- Email: [email protected]
Testis–brain RNA-binding protein (Translin) is primarily expressed in neurons of the mouse brain
Brain Research 819 Ž1999. 174–178
Short communication
Testis–brain RNA-binding protein ŽTranslin . is primarily expressed in neurons of the mouse brain Xin Qi Wu a , Peter Petrusz b , Norman B. Hecht a
a, )
Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology, UniÕersity of PennsylÕania Medical Center, 752b Clinical Research Building, 415 Curie BouleÕard, Philadelphia, PA 19104, USA b Department of Cell Biology and Anatomy, UniÕersity of North Carolina School of Medicine, Chapel Hill, NC 27599-7090, USA Accepted 8 December 1998
Abstract The subcellular locationŽs. of the DNA- and RNA-binding protein, Testis–Brain RNA-Binding Protein ŽTB-RBP.rTranslin in mouse brain has been determined in paraffin sections by immunocytochemistry with an affinity purified antibody to mouse recombinant TB-RBP. Nuclear staining was frequently seen in neurons throughout the brain, but no TB-RBPrTranslin was detected in many of the neurons in superficial layers of the cerebral cortex and in some cells of the cerebellum. Cytoplasmic staining extending into the dendrites was seen in large neurons such as pyramidal neurons in Layer 5 of the cortex and magnocellular neurons of the hypothalamus or the brainstem raphe. q 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA-binding protein; RNA-binding protein; Brain
In the mammalian testis, the RNA-binding protein Testis–Brain RNA-Binding Protein ŽTB-RBP. binds to highly conserved sequences ŽY and H elements. present in the 3X untranslated regions of a number of ‘paternal’ stored mRNAs including the protamines w7,8x. TB-RBP represses cell-free translation of mRNA constructs containing these conserved sequences. In in vitro microtubule reconstitution assays, TB-RBP binds specific testicular and brain mRNAs including myelin basic protein mRNA to microtubules w3,4x. The conserved Y and H sequences are present in the mRNAs of many other genes expressed in the brain w3x. Recently, TB-RBP was isolated and cloned and shown to be the 26 kDa mouse homologue of the human protein Translin w16x, a protein shown to bind to single-stranded DNA sequences present at breakpoint junctions of chromosomal translocations w1,6x. These findings suggest that TB-RBP has distinct nuclear and cytoplasmic functions in cells. RNA-binding assays with cytosolic extracts from diverse tissues revealed high levels of TB-RBP binding activity in mouse brain extracts w3x. Here we use Western
) Corresponding author. [email protected]
Fax:
q 1-215-573-5408;
E-mail:
blotting to confirm the presence of high levels of TB-RBP protein and employ immunocytochemistry to localize TBRBP in paraffin sections of mouse brain. We conclude that TB-RBP is primarily present in the nuclei of neurons and is not readily detectable in paraffin sections in glial cells. In the cytoplasm, TB-RBP appears frequently restricted to a perinuclear region. However, in large neurons, TB-RBP staining extends into the processes. Cytosolic extracts from brain and testes were prepared as described previously w3,4,16x. For immunoprecipitations, extracts Ž1 mg. were incubated with antibody Ž4 ug. and Protein A agarose beads Ž10 mg. in 400 ml TBS containing 0.1% NP-40 for 4 h at 48C. Two antibodies, Ab-KNDS, a non-precipitating antibody prepared against a 16-amino-acid peptide from TB-RBP or a precipitating antibody to recombinant mouse TB-RBP, Ab-TB-RBP, were used w16x. The agarose beads were collected by centrifugation at 2000 rpm for 2 min and washed 4 times with 1 ml TBS containing 0.1% NP-40. The proteins were released from the beads by boiling in SDS-PAGE loading buffer Ž20 ml. for 3 min. For Western blotting, cytosolic extracts Ž30 mg. or the antibody immunoprecipitates were electrophoresed on 10% SDS polyacrylamide gel. The proteins were transferred from the gels onto nitrocellulose membranes. The membranes were blocked overnight at
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 2 2 - 5
X.Q. Wu et al.r Brain Research 819 (1999) 174–178
Fig. 1. Immunoprecipitation and Western blot analysis of TB-RBP. Lane 1: cytosolic extract Ž30 ug. of testis; Lane 2: cytosolic extract Ž30 ug. of brain. Lanes 3 and 4: immunoprecipitation of testicular cytosolic extract with Ab-TB-RBP and Ab-KNDS, respectively. Lanes 5 and 6: immunoprecipitation of brain cytosolic extract with Ab-TB-RBP and Ab-KNDS, respectively. Proteins were resolved on a 10% SDS polyacrylamide gel.
