Browsing by keyword "caenorhabditis elegans"
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Pathogen infection and cholesterol deficiency activate the C. elegans p38 immune pathway through a TIR-1/SARM1 phase transitionIntracellular signaling regulators can be concentrated into membrane-free, higher ordered protein assemblies to initiate protective responses during stress - a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD(+) glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD(+) glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD(+) glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that a loss-of-function mutation in nhr-8, which alters cholesterol metabolism and is used to study conditions of sterol deficiency, causes TIR-1/SARM1 to oligomerize into puncta in intestinal epithelial cells. Cholesterol scarcity increases p38 PMK-1 phosphorylation, primes immune effector induction in a manner that requires TIR-1/SARM1 oligomerization and its intrinsic NAD(+) glycohydrolase activity, and reduces pathogen accumulation in the intestine during a subsequent infection. These data reveal a new adaptive response that allows a metazoan host to anticipate pathogen threats during cholesterol deprivation, a time of relative susceptibility to infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD(+) glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.
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PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactionsInteractions between RNA binding proteins (RBPs) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using 'protein-centered' (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest-in a 'gene-centered' manner-yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA 'bait' can be tested versus multiple RBP 'preys' in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3' UTRs. PRIMA faithfully recapitulated numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.
