA methylation-associated inactivation of TGFβ-related genes DRM/Gremlin, RUNX3, and HPP1 in human cancers

The transforming growth factor β (TGFβ)-signalling pathway is deregulated in many cancers. We examined the role of gene silencing via aberrant methylation of DRM/Gremlin and HPP1, which inhibit TGFβ signalling, and RUNX3, which facilitates TGFβ-signalling, of all genes that are thought to be tumour suppressors, are aberrantly expressed, and are thus thought to have important role in human cancers. We examined DRM/Gremlin mRNA expression in 44 cell lines and the promoter methylation status of DRM/Gremlin, HPP1, and RUNX3 in 44 cell lines and 511 primary tumours. The loss of DRM/Gremlin mRNA expression in human cancer cell lines is associated with DNA methylation, and treatment with the methylation inhibitor-reactivated mRNA expression (n=13). Methylation percentages of the three genes ranged from 0–83% in adult tumours and 0–50% in paediatric tumours. Methylation of DRM/Gremlin was more frequent in lung tumours in smokers, and methylation of all three genes was inversely correlated with prognosis in patients with bladder or prostate cancer. Our results provide strong evidence that these TGFβ-related genes are frequently deregulated through aberrant methylation in many human malignancies

The DOCK Protein Sponge Binds to ELMO and Functions in Drosophila Embryonic CNS Development

Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegans Ced-5, human DOCK180, Drosophila Myoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of N-cadherin in neuronal development

A trematode parasite derived growth factor binds and exerts influences on host immune functions via host cytokine receptor complexes

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allow- ing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory recep- tor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is depen- dent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody- dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for damp- ened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targetingjuvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection

MISC-1/OGC Links Mitochondrial Metabolism, Apoptosis and Insulin Secretion

We identified MISC-1 (Mitochondrial Solute Carrier) as the C. elegans orthologue of mammalian OGC (2-oxoglutarate carrier). OGC was originally identified for its ability to transfer α-ketoglutarate across the inner mitochondrial membrane. However, we found that MISC-1 and OGC are not solely involved in metabolic control. Our data show that these orthologous proteins participate in phylogenetically conserved cellular processes, like control of mitochondrial morphology and induction of apoptosis. We show that MISC-1/OGC is required for proper mitochondrial fusion and fission events in both C. elegans and human cells. Transmission electron microscopy reveals that loss of MISC-1 results in a decreased number of mitochondrial cristae, which have a blebbed appearance. Furthermore, our pull-down experiments show that MISC-1 and OGC interact with the anti-apoptotic proteins CED-9 and Bcl-xL, respectively, and with the pro-apoptotic protein ANT. Knock-down of misc-1 in C. elegans and OGC in mouse cells induces apoptosis through the caspase cascade. Genetic analysis suggests that MISC-1 controls apoptosis through the physiological pathway mediated by the LIN-35/Rb-like protein. We provide genetic and molecular evidence that absence of MISC-1 increases insulin secretion and enhances germline stem cell proliferation in C. elegans. Our study suggests that the mitochondrial metabolic protein MISC-1/OGC integrates metabolic, apoptotic and insulin secretion functions. We propose a novel mechanism by which mitochondria integrate metabolic and cell survival signals. Our data suggest that MISC-1/OGC functions by sensing the metabolic status of mitochondria and directly activate the apoptotic program when required. Our results suggest that controlling MISC-1/OGC function allows regulation of mitochondrial morphology and cell survival decisions by the metabolic needs of the cell

The Drosophila TRPP Cation Channel, PKD2 and Dmel/Ced-12 Act in Genetically Distinct Pathways during Apoptotic Cell Clearance

Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway

E4 ligase–specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis

Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response

C. elegans EIF-3.K Promotes Programmed Cell Death through CED-3 Caspase

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution