RESUMO
The mammalian proteins hnRNP A1 and hnRNP H control many splicing decisions in viral and cellular primary transcripts. To explain some of these activities, we have proposed that self-interactions between bound proteins create an RNA loop that represses internal splice sites while simultaneously activating the external sites that are brought in closer proximity. Here we show that a variety of hnRNP H binding sites can affect 5' splice site selection. The addition of two sets of hnRNP H sites in a model pre-mRNA modulates 5' splice site selection cooperatively, consistent with the looping model. Notably, binding sites for hnRNP A1 and H on the same pre-mRNA can similarly collaborate to modulate 5' splice site selection. The C-terminal portion of hnRNP H that contains the glycine-rich domains (GRD) is essential for splicing activity, and it can be functionally replaced by the GRD of hnRNP A1. Finally, we used the bioluminescence resonance energy transfer (BRET) technology to document the existence of homotypic and heterotypic interactions between hnRNP H and hnRNP A1 in live cells. Overall, our study suggests that interactions between different hnRNP proteins bound to distinct locations on a pre-mRNA can change its conformation to affect splicing decisions.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/fisiologia , Sítios de Splice de RNA , Sequência de Bases/fisiologia , Sítios de Ligação/genética , Células Cultivadas , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica/fisiologia , Sítios de Splice de RNA/genética , Sítios de Splice de RNA/fisiologia , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato/genética , TransfecçãoRESUMO
Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Humanos , Imunoprecipitação , Medições Luminescentes , Multimerização Proteica , Ribonucleoproteínas/metabolismoRESUMO
Staufen1 is a component of transported ribonucleoprotein complexes. Genetic work in Drosophila has suggested that Staufen plays a role in the de-repression of translation of oskar mRNA following localization. To determine whether Staufen1 can play a similar role in mammals, we studied translation of transcripts in the presence or in the absence of Staufen1. Translationally repressed mRNAs were generated by fusing the structured human immunodeficiency virus type 1 trans-activating response (TAR) element to the 5' end of a reporter transcript. In rabbit reticulocyte lysates and in mammalian cultured cells, the addition of Staufen1 resulted in the up-regulation of reporter activity when translation was driven by the TAR-bearing RNA. In contrast, Staufen1 had no effect on translation of efficiently translated mRNAs lacking an apparent structured 5' end, suggesting that Staufen1-binding to the 5' end is required for enhanced translation. Consistently, Staufen1 RNA-binding activity is necessary for this translational effect. In addition, similar up-regulation of translation was observed when Staufen1 was tethered to the 5' end of mRNAs via other structured RNAs, the highest level of translational increase being obtained with the bona fide Staufen1-binding site of the Arf1 transcript. The expression of Staufen1 promoted polysomal loading of TAR-luciferase transcripts resulting in enhanced translation. Our results support a model in which the expression of Staufen1 and its interaction with the 5' end of RNA and ribosomes facilitate translation initiation.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Repetição Terminal Longa de HIV , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Polirribossomos/química , RNA Mensageiro/química , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Coelhos , eIF-2 Quinase/metabolismoRESUMO
UNLABELLED: Since HbA1c became a diagnostic criterion for type 2 diabetes, assays measuring this analyte were required to be analytically stronger. Although precision of Sebia Capillarys 2 Flex Piercing® was previously evaluated with standard methods and material, we wanted to document the long-term performance of this analyzer in two different centers. DESIGN AND METHODS: In this study, we evaluated, with a non-conventional methodology, the analytical precision of the Sebia Capillarys 2 Flex Piercing® using HbA1c patient data and compared it with two different common commercial immunoassays. RESULTS: CV of 1.33% and 1.32% was obtained in the two centers for the electrophoretic technique compared to 2.66% and 3.92% for the Roche Cobas Integra 800® and Beckman Synchron DxC® immunoassays technique respectively. CONCLUSIONS: Our results show that capillary electrophoresis is more robust than the 2 immunoassays tested for long-term precision and even exceeds minimal requirements recommended.