48C in TBS containing 5% nonfat milk and then incubated with Ab-TB-RBP Ž1:1000. in TBS containing 0.25% nonfat milk for 1 h at RT. After washing 3 times Ž10 min each., the membranes were incubated with Protein A linked with horseradish peroxidase Ž1:5000. for 1 h at RT. The membranes were washed 3 times Ž10 min each. and TB-RBP was detected with the ECL system ŽAmersham.. Adult male and female mice were obtained from a colony at the University of North Carolina derived from mating CD1 females ŽCharles River. with C57BLr6J= DBAr2J males ŽJackson.. For immunolocalization, animals were deeply anesthetized with ether and perfused transcardially with a fixative of 4% paraformaldehyde in 0.1 M, pH 7.4 phosphate buffer. Tissues were post-fixed by immersion in the same fixative overnight at 48C, washed for 2 days in several changes of 0.01 M phosphate-buffered saline ŽPBS. pH 7.4, embedded in paraffin, sectioned serially at 8 mm and mounted on standard microscope slides. For immunostaining, the sections were deparaffinized and stained according to the ‘double PAP’ immunoperoxidase method as described by Ordronneau et al. w12x with diaminobenzidine ŽAldrich. as chromogen. Toluidine blue was used as a counterstain. The anti-TB-RBP antibodies were raised in rabbits as previously described w16x, affinity purified, and used at a working dilution of 1:1000. Sheep anti-rabbit IgG and rabbit PAP were purchased from Arnel Chemical ŽNew York, NY.. Controls included progressive dilutions of the primary antibody, staining with antibody pre-adsorbed with the purified antigen, and staining testis sections as positive controls. Based upon a number of biochemical assays including specific RNA-binding, UV cross-linking, and V8 digestion
175
of UV cross-linked protein–RNA complexes, TB-RBP appears to be identical in testis and brain w3,4x. Here we show by Western blotting with an affinity purified antibody to recombinant TB-RBP, TB-RBP is present in both testis and brain extracts ŽFig. 1, lanes 1 and 2.. Moreover, when TB-RBP is specifically immunoprecipitated from testis and brain extracts, TB-RBP is detected in the pellets ŽFig. 2, lanes 3 and 5.. When a control non-precipitating antibody prepared against a 16 amino acid peptide of mouse TB-RBP is used, no TB-RBP is detected in the precipitates ŽFig. 1, lanes 4 and 6.. To define the locationŽs. of TB-RBP in the brain of adult mice, immunostaining was performed with the same affinity purified antibody against recombinant mouse TBRBP. Dilution of the primary antibody resulted in a gradual reduction of staining intensity. At a dilution of 1:100 000, no staining could be discerned. Incubation of the primary antibody with recombinant TB-RBP Ž10 mgrml. abolished all staining. Sections of testes stained with the antibody diluted 1:1000 stained intensely as described earlier for Western blotting of testes extracts w16x. The anti-TB-RBP serum stained nuclei of neurons throughout the brain. In most areas, all identifiable neuronal nuclei were stained, albeit with variable intensity ŽFig. 2A.. However, certain nuclei appeared unstained, including those in the superficial layers of the cerebral cortex ŽFig. 2A. and most of the nuclei in Purkinje cells of the cerebellum. Cytoplasmic staining was also variable in intensity and was mostly confined to the perikaryon ŽFig. 2B.. In some of the largest neurons, such as pyramidal neurons in the cortex, and magnocellular neurons of the hypothalamus or the brainstem raphe, and motoneurons of the spinal cord, the cytoplasmic staining was more diffuse and extended into dendrites ŽFig. 2C and D.. In some larger neurons, an unusual concentric pattern of staining was seen: darkly stained nuclei were surrounded by a lighter area, which, in turn, was surrounded by a darker area showing course granular staining ŽFig. 2C.. Staining was not detected in glial cells, in white matter Že.g., Fig. 2B. or in the ependyma lining the brain ventricles, vascular smooth muscle and endothelium. We conclude from our selective detection of TB-RBP in brain cells that it is differentially expressed although epitope masking cannot be totally excluded. The human protein Translin was initially characterized as a DNA-binding protein which specifically binds to
Fig. 2. ŽA. Low-power view of portions of the cerebral cortex and hippocampus demonstrating the distribution of immunoreactive TB-RBP. Labeling Žbrown. is localized largely to neuronal perikarya. However, many unstained neurons Žblue. are present in superficial layers of the cortex Ž).. Magnification: 16 = . ŽB. Higher-power view of a portion of the hippocampus ŽCA1; lower left., the corpus callosum Ž)., and Layer VI of the cerebral cortex. Note the lack of immunostaining in the white matter of the corpus callosum, including glial cell nuclei Žarrowheads.. Nuclear staining of variable intensity is seen in hippocampal and cortical neurons. Magnification: 40 = . ŽC. Ventral horn of the spinal cord. Most, if not all neurons are stained, note peculiar circular pattern of labeling in some of the larger perikarya and lack of staining in glial cells. Magnification: 40 = . ŽD. Higher-power view of the ventral horn of the spinal cord. Note strong labeling in large motoneuron and the lighter but distinct labeling in its proximal dendrites Žarrowheads.. Magnification: 100 = .
