Crystal Structure of TB-RBP, a Novel RNA-binding and Regulating Protein

doi:10.1016/S0022-2836(02)00364-9

Journal of Molecular Biology

Volume 319, Issue 5, 21 June 2002, Pages 1049-1057

https://doi.org/10.1016/S0022-2836(02)00364-9 Get rights and content

Abstract

The testis/brain-RNA-binding protein (TB-RBP) spatially and temporally controls the expression of specific mRNAs in developing male germ cells and brain cells, and is implicated in DNA recombination and repair events. We report the 2.65 Å crystal structure of mouse TB-RBP. The structure is predominantly α-helical and exhibits a novel protein fold and mode of assembly. Crystal symmetry and molecular symmetry combine to form an octet of TB-RBP monomers in the shape of an elongated spherical particle with a large cavity at its center. Amino acid residues that affect RNA and DNA binding are located on the interior surface of the assembled particle, and a putative nucleotide-binding domain that controls RNA binding is located at a dimer interface. Other modes of assembly are suggested for TB-RBP based on our structure and recently reported electron microscopic reconstructions of human TB-RBP.

Introduction

Mammalian spermatogenesis proceeds in stages that require various forms of gene expression.¹ Early stages are frequently controlled at the level of transcription. However, transcription ceases mid-way through spermiogenesis, the haploid phase of spermatogenesis. As a result, gene expression in the later phases of male germ cell development is controlled at the level of mRNA translation. Stored mRNAs are translationally suppressed until the proteins for which they code are developmentally required. This vital process requires the action of proteins that can specifically recognize certain mRNAs and, by binding to them, prevent expression. These silencing proteins must also possess temporal and/or spatial sensors to allow appropriate release of mRNA.

Testis/brain-RNA-binding protein (TB-RBP) was identified initially as a protein able to suppress the translation of stored mRNAs that contained sequences called H and Y elements, of 14 and 15 residues, respectively, in their 3′ untranslated region or UTR.² The gene coding for TB-RBP has been cloned and expressed in bacteria.³ It was found to code for a 228 residue, 26 kDa protein. In addition to exerting an influence on the temporal expression of mRNA, it was discovered that TB-RBP can influence the spatial expression of mRNA. TB-RBP can bind to and link its associated mRNAs to microtubules to facilitate movement within cells.⁴ One example of this spatial control-function occurs during the haploid phases of spermatogenesis. Each spermatid has either an X or a Y chromosome that contains essential genes. Therefore, there is a need to share genetic information between haploid spermatids. TB-RBP has been shown to cross through intercellular bridges between developing haploid spermatids and is thus thought to be involved in maintaining genetic equivalence between spermatids.⁵ TB-RBP appears to act as an adaptor molecule in tubulin-mediated transport of mRNA. It is abundant in brain cells, where it binds to the 3′ UTR of translationally suppressed and transported brain mRNAs.⁶

Translin is the human orthologue of TB-RBP; they are nearly 99% identical in sequence, differing at just three amino acid positions. Translin binds single-stranded DNA (ssDNA) sequences found near breakpoint regions in recombination hot spots.3., 7. Such binding has been implicated in gene translocation events in myeloid leukemia cell lines, where high levels of TB-RBP are found in the nucleus.⁷ The exact nature of the ssDNA binding by TB-RBP/translin is not known but the localization of TB-RBP/translin to the nucleus in pachytene spermatocytes implicates its function in meiotic DNA recombination and repair events.⁸ The ability of TB-RBP, and translin, to bind to mRNA and DNA is mediated by a conserved basic sequence RFHEH, which lies between residues 86 and 90.9., 10.

In accomplishing its varied functions, TB-RBP interacts with mRNA and with a variety of signal molecules and proteins. It now appears that TB-RBP binding of mRNA may be regulated by GTP. It has been shown that a TB-RBP·GDP complex will bind RNA, but binding of GTP releases it. Mutagenic alteration of the guanine nucleotide site prevents RNA binding, presumably because it prevents GDP binding.¹¹ TB-RBP/translin is known to interact with TRAX, a structural homologue that does not bind mRNA.¹² Formation of hetero-oligomers between TB-RBP and TRAX releases bound mRNA.¹⁰ Immunoprecipitation with anti-TB-RBP antibodies precipitated complexes with cytoskeletal γ-actin.¹³ This is consistent with the notion that TB-RBP serves as an adaptor during transport of mRNA through cytosolic bridges between developing male germ cells. Very recently, the Hecht group used immunoprecipitation to demonstrate that TB-RBP associates with a microtubular motor protein that is a KIF isoform (unpublished results). KIF proteins are members of the kinesin family,¹⁴ and this observation suggests strongly that TB-RBP can act as an adapter to link specific mRNAs to a neuronal motor transport system. To date, no high-resolution structure of TB-RBP, translin, or any related protein has been solved. Here, we present the 2.65 Å crystal structure of TB-RBP and describe its novel fold and mode of assembly.

