TY - JOUR T1 - Genome-wide analysis reveals novel genes essential for heme homeostasis in Caenorhabditis elegans. JF - PLoS Genet Y1 - 2010 A1 - Severance, Scott A1 - Rajagopal, Abbhirami A1 - Rao, Anita U A1 - Cerqueira, Gustavo C A1 - Mitreva, Makedonka A1 - El-Sayed, Najib M A1 - Krause, Michael A1 - Hamza, Iqbal KW - Animals KW - Caenorhabditis elegans KW - Dose-Response Relationship, Drug KW - Gene Expression Profiling KW - Gene Expression Regulation KW - genes KW - Genome-Wide Association Study KW - Heme KW - Homeostasis KW - HUMANS KW - Leishmania KW - Nematoda KW - Trypanosoma AB -

Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme--a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 microM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA-mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival.

VL - 6 CP - 7 M3 - 10.1371/journal.pgen.1001044 ER - TY - JOUR T1 - Sex-lethal splicing autoregulation in vivo: interactions between SEX-LETHAL, the U1 snRNP and U2AF underlie male exon skipping. JF - Development Y1 - 2003 A1 - Nagengast, Alexis A A1 - Stitzinger, Shane M A1 - Tseng, Chin-Hsiu A1 - Mount, Stephen M A1 - Salz, Helen K KW - Alternative Splicing KW - Amino Acid Sequence KW - Animals KW - Animals, Genetically Modified KW - Drosophila melanogaster KW - Drosophila Proteins KW - Exons KW - Female KW - Gene Expression Regulation, Developmental KW - Genes, Insect KW - Homeostasis KW - Male KW - Models, Genetic KW - Molecular Sequence Data KW - Nuclear Proteins KW - Point Mutation KW - Ribonucleoprotein, U1 Small Nuclear KW - Ribonucleoproteins KW - RNA Splicing KW - RNA-Binding Proteins KW - Sequence Homology, Amino Acid KW - Sex Differentiation AB -

Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific SEX-LETHAL RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is sans-fille (snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether SNF acts as part of an intact snRNP or a free protein. We provide evidence that SEX-LETHAL interacts with SANS-FILLE in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with SEX-LETHAL. Moreover, we find that SEX-LETHAL associates with other integral U1 snRNP components, and we provide genetic evidence to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link SEX-LETHAL with the heterodimeric splicing factor, U2AF. These studies point specifically to a mechanism by which SEX-LETHAL represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, our studies are difficult to reconcile with the current model that proposes that the SEX-LETHAL blocks splicing at the second catalytic step, and instead argue that the SEX-LETHAL protein acts after splice site recognition, but before catalysis begins.

VL - 130 CP - 3 ER -