176
X.Q. Wu et al.r Brain Research 819 (1999) 174–178
X.Q. Wu et al.r Brain Research 819 (1999) 174–178
consensus sequences at breakpoint junctions of chromosomal translocations in lymphoid malignancies w1x. Although nuclear localization of Translin was limited to lymphoid lineage cells, it was also detected in the cytosol of nonlymphoid, lymphoid, and non-hematopoetic cells. The mouse homologue of Translin, TB-RBP, was initially identified as a RNA-binding protein that recognizes conserved sequences in the 3X untranslated regions of stored male germ cell mRNAs w7,8x. Additional studies revealed TBRBP binds specific mRNAs to microtubules w4x. Following its cloning and expression, TB-RBP was demonstrated to bind to the same single stranded DNA sequences as does its nearly identical human homologue, Translin w16x. With the affinity purified antibody to TB-RBP used in these studies, immunolocalization analyses have demonstrated TB-RBP to be present in the nuclei of male germ cells undergoing meiosis and in the cytoplasm of post-meiotic male germ cells where large amounts of ‘paternal’ mRNAs are stored before translation w2,5,9x. The abundance of TB-RBP in intercellular bridges of male germ cells suggests TB-RBP is involved in mRNA transport between cells in the testis as well as between cellular compartments w9x. What might be the function Žs. of TB-RBPrTranslin in the adult mouse brain? Here we show that TB-RBP is primarily in the nuclei of neurons as well as in dendrites, arguing for both nuclear and cytoplasmic functions in the brain. Taira et al. w14x have identified Translin as a component of a single stranded DNA binding complex in rat brain which they propose has a role in DNA repair. Muramatsu et al. w10x report Translin binds to BC1 RNA, a neural RNA which is expressed in neuronal dendrites as ribonucleoprotein particles. As expected, Translin binds to two conserved binding sequences in BC1 RNA that are similar to the binding sites of translationally regulated testicular mRNAs and brain mRNAs that are attached to microtubules by TB-RBP. Recent studies with cultured hippocampal neurons argue for a role for TB-RBP in the spatial localization of mRNAs in neurons w13x. When the ribonucleoprotein complexes of rat hippocampal cells are disrupted with antisense oligodeoxynucleotides, two regulatory proteins, TB-RBP and a kinase heavy chain, have been identified. Inactivation of TB-RBP causes the mislocalization of mRNAs encoding a-calmodulin dependent kinase II and ligatin, whereas suppression of the kinase heavy chain motor protein selectively alters localization of the a-calmodulin dependent kinase II mRNA. These studies suggest TB-RBP also functions in mRNA transport in hippocampal cells. In the brain as in the testis, TB-RBP appears to be a multifunctional protein. The presence of TB-RBP in many, but not all, brain cells suggests neuron-specific aspects of post-transcriptional RNA regulation for this 26 kDa protein in the brain as has been reported for the Hu gene family of neuron-specific RNA-binding proteins w11x. Similarly, three related RNA-binding proteins FMR1, FXR1,
177
and FXR2 are coexpressed in the cytoplasm of specific differentiated neurons, but show a differential expression during brain development w15x. We propose that in the nuclei of specific neurons and in male germ cells during meiosis, TB-RBP functions in maintaining DNA integrity andror in binding to specific mRNAs for transport to the cytoplasm. Once in the cytoplasm, TB-RBP helps store, transport and localize specific mRNAs, often in translationally inactive forms.