Section snippets

TB-RBP structure determination

Multi-wavelength anomalous diffraction (MAD) data from a methyl mercury derivative crystal provided experimental phases for TB-RBP structure determination (Table 1). There are four TB-RBP monomers in an asymmetric unit, and this 4-fold molecular symmetry was used to modify and improve experimental phases. Electron density maps produced with the modified experimental phases were of high quality and allowed a majority of the atomic model to be constructed (Figure 1(a)). The final model consists

Crystal preparation and data collection

TB-RBP was purified and crystallized as described.¹⁸ The crystals belong to orthorhombic space group P2₁2₁2 with the following cell constants: $a=97.2 A ̊,$ $b=135.5 A ̊,$ $c=92.2 A ̊ .$ Prior to collecting diffraction data, crystals were transferred from artificial mother liquor (0.8 M NH₄SO₄, 100 mM sodium acetate, pH 4.6) to cryo-solution (10% (w/v) PEG 4000, 30% (v/v) glycerol, 100 mM sodium acetate, pH 4.6), and then flash-cooled in liquid N₂. A methyl mercury derivative was prepared by including 0.2 mM

Acknowledgements

This work was supported by grant GM 30048 from the National Institutes of Health, and by grants from the Foundation for Research and the Welch Foundation. We thank A. Monzingo for careful manuscript editing, and T. Langdon and D. Cascio for assistance in synchrotron data collection.

