Functional characterization of testis-brain RNA-binding protein, TB-RBP/Translin, in translational regulation.

Testis-brain RNA-binding protein (TB-RBP/Translin) is known to contribute to the translational repression of a subset of haploid cell-specific mRNAs, including protamine 2 (Prm2) mRNA. Mutant mice lacking TB-RBP display abnormal spermatogenesis, despite normal male fertility. In this study, we carried out functional analysis of TB-RBP in mammalian cultured cells to understand the mechanism of translational repression by this RNA-binding protein. Although the amino acid sequence contained a eukaryotic translation initiation factor 4E (EIF4E)-recognition motif, TB-RBP failed to interact with EIF4E. In cultured cells, TB-RBP was unable to reduce the activity of luciferase encoded by a reporter mRNA carrying the 3'-untranslated region of Prm2. However, λΝ-BoxB tethering assay revealed that the complex of TB-RBP with its binding partner, Translin-associated factor X (TRAX), exhibits the ability to reduce the luciferase reporter activity by degrading the mRNA. These results suggest that TB-RBP may play a regulatory role in determining the sequence specificity of TRAX-catalyzed mRNA degradation.

Role of amino acid residues important for nucleic acid binding in human Translin.

Translin is a multifunctional DNA/RNA binding protein involved in DNA repair and RNA metabolism. It has two basic regions and involvement of some residues in these regions in nucleic acid binding is established experimentally. Here we report the functional role of four residues of basic region II, Y85, R86, H88, R92 and one residue of C terminal region, K193 in nucleic acid binding using substitution mutant variants. CD analysis of the mutant proteins showed that secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Octameric state was maintained in all the mutants of basic region as evidenced by TEM, DLS, native PAGE and gel filtration analyses. However, K193G mutation completely abolished the octameric state of Translin protein and consequently its ability to bind ssDNA/ssRNA. The mutants of the basic region II exhibited a differential effect on nucleic acid binding, with R86A and R92G as most deleterious. Interestingly, H88A mutant showed higher nucleic acid binding affinity in comparison to the wild type Translin. An in silico analysis of the mutant variant sequences predicted all the mutations to be destabilizing, causing increase in flexibility and also leading to disruption of local interactions. The differential effect of mutations on DNA/RNA binding where octameric state is maintained could be attributed to these predicted disturbances.

C-terminal residues of rice translin are essential for octamer formation and nucleic acid binding.

Translin is a DNA/RNA binding protein involved in DNA repair and RNA metabolism. Previously, we had shown that rice translin (221 amino acids) exhibits biochemical activities similar to that of the human translin protein. Here we report the role of the C-terminal random coil in rice translin function by analyzing truncation (after 215th residue, Tra - 215) and substitution mutant proteins (Ser216Ala, Lys217Ala, Gln218Ala, Glu219Ala). Circular Dichroism (CD) analysis of Tra-215 showed deviations in comparison to Tra-WT. Truncation abolished the DNA binding activity and octamer formation as evidenced by the absence of ring like structures from TEM analysis. CD analysis of the substitution mutant proteins showed that the secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Native PAGE and TEM analysis of the substitution mutants showed that Lys217Ala mutation completely abolished the octamer formation as rings and nucleic acid binding. Glu219Ala mutation also affected oligomerization but exhibited marginal RNA binding at higher protein concentrations and interestingly, failed to bind to DNA. However, Ser216Ala and Gln218Ala substitutions did not affect above mentioned activities of translin. Our results indicate that the C-terminal residues are one of the determinants of octamer formation in rice translin, with lysine at 217th position being the most important. Therefore, in conclusion, although the C-terminal residues do not form any defined secondary structure in the translin monomer, they are definitely involved in octamer formation and hence important for its molecular function. We have attempted to find the critical residues in translin function, which will advance our understanding of translin in DNA repair process in general and of rice translin in particular.