Acknowledgements This research was supported by HD 28832.
References w1x K. Aoki, K. Suzuki, T. Sugano, K. Nakahara, O. Kuge, A. Omori, M. Kasai, A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocations, Nat. Genet. 10 Ž1995. 167–174. w2x W. Gu, X.-Q. Wu, X.H. Meng, M. El-Alfy, C. Morales, N.B. Hecht, The RNA- and DNA-binding protein TB-RBP, is spatially and developmentally regulated during spermatogenesis, Mol. Reprod. Dev. 49 Ž1998. 219–228. w3x J. Han, W. Gu, N.B. Hecht, TB-RBP, a testicular translational regulatory RNA-binding protein that is abundant in the brain and X binds to the 3 UTR of Tau mRNA, a transported brain mRNA, Biol. Reprod. 53 Ž1995. 707–717. w4x J. Han, G. Yiu, N.B. Hecht, Testis–brain RNA-binding protein ŽTB-RBP. is a microtubule associated protein that attaches translationally repressed and transported mRNAs to microtubules, Proc. Natl. Acad. Sci. USA 92 Ž1995. 9550–9554. w5x N.B. Hecht, Molecular basis of male germ cell differentiation, BioEssays 20 Ž1998. 555–561. w6x M. Kasai, T. Matsuzaki, K. Katayanagi, A. Omori, R.T. Maziarz, J.L. Strominger, K. Aoki, K. Suzuki, The Translin ring specifically recognizes DNA ends at recombination hot spots in the human genome, J. Biol. Chem. 272 Ž1997. 11402–11407. w7x Y.K. Kwon, N.B. Hecht, Cytoplasmic protein binding to highly X conserved sequences in the 3 untranslated region of mouse protamine 2 mRNA, a translationally regulated gene of male germ cells, Proc. Natl. Acad. Sci. USA 88 Ž1991. 3584–3588. w8x Y.K. Kwon, N.B. Hecht, Binding of a phosphoprotein to the 3X untranslated region of the mouse protamine 2 mRNA temporally represses its translation, Mol. Cell Biol. 13 Ž1993. 6547–6557. w9x C.R. Morales, X.-Q. Wu, N.B. Hecht, The DNArRNA-Binding Protein, TB-RBP, moves from the nucleus to the cytoplasm and through intercellular bridges in male germ cells, Dev. Biol. 201 Ž1998. 113–123. w10x T. Muramatsu, A. Ohmae, K. Anzai, BC1 RNA protein particles in mouse brain contain two Y–H-element-binding proteins, Translin and a 37 kDa protein, Biochem. Biophys. Res. Commun. 247 Ž1998. 7–11. w11x H. Okano, R. Darnell, A hierarchy of Hu RNA binding proteins in developing and adult neurons, J. Neurol. 17 Ž1997. 3024–3037. w12x P. Ordronneau, P.B.M. Lindstrom, P. Petrusz, Four unlabeled antibody bridge techniques: a comparison, J. Histochem. Cytochem. 29 Ž1981. 1397–1404. w13x W. Severt, T. Biber., X.-Q. Wu, N.B. Hecht, R. DeLorenzo, E.
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Jakoi, Sorting of messenger RNAs to dendritic synapses involves the testis–brain RNA binding-protein and kinesin heavy chain, submitted. w14x E. Taira, P.M. Finkenstadt, J.M. Baraban, Identification of Translin and Trax as components of the GS1 strand-specific DNA binding complex enriched in brain, J. Neurochem. 71 Ž1998. 471–477. w15x F. Tamanini, R. Willemsen, L. van Unen, C. Bontekoe, H. Galjaard,
B. Oostra, A. Hoogevenn, Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and testis, Hum. Mol. Genet. 6 Ž1997. 1315–1322. w16x X.-Q. Wu, W. Gu, X.-H. Meng, N.B. Hecht, The RNA-binding protein TB-RBP is the mouse homologue of Translin, a recombination protein associated with chromosomal translocations, Proc. Natl. Acad. Sci. USA 94 Ž1997. 5640–5645.
