Abstract
Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP.
Highlights
Spermatogenesis is the dynamic developmental process in the testis where the male gamete differentiates into spermatozoa
Translin-associated factor X (TRAX) was identified as a protein that interacts with Testis brain RNA-binding protein (TB-RBP) and is dependent upon TB-RBP for stabilization
Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal
Summary
Spermatogenesis is the dynamic developmental process in the testis where the male gamete differentiates into spermatozoa. One of the nucleic acid-binding proteins, testis brain RNAbinding protein (TB-RBP), the mouse orthologue of human translin [7,8,9], has emerged as a highly versatile molecule. As a DNA-binding protein, TB-RBP/ translin has been proposed to bind to breakpoint junctions of chromosomal translocations [7,8,9, 15,16,17] and regulate cell proliferation rates [18, 19]. The leucine zipper domain of TRAX is essential for interactions with TBRBP and with C1D, an activator of the DNA-dependent protein kinase involved in the repair of DNA-double strand breaks and V(D)J recombination [22, 23]. The official nomenclature for the mouse TRAX gene is Tsnax
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