RESUMO
We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.
Assuntos
Proteínas de Transporte/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação a DNA , Células HeLa , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análogos & derivados , Ligação Proteica , RNA Mensageiro/química , Proteínas de Ligação a RNA , Fatores de TranscriçãoRESUMO
CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of "regulated instability" of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3' untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Delta1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Delta1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.
Assuntos
Ligante de CD40/genética , Ligante de CD40/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/análogos & derivados , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células Jurkat , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Elementos de Resposta/fisiologia , Deleção de SequênciaRESUMO
Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT-PCR, we investigated the regulation of GlyR alpha-subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR alpha2 subunit, alpha2A and alpha2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named "alpha2N." Examination of the RNA from spinal cords of different-aged rats showed a dramatic down-regulation of alpha2N during prenatal development: alpha2N mRNA formed a significant portion of the alpha2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR alpha2 pre-mRNA, the neurooncological ventral antigen-1 (Nova-1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of alpha2N, calling into question their involvement in the developmental regulation of alpha2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova-1 variants significantly altered the relative level of GlyR alpha2N, showing that Nova-1 isoforms can regulate GlyR alpha2 pre-mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR alpha2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova-1 and brPTB in the developing spinal cord, and suggest that Nova-1 plays a role in regulating GlyR alpha2N in developing neurons.
