RESUMO
The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes Dominantes/genética , Guanosina Trifosfato/metabolismo , Mutação/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células 3T3 , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Morte Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Testículo/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
TB-RBP (testis-brain RNA-binding protein) is a mouse RNA-binding protein that controls the temporal and spatial expression of mRNA. Found most abundantly in brain and male germ cells in the testis, TB-RBP is known to suppress the translation of specific testicular and brain mRNAs as part of cell development. TB-RBP-mRNA complexes can bind microtubules and thereby facilitate RNA translocation. Translin is the human orthologue of TB-RBP which binds to single-stranded ends of DNA sequences in breakpoint regions of chromosomal translocations. TB-RBP/translin has been crystallized in space group P2(1)2(1)2. The expression, purification, and crystallization of TB-RBP are described as well as preliminary X-ray diffraction data. The multimeric state of TB-RBP is addressed using dynamic light-scattering results.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas de Ligação a RNA/química , Animais , Cristalização , Cristalografia por Raios X , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/biossíntese , Conformação Proteica , Proteínas de Ligação a RNA/biossínteseRESUMO
Trax (Translin-associated factor X) has been shown to interact with TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays. Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA, but forms heterodimers with TB-RBP under reducing conditions. The heterodimer of TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to specific single stranded DNA sequences. The in vitro interactions between TB-RBP and Trax are confirmed by similar interactions in the yeast two-hybrid system. Cell fractionation and confocal microscope studies reveal that Trax is predominantly cytoplasmic. In contrast, TB-RBP is present in both the nuclei and cytoplasm of transfected cells and uses a highly conserved nuclear export signal to exit nuclei. In addition to a leucine zipper, two basic domains in TB-RBP are essential for RNA binding, but only one of these domains is needed for DNA binding. Trax restores DNA binding to TB-RBP containing an altered form of this domain. These data suggest that Trax-TB.RBP interactions modulate the DNA- and RNA-binding activity of TB-RBP.
Assuntos
Proteínas de Transporte/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células 3T3 , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Clonagem Molecular , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Transporte Proteico , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The single copy mouse Testis Brain RNA-Binding Protein (TB-RBP) gene encodes three mRNAs of 3.0, 1.7, and 1.0 kb which only differ in their 3' UTRs. The 1 kb TB-RBP mRNA predominates in testis, while somatic cells preferentially express the 3.0 kb TB-RBP mRNA. Here we show that the 1 kb mRNA is translated several-fold more efficiently than the 3 kb TB-RBP in rabbit reticulocyte lysates and cells with elevated levels of the 1 kB TB-RBP mRNA express high levels of TB-RBP. To determine if the cleavage stimulatory factor CstF 64 can modulate the alternative splicing of the TB-RBP pre-mRNA and therefore TB-RBP expression, CstF 64 levels and binding to alternative polyadenylation sites were examined. CstF 64 is abundant in the testis and preferentially binds to a distal site in the TB-RBP pre-mRNA that produces the 3 kb TB-RBP. Moreover, upregulation or overexpression of CstF 64 increases the poly(A) site selection for the 1 kb TB-RBP mRNA. We propose that the level of the polyadenylation factor CstF 64 modulates the level of TB-RBP synthesis in male germ cells by an alternative processing of the TB-RBP pre-mRNA.
