RESUMO
In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as "basal-body cages." The major protein comprising Euplotes cages has been previously identified and termed "cagein." Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear "nanochromosome" of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.
Assuntos
Cilióforos , Euplotes , Corpos Basais , Cilióforos/genética , Euplotes/genética , Organelas , ProteínasRESUMO
Mutations that enhance the response to double-stranded RNA (dsRNA) have revealed components of the RNA interference (RNAi) pathway or related small RNA pathways. To explore these small RNA pathways, we screened for Caenorhabditis elegans mutants displaying an enhanced response to exogenous dsRNAs. Here we describe the isolation of mutations in two adjacent, divergently transcribed open reading frames (eri-6 and eri-7) that fail to complement. eri-6 and eri-7 produce separate pre-messenger RNAs (pre-mRNAs) that are trans-spliced to form a functional mRNA, eri-6/7. Trans-splicing of eri-6/7 is mediated by a direct repeat that flanks the eri-6 gene. Adenosine to inosine editing within untranslated regions of eri-6 and eri-7 pre-mRNAs reveals a double-stranded pre-mRNA intermediate, forming in the nucleus before splicing occurs. The ERI-6/7 protein is a superfamily I helicase that both negatively regulates the exogenous RNAi pathway and functions in an endogenous RNAi pathway.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Genes de Helmintos/genética , Interferência de RNA/fisiologia , Trans-Splicing , Animais , Caenorhabditis elegans/classificação , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Fases de Leitura Aberta/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.