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The single-stranded DNA (ssDNA)/RNA binding protein translin was suggested to be involved in chromosomal translocations, telomere metabolism, and mRNA transport and translation. Oligonucleotide binding surfaces map within a closed cavity of translin octameric barrels, raising the question as to how DNA/RNA gain access to this inner cavity, particularly given that, to date, none of the barrel structures reported hint to an entryway. Here, we argue against a mechanism by which translin octamers may “dissociate and reassemble” upon RNA binding and report a novel “open”-barrel structure of human translin revealing a feasible DNA/RNA entryway into the cavity. Additionally, we report that translin not only is confined to binding of ssDNA oligonucleotides, or single-stranded extensions of double-stranded DNA (dsDNA), but also can bind single-stranded sequences internally embedded in dsDNA molecules.
Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe
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Citation Excerpt :
The 3D structures of the human and mouse Translins were solved by X-ray crystallography at resolutions of 2.65 Å and 2.2 Å, respectively, and were found to be virtually identical [15,16]. The crystals had a tetramer as an asymmetric unit, but an octameric barrel structure is generated by rotation of the tetramer about a 2-fold crystallographic axis [15,16]. The individual Translin subunits consist of 228 amino acids and contain seven α helices that constitute > 70% of the amino acid residues.
Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin–TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin–TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein–protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.
Low-resolution structure of Drosophila translin
2012, FEBS Open Bio
Citation Excerpt :
Likewise, a DNA-binding incompetent P168S mutant of drosophila translin exists as a tetramer, both in solution and in crystals [12]. Currently, crystal structures of human and mouse translin proteins, which share more than 98% sequence identity [13,14] and that of drosophila P168S mutant translin [12] are known. The crystal structures of translin–TRAX heteromeric complexes of human and drosophila have also been resolved recently [15,16].
Crystals of native Drosophila melanogaster translin diffracted to 7 Å resolution. Reductive methylation of the protein improved crystal quality. The native and methylated proteins showed similar profiles in size-exclusion chromatography analyses but the methylated protein displayed reduced DNA-binding activity. Crystals of the methylated protein diffracted to 4.2 Å resolution at BM14 of the ESRF synchrotron. Crystals with 49% solvent content belonged to monoclinic space group P2₁ with eight protomers in the asymmetric unit. Only 2% of low-resolution structures with similar low percentage solvent content were found in the PDB. The crystal structure, solved by molecular replacement method, refined to R_work (R_free) of 0.24 (0.29) with excellent stereochemistry. The crystal structure clearly shows that drosophila protein exists as an octamer, and not as a decamer as expected from gel-filtration elution profiles. The similar octameric quaternary fold in translin orthologs and in translin–TRAX complexes suggests an up-down dimer as the basic structural subunit of translin-like proteins. The drosophila oligomer displays asymmetric assembly and increased radius of gyration that accounts for the observed differences between the elution profiles of human and drosophila proteins on gel-filtration columns. This study demonstrates clearly that low-resolution X-ray structure can be useful in understanding complex biological oligomers.
Translin. TRAX Complex (C3PO), a Novel Ribonuclease for the Degradation of Small RNAs.
2012, Enzymes
C3PO is a heteromeric complex of two evolutionarily related proteins, translin and TRAX. C3PO was recently discovered to possess ribonuclease activity and promote the activation of RNA-induced silencing complex (RISC) in Drosophila and human by degrading the passenger strand fragments of duplex siRNA. C3PO is also involved in tRNA processing in the filamentous fungus Neurospora crassa and degrades the 5′-leader fragment that is cleaved off the precursor tRNA (pre-tRNA) by RNase P. Structural and biochemical studies revealed that active C3PO adopts a football-shaped hetero-octamer configuration containing six translin and two TRAX subunits. The RNA binding and catalytic residues are located at the interface between TRAX and translin subunits facing the hollow interior of C3PO octamer, indicating that C3PO binds and cleaves RNA inside its largely closed barrel. The ability of translin and TRAX to form complexes of different oligomeric states, such as tetramers, hexamers, and octamers, suggests a novel mechanism for the recruitment of RNA substrate and the assembly of active C3PO. Further mechanistic and functional studies are required to fully understand the role of C3PO in a number of biological processes.
Analysis of nucleic acid binding by a recombinant translin-trax complex
2010, Biochemical and Biophysical Research Communications
Translin is a highly conserved mammalian RNA and DNA-binding protein involved in DNA recombination and RNA trafficking. Crystal structures of mouse and human translin have been solved, but do not provide information about nucleic acid binding or recognition. Translin has a partner protein, translin-associated factor x (trax), which is believed to regulate translin’s subcellular locale and affinity for certain RNA and DNA sequences. Here we present a comparative study of recombinant translin and translin–trax complex binding to specific RNA and DNA sequences. It was observed that translin preferentially binds to G-rich RNA sequences whereas translin–trax preferentially binds G-rich DNA sequences. Translin can bind mRNA sequences with sub-micromolar K_d values, and the complex with trax can bind G-rich DNA with similar affinity. We conclude that trax acts to regulate translin’s RNA and DNA binding affinities as part of a cellular RNA trafficking mechanism.
Expression pattern of Drosophila translin and behavioral analyses of the mutant
2007, European Journal of Cell Biology
Translin is an evolutionarily conserved ∼27-kDa protein that binds to specific DNA and RNA sequences and has diverse cellular functions. Here, we report the cloning and characterization of the translin orthologue from the fruit fly Drosophila melanogaster. Under protein-denaturing conditions, purified Drosophila translin exists as a mixture of dimers and monomers just like human translin. In contrast to human translin, the Drosophila translin dimers do not appear to be stabilized by disulfide interactions. Drosophila translin shows a ubiquitous cytoplasmic localization in early embryonal syncytial stage, with an enhanced staining in ventral neuroblasts at later stages (8–9), which are probably at metaphase. An elevated expression was seen in several other cell types, such as cells around the tracheal pits in the embryo and oenocytes in the third instar larva. RNA in situ hybridization showed an increased expression in the ventral midline cells of the larval brain, suggesting a neuronal expression, which was corroborated by protein immunostaining. In adult flies, Drosophila translin is localized in the brain neuronal cell bodies and in early spermatocytes. Interestingly, Drosophila translin mutants exhibit an impaired motor response which is sex specific. Taken together, the multiple cellular localizations, the high neuronal expression and the attendant locomotor defect of the Drosophila translin mutant suggest that Drosophila translin may have roles in neuronal development and behavior analogous to that of mouse translin.

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Journal of Molecular Biology

Crystal Structure of TB-RBP, a Novel RNA-binding and Regulating Protein

Abstract

Introduction

Section snippets

TB-RBP structure determination

Crystal preparation and data collection

Acknowledgements

Dev. Biol.

FEBS Letters

J. Biol. Chem.

FEBS Letters

J. Struct. Biol.

Methods Enzymol.

Molecular mechanisms of male germ cell differentiation

BioEssays

Cytoplasmic protein binding to highly conserved sequences in the 3′ untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

Proc. Natl Acad. Sci. USA

The RNA-binding protein, TB-RBP, is the mouse homologue of translin, a recombination protein associated with chromosomal translocations

Proc. Natl Acad. Sci. USA

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules

Proc. Natl Acad. Sci. USA

Testis-brain RNA-binding protein, a testicular translational regulatory RNA-binding protein, is present in the brain and binds to the 3′ untranslated regions of transported brain mRNAs

Biol. Reprod.

A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocations

Nature Genet.

Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP)

Mol. Reprod. Dev.

Altering the GTP binding domain of the DNA/RNA-binding protein, translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype

Nucl. Acids Res.

Protein–protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain

Biochemistry