Short communication
Testis–brain RNA-binding protein ŽTranslin . is primarily expressed in neurons of the mouse brain Xin Qi Wu a , Peter Petrusz b , Norman B. Hecht a
a, )
Center for Research on Reproduction and Women’s Health and Department of Obstetrics and Gynecology, UniÕersity of PennsylÕania Medical Center, 752b Clinical Research Building, 415 Curie BouleÕard, Philadelphia, PA 19104, USA b Department of Cell Biology and Anatomy, UniÕersity of North Carolina School of Medicine, Chapel Hill, NC 27599-7090, USA Accepted 8 December 1998
Abstract The subcellular locationŽs. of the DNA- and RNA-binding protein, Testis–Brain RNA-Binding Protein ŽTB-RBP.rTranslin in mouse brain has been determined in paraffin sections by immunocytochemistry with an affinity purified antibody to mouse recombinant TB-RBP. Nuclear staining was frequently seen in neurons throughout the brain, but no TB-RBPrTranslin was detected in many of the neurons in superficial layers of the cerebral cortex and in some cells of the cerebellum. Cytoplasmic staining extending into the dendrites was seen in large neurons such as pyramidal neurons in Layer 5 of the cortex and magnocellular neurons of the hypothalamus or the brainstem raphe. q 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA-binding protein; RNA-binding protein; Brain
In the mammalian testis, the RNA-binding protein Testis–Brain RNA-Binding Protein ŽTB-RBP. binds to highly conserved sequences ŽY and H elements. present in the 3X untranslated regions of a number of ‘paternal’ stored mRNAs including the protamines w7,8x. TB-RBP represses cell-free translation of mRNA constructs containing these conserved sequences. In in vitro microtubule reconstitution assays, TB-RBP binds specific testicular and brain mRNAs including myelin basic protein mRNA to microtubules w3,4x. The conserved Y and H sequences are present in the mRNAs of many other genes expressed in the brain w3x. Recently, TB-RBP was isolated and cloned and shown to be the 26 kDa mouse homologue of the human protein Translin w16x, a protein shown to bind to single-stranded DNA sequences present at breakpoint junctions of chromosomal translocations w1,6x. These findings suggest that TB-RBP has distinct nuclear and cytoplasmic functions in cells. RNA-binding assays with cytosolic extracts from diverse tissues revealed high levels of TB-RBP binding activity in mouse brain extracts w3x. Here we use Western
) Corresponding author. [email protected]
Fax:
q 1-215-573-5408;
E-mail:
blotting to confirm the presence of high levels of TB-RBP protein and employ immunocytochemistry to localize TBRBP in paraffin sections of mouse brain. We conclude that TB-RBP is primarily present in the nuclei of neurons and is not readily detectable in paraffin sections in glial cells. In the cytoplasm, TB-RBP appears frequently restricted to a perinuclear region. However, in large neurons, TB-RBP staining extends into the processes. Cytosolic extracts from brain and testes were prepared as described previously w3,4,16x. For immunoprecipitations, extracts Ž1 mg. were incubated with antibody Ž4 ug. and Protein A agarose beads Ž10 mg. in 400 ml TBS containing 0.1% NP-40 for 4 h at 48C. Two antibodies, Ab-KNDS, a non-precipitating antibody prepared against a 16-amino-acid peptide from TB-RBP or a precipitating antibody to recombinant mouse TB-RBP, Ab-TB-RBP, were used w16x. The agarose beads were collected by centrifugation at 2000 rpm for 2 min and washed 4 times with 1 ml TBS containing 0.1% NP-40. The proteins were released from the beads by boiling in SDS-PAGE loading buffer Ž20 ml. for 3 min. For Western blotting, cytosolic extracts Ž30 mg. or the antibody immunoprecipitates were electrophoresed on 10% SDS polyacrylamide gel. The proteins were transferred from the gels onto nitrocellulose membranes. The membranes were blocked overnight at
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 2 2 - 5
X.Q. Wu et al.r Brain Research 819 (1999) 174–178
Fig. 1. Immunoprecipitation and Western blot analysis of TB-RBP. Lane 1: cytosolic extract Ž30 ug. of testis; Lane 2: cytosolic extract Ž30 ug. of brain. Lanes 3 and 4: immunoprecipitation of testicular cytosolic extract with Ab-TB-RBP and Ab-KNDS, respectively. Lanes 5 and 6: immunoprecipitation of brain cytosolic extract with Ab-TB-RBP and Ab-KNDS, respectively. Proteins were resolved on a 10% SDS polyacrylamide gel.
