RESUMO
The 5'-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5'-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stem-loop element. Taken together, our study describes the functional relevance of two alternative 5'-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sítios Internos de Entrada Ribossomal , RNA Mensageiro/química , Regiões 5' não Traduzidas , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Processamento Alternativo , Contagem de Células , Quadruplex G , Células HEK293 , Humanos , Células MCF-7 , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
Sialuria is a rare autosomal dominant disorder of mammalian metabolism, caused by defective feedback inhibition of the UDP-N-acetylglucosamine-2-epimerase N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. Sialuria is characterized by overproduction of free sialic acid in the cell cytoplasm. Patients exhibit vastly increased urinary excretion of sialic acid and show differently pronounced developmental delays. The physiopathology of sialuria is not well understood. Here we established a transgenic mouse line that expresses GNE containing the sialuria mutation R263L, in order to investigate the influence of an altered sialic acid concentration on the organism. The transgenic mice that expressed the mutated RNA excreted up to 400 times more N-acetylneuraminic acid than wild-type mice. Additionally, we found higher sialic acid concentration in the brain cytoplasm. Analyzing the (poly)sialylation of neural cell adhesion molecule (NCAM) revealed increased polysialylation in brains of transgenic mice compared to wild-type. However, we found only minor changes in membrane-bound sialylation in various organs but, surprisingly, a significant increase in surface sialylation on leukocytes. Our results suggest that the intracellular sialic acid concentration regulates polysialylation on NCAM in vivo; this could play a role in the manifestation of the developmental delays in sialuria patients.
Assuntos
Leucócitos/metabolismo , Complexos Multienzimáticos/genética , Ácido N-Acetilneuramínico/urina , Moléculas de Adesão de Célula Nervosa/metabolismo , Processamento de Proteína Pós-Traducional , Doença do Armazenamento de Ácido Siálico/metabolismo , Fatores Etários , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Retroalimentação Fisiológica , Humanos , Leucócitos/patologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Complexos Multienzimáticos/deficiência , Mutação , Moléculas de Adesão de Célula Nervosa/química , Moléculas de Adesão de Célula Nervosa/genética , Especificidade de Órgãos , Doença do Armazenamento de Ácido Siálico/genética , Doença do Armazenamento de Ácido Siálico/patologiaRESUMO
UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids. Sialic acids are terminal monosaccharides of glycoconjugates and gangliosides, which have an essential influence on various cell interactions. The sialylation of proteins varies during development, aging, and pathogenesis of degenerative diseases such as Morbus Alzheimer, diabetes mellitus type II, or myopathies. Mutation of methionine 743 in the GNE leads to a 30% reduction of the enzyme activity and is responsible for an aggressive form of GNE myopathy. GNE myopathy or hereditary inclusion body myopathy (HIBM) is an age-dependent muscular dystrophy. Here, we analyzed the impact of the exchange of methionine to threonine at position 743 which introduces an additional potential phosphorylation/O-GlcNAcylation site. We found increased O-GlcNAcylation of the M743T variant compared to the wild-type GNE. In addition, removal of the O-GlcNAc of the M743T variant resulted in an increased activity comparable to activity of the wild-type GNE. Furthermore, the half-life of the M743T variant is two times longer than for the wild-type GNE protein. This study provides that the balance of phosphorylation and O-GlcNAcylation is decisive involved in efficiency and regulation of GNE.