RESUMO
Serine/arginine-rich splicing factor 3 (SRSF3), a member of the serine/arginine (SR)-rich family of proteins, regulates both alternative splicing of pre-mRNA and export of mature mRNA from the nucleus. Although its role in nuclear mRNA processing is well understood, the mechanism by which it alters the fate of cytoplasmic mRNA molecules remains elusive. Here, we provide evidence that SRSF3 not only regulates the alternative splicing pattern of programmed cell death 4 (PDCD4) mRNA, but also modulates its translational efficiency in the cytoplasm by lowering translation levels. We observed a marked increase in PDCD4 mRNA in translating polysome fractions upon silencing of SRSF3, and, conversely, ectopic overexpression of SRSF3 shifted PDCD4 mRNA into non-translating ribosomal fractions. In live cells, SRSF3 colocalized with PDCD4 mRNA in P-bodies (PBs), where translationally silenced mRNAs are deposited, and this localization was abrogated upon SRSF3 silencing. Furthermore, using two different reporter systems, we showed that SRSF3 interacts directly with PDCD4 mRNA and mediates translational repression by binding to the 5'-untranslated region (5'-UTR). In summary, our data suggest that the oncogenic potential of SRSF3 might be realized, in part, through the translational repression of PDCD4 mRNA.
Assuntos
Regiões 5' não Traduzidas , Proteínas Reguladoras de Apoptose/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Apoptose/genética , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina , TransfecçãoRESUMO
Sequencing of the Moraxella sp. CK-1 autolysin (cell wall hydrolases) gene showed the presence of an open reading frame which encodes a polypeptide of 273 amino acids with a molecular mass of 33,316 Da. A presumed ribosomal binding site, a possible -10 and -35 region, and rho-dependent terminators were found. The C-terminal region of the mature protein showed considerable homology with the Thermus sp. serine proteinase. Enzyme assay suggests that the recombinant autolysin has amidase or endopeptidase activity. Analysis of the peptidoglycan fragments, following the treatment with the autolysin, indicates that this protein is an N-acetylmuramyl-L-alanine amidase. Insertional inactivation of the autolysin of Moraxella sp. CK-1 chromosome led to a decrease in cell wall hydrolytic activity, clumping of the cells, and color change. No lytic band present in inactivated magA mutant by renaturing SDS-PAGE.
Assuntos
Parede Celular/enzimologia , Moraxella/enzimologia , Mutagênese Insercional , N-Acetil-Muramil-L-Alanina Amidase/genética , Sequência de Aminoácidos , Bacteriólise , Sequência de Bases , Dados de Sequência Molecular , Moraxella/genética , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A gene of Moraxella sp. CK-1 encoding cell wall lytic activity has been cloned and expressed in E. coli. A genomic library of Moraxella sp. CK-1 was constructed in the multifunctional phagemid vector pT7T3 19u, with partial Sau3A digests of Moraxella sp. CK-1 genomic DNA inserted at the BamHI restriction site. Screening of about 5,000 transformants for cell lysis activity in LB agar plates containing Micrococcus Iuteus cells gave one positive clone harboring the 3.7 kb insert (pMXA282). Restriction mapping and deletion analysis of the recombinant plasmid carrying a 3.7 kb insert suggested that the autolysin gene was located within a 1.1 kb BamHI-PstI fragment. Analysis of extracts of E. coli clone harboring recombinant plasmids on renaturing SDS-polyacrylamide gels containing heat-killed Micrococcus luteus cells showed a clear zone around a polypeptide of about 32 kDa. Lytic activity against Micrococcus luteus cell walls by the cloned autolysin was maximum at pH 9.0. Even in conditions of over pH 10.0, this cloned autolysin showed a vigrous lytic activity. Southern blot analysis suggested the existence of other homologous regions in Moraxella sp. CK-1 genome.