48C in TBS containing 5% nonfat milk and then incubated with Ab-TB-RBP Ž1:1000. in TBS containing 0.25% nonfat milk for 1 h at RT. After washing 3 times Ž10 min each., the membranes were incubated with Protein A linked with horseradish peroxidase Ž1:5000. for 1 h at RT. The membranes were washed 3 times Ž10 min each. and TB-RBP was detected with the ECL system ŽAmersham.. Adult male and female mice were obtained from a colony at the University of North Carolina derived from mating CD1 females ŽCharles River. with C57BLr6J= DBAr2J males ŽJackson.. For immunolocalization, animals were deeply anesthetized with ether and perfused transcardially with a fixative of 4% paraformaldehyde in 0.1 M, pH 7.4 phosphate buffer. Tissues were post-fixed by immersion in the same fixative overnight at 48C, washed for 2 days in several changes of 0.01 M phosphate-buffered saline ŽPBS. pH 7.4, embedded in paraffin, sectioned serially at 8 mm and mounted on standard microscope slides. For immunostaining, the sections were deparaffinized and stained according to the ‘double PAP’ immunoperoxidase method as described by Ordronneau et al. w12x with diaminobenzidine ŽAldrich. as chromogen. Toluidine blue was used as a counterstain. The anti-TB-RBP antibodies were raised in rabbits as previously described w16x, affinity purified, and used at a working dilution of 1:1000. Sheep anti-rabbit IgG and rabbit PAP were purchased from Arnel Chemical ŽNew York, NY.. Controls included progressive dilutions of the primary antibody, staining with antibody pre-adsorbed with the purified antigen, and staining testis sections as positive controls. Based upon a number of biochemical assays including specific RNA-binding, UV cross-linking, and V8 digestion
175
of UV cross-linked protein–RNA complexes, TB-RBP appears to be identical in testis and brain w3,4x. Here we show by Western blotting with an affinity purified antibody to recombinant TB-RBP, TB-RBP is present in both testis and brain extracts ŽFig. 1, lanes 1 and 2.. Moreover, when TB-RBP is specifically immunoprecipitated from testis and brain extracts, TB-RBP is detected in the pellets ŽFig. 2, lanes 3 and 5.. When a control non-precipitating antibody prepared against a 16 amino acid peptide of mouse TB-RBP is used, no TB-RBP is detected in the precipitates ŽFig. 1, lanes 4 and 6.. To define the locationŽs. of TB-RBP in the brain of adult mice, immunostaining was performed with the same affinity purified antibody against recombinant mouse TBRBP. Dilution of the primary antibody resulted in a gradual reduction of staining intensity. At a dilution of 1:100 000, no staining could be discerned. Incubation of the primary antibody with recombinant TB-RBP Ž10 mgrml. abolished all staining. Sections of testes stained with the antibody diluted 1:1000 stained intensely as described earlier for Western blotting of testes extracts w16x. The anti-TB-RBP serum stained nuclei of neurons throughout the brain. In most areas, all identifiable neuronal nuclei were stained, albeit with variable intensity ŽFig. 2A.. However, certain nuclei appeared unstained, including those in the superficial layers of the cerebral cortex ŽFig. 2A. and most of the nuclei in Purkinje cells of the cerebellum. Cytoplasmic staining was also variable in intensity and was mostly confined to the perikaryon ŽFig. 2B.. In some of the largest neurons, such as pyramidal neurons in the cortex, and magnocellular neurons of the hypothalamus or the brainstem raphe, and motoneurons of the spinal cord, the cytoplasmic staining was more diffuse and extended into dendrites ŽFig. 2C and D.. In some larger neurons, an unusual concentric pattern of staining was seen: darkly stained nuclei were surrounded by a lighter area, which, in turn, was surrounded by a darker area showing course granular staining ŽFig. 2C.. Staining was not detected in glial cells, in white matter Že.g., Fig. 2B. or in the ependyma lining the brain ventricles, vascular smooth muscle and endothelium. We conclude from our selective detection of TB-RBP in brain cells that it is differentially expressed although epitope masking cannot be totally excluded. The human protein Translin was initially characterized as a DNA-binding protein which specifically binds to
Fig. 2. ŽA. Low-power view of portions of the cerebral cortex and hippocampus demonstrating the distribution of immunoreactive TB-RBP. Labeling Žbrown. is localized largely to neuronal perikarya. However, many unstained neurons Žblue. are present in superficial layers of the cortex Ž).. Magnification: 16 = . ŽB. Higher-power view of a portion of the hippocampus ŽCA1; lower left., the corpus callosum Ž)., and Layer VI of the cerebral cortex. Note the lack of immunostaining in the white matter of the corpus callosum, including glial cell nuclei Žarrowheads.. Nuclear staining of variable intensity is seen in hippocampal and cortical neurons. Magnification: 40 = . ŽC. Ventral horn of the spinal cord. Most, if not all neurons are stained, note peculiar circular pattern of labeling in some of the larger perikarya and lack of staining in glial cells. Magnification: 40 = . ŽD. Higher-power view of the ventral horn of the spinal cord. Note strong labeling in large motoneuron and the lighter but distinct labeling in its proximal dendrites Žarrowheads.. Magnification: 100 = .
