RESUMO
The closely related mammalian proteins IA-2 and phogrin are protein tyrosine phosphatase-like receptor proteins spanning the membrane of dense core vesicles of neuroendocrine tissues. They are of interest as molecular components of the secretory machinery and as major targets of autoimmunity in type I diabetes mellitus. The Caenorhabditis elegans genome has a single copy of an IA-2/phogrin homolog ida-1 III (islet cell diabetic autoantigen), which encodes the ida-1 (B0244.2) gene product as a series of 12 exons over a 10-kb region of chromosome III. The full-length sequence of the ida-1 cDNA encoded a 767-amino acid type 1 transmembrane protein of 87 kDa. The PTP catalytic site consensus sequence of IDA-1, like IA-2 and phogrin, diverged and would not be active. Expression of green fluorescent protein (GFP) under the ida-1 gene promoter showed activity in a subset of around 30 neurons with sensory functions and the uv1 cells of the vulva in hermaphrodites. Males showed additional expression in male-specific neurons. In situ experiments in rat brain showing the distribution of IA-2 and phogrin suggested a complimentary and overlapping pattern compared with the proprotein convertases PC1 and PC2. In C. elegans, IDA-1-expressing cells comprised a subset of those expressing the PC2 homolog KPC-2 (C51E3. 7), consistent with IDA-1 being a component of neuropeptide-containing dense core vesicles. The results support the hypothesis that C. elegans IDA-1 is the functional homolog of IA-2 and phogrin in mammals. Analysis of the function of IDA-1 should contribute to our understanding of the function of these proteins in signal transduction, vesicle locomotion, and exocytosis.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Sistema Nervoso/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteínas Tirosina Fosfatases/genética , Sequência de Aminoácidos/genética , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Autoantígenos , Encéfalo/metabolismo , Caenorhabditis elegans/citologia , Clonagem Molecular/métodos , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde , Insulina/metabolismo , Secreção de Insulina , Proteínas Luminescentes , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Sistema Nervoso/citologia , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Pró-Proteína Convertase 2 , Pró-Proteína Convertases , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Vesículas Secretórias/metabolismo , Homologia de Sequência de Aminoácidos , Subtilisinas/genética , Subtilisinas/metabolismoRESUMO
In Caenorhabditis elegans, pre-mRNA for the essential splicing factor U2AF65 sometimes is spliced to produce an RNA that includes an extra 216-bp internal exon, exon 3. Inclusion of exon 3 inserts an in-frame stop codon, yet this RNA is not subject to SMG-mediated RNA surveillance. To test whether exon 3 causes RNA to remain nuclear and thereby escape decay, we inserted it into the 3' untranslated region of a gfp reporter gene. Although exon 3 did not affect accumulation or processing of the mRNA, it dramatically suppressed expression of green fluorescent protein (GFP). We showed by in situ hybridization that exon 3-containing gfp RNA is retained in the nucleus. Intriguingly, exon 3 contains 10 matches to the 8-bp 3' splice-site consensus. We hypothesized that U2AF might recognize this octamer and thereby prevent export. This idea is supported by RNA interference experiments in which reduced levels of U2AF resulted in a small burst of gfp expression.