176
X.Q. Wu et al.r Brain Research 819 (1999) 174–178
X.Q. Wu et al.r Brain Research 819 (1999) 174–178
consensus sequences at breakpoint junctions of chromosomal translocations in lymphoid malignancies w1x. Although nuclear localization of Translin was limited to lymphoid lineage cells, it was also detected in the cytosol of nonlymphoid, lymphoid, and non-hematopoetic cells. The mouse homologue of Translin, TB-RBP, was initially identified as a RNA-binding protein that recognizes conserved sequences in the 3X untranslated regions of stored male germ cell mRNAs w7,8x. Additional studies revealed TBRBP binds specific mRNAs to microtubules w4x. Following its cloning and expression, TB-RBP was demonstrated to bind to the same single stranded DNA sequences as does its nearly identical human homologue, Translin w16x. With the affinity purified antibody to TB-RBP used in these studies, immunolocalization analyses have demonstrated TB-RBP to be present in the nuclei of male germ cells undergoing meiosis and in the cytoplasm of post-meiotic male germ cells where large amounts of ‘paternal’ mRNAs are stored before translation w2,5,9x. The abundance of TB-RBP in intercellular bridges of male germ cells suggests TB-RBP is involved in mRNA transport between cells in the testis as well as between cellular compartments w9x. What might be the function Žs. of TB-RBPrTranslin in the adult mouse brain? Here we show that TB-RBP is primarily in the nuclei of neurons as well as in dendrites, arguing for both nuclear and cytoplasmic functions in the brain. Taira et al. w14x have identified Translin as a component of a single stranded DNA binding complex in rat brain which they propose has a role in DNA repair. Muramatsu et al. w10x report Translin binds to BC1 RNA, a neural RNA which is expressed in neuronal dendrites as ribonucleoprotein particles. As expected, Translin binds to two conserved binding sequences in BC1 RNA that are similar to the binding sites of translationally regulated testicular mRNAs and brain mRNAs that are attached to microtubules by TB-RBP. Recent studies with cultured hippocampal neurons argue for a role for TB-RBP in the spatial localization of mRNAs in neurons w13x. When the ribonucleoprotein complexes of rat hippocampal cells are disrupted with antisense oligodeoxynucleotides, two regulatory proteins, TB-RBP and a kinase heavy chain, have been identified. Inactivation of TB-RBP causes the mislocalization of mRNAs encoding a-calmodulin dependent kinase II and ligatin, whereas suppression of the kinase heavy chain motor protein selectively alters localization of the a-calmodulin dependent kinase II mRNA. These studies suggest TB-RBP also functions in mRNA transport in hippocampal cells. In the brain as in the testis, TB-RBP appears to be a multifunctional protein. The presence of TB-RBP in many, but not all, brain cells suggests neuron-specific aspects of post-transcriptional RNA regulation for this 26 kDa protein in the brain as has been reported for the Hu gene family of neuron-specific RNA-binding proteins w11x. Similarly, three related RNA-binding proteins FMR1, FXR1,
177
and FXR2 are coexpressed in the cytoplasm of specific differentiated neurons, but show a differential expression during brain development w15x. We propose that in the nuclei of specific neurons and in male germ cells during meiosis, TB-RBP functions in maintaining DNA integrity andror in binding to specific mRNAs for transport to the cytoplasm. Once in the cytoplasm, TB-RBP helps store, transport and localize specific mRNAs, often in translationally inactive forms.
Acknowledgements This research was supported by HD 28832.
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