Translin-associated Factor X Is Post-transcriptionally Regulated by Its Partner Protein TB-RBP, and Both Are Essential for Normal Cell Proliferation

Abstract

slow cell growth rates. We conclude that TRAX is posttranscriptionally stabilized by TB-RBP and both proteins are needed for normal cell proliferation.The human protein Translin and its mouse orthologue, testis-brain RNA-binding protein (TB-RBP), 1 are single-stranded DNA-and RNA-binding proteins with proposed functions in chromosomal translocations in lymphoid cells and mRNA transport and storage in brain and testis (1-4). TB-RBP is a highly conserved protein with mouse and human proteins differing in 3 of 228 amino acids (5, 6). In in vitro assays, TB-RBP/Translin binds to consensus chromosomal DNA breakpoint junctions as an octameric ring and recognizes DNA breaks at genomic hotspots (2,7,8). TB-RBP also binds to consensus RNA sequences present in many brain and testis mRNAs (6, 9 -11) and links specific mRNAs to microtubules (10,12,13). In the testis TB-RBP forms a complex with the Ter ATPase to transport specific mRNAs from nuclei to cytoplasm and through intercellular bridges (4). This ribonucleoprotein complex contains the kinesin KIF 17b, a testicular motor protein controlling transcription of CREM-regulated male germ cell mRNAs (14). The level of Translin has been demonstrated to closely parallel the proliferative state of somatic cells with its induced overexpression in HEK 293 cells accelerating cell proliferation (15). TRAX, first characterized as a Translin-like protein (16), forms DNA and RNA binding complexes with Translin (17,18). TRAX has been proposed to function in DNA repair in conjunction with the nuclear matrix protein C1D (19).Recently, we generated mice with a functional deletion of the TB-RBP gene (20). The mice are growth retarded and show defects in behavior and fertility. Many of the germ cells in the testis cannot proceed beyond first meiotic metaphase suggesting a defect in chromosome segregation and cytokinesis. Using 14.5-day-old embryos from heterozygous crosses, we have developed lines of MEFs from TB-RBP null animals and from their littermates. As in TB-RBP-deficient mice, these cells lack both TB-RBP and TRAX, although TRAX mRNA levels are not reduced. We find that early passage null MEFs have slower rates of proliferation with a block in the G 2 stage of the cell cycle. Reintroduction of TB-RBP increases the rate of cell proliferation and stabilizes the TRAX protein. Transgenic and transfection experiments indicate that TRAX protein is dependent upon TB-RBP for stabilization and both TB-RBP and TRAX are needed for normal cell proliferation. EXPERIMENTAL PROCEDURES Generation of MEFs-TB-RBP-deficientMEFs were derived from TB-RBP null mice generated by gene trapping (20). MEFs were produced using established protocols (21). Briefly, embryos were removed from pregnant heterozygous mice at 14.5 days and rinsed separately in sterile PBS. Fetal membranes, placenta, head, and soft tissues were discarded, and the remaining tissues were rinsed in fresh PBS. Each embryo was minced and incubated at 37°C for 5 min in 2 ml of 0.25% trypsin/EDTA. Debris was removed by passing the cell susp...

Translin associated protein X is essential for cellular proliferation

“…Compared to control Hela cells, the mRNA levels of the interferon markers in Hela cell lines expressing GFP or TRAX shRNAs only fluctuated slightly ( Table 1). The decreases of MX1, IF144 and IFIT1 mRNAs in cell lines expressing TRAX shRNAs are likely due to the slower growth rate of the cells [11]. The non-induction of the interferon mRNAs indicates that the reduction in TRAX was not part of a global interferon response in the infected cells.…”

“…However, upon reintroduction of TB-RBP, TRAX also began to accumulate. TB-RBP is needed to stabilize TRAX by forming hetero-oligomers because in the absence of TB-RBP, TRAX becomes ubiquitinated and degraded [11]. Thus, the concomitant loss of both TB-RBP and TRAX in TB-RBP deficient mice makes it difficult to relate specific cellular functions to TB-RBP, to TRAX, or to both proteins.…”

“…Embryonic fibroblasts (MEFs) from TB-RBP null mice proliferated more slowly than MEFs isolated from their heterozygous or wild type littermates. Reintroduction of TB-RBP into the null MEFs remedied this defect [11]. However, upon reintroduction of TB-RBP, TRAX also began to accumulate.…”

“…Of 15 lines of Hela cells expressing TRAX shRNAs, 14 out of 15 had a slower growth rate (data not shown). A similar slower growth rate of NIH 3T3 cells was observed when TRAX levels were transiently reduced with TRAX siRNAs [11]. To confirm that the reduction of TRAX expression was due to the specific loss of TRAX mRNAs, we measured TRAX mRNA levels by real-time RT-PCR.…”

“…The CMV-shRNA vector selectively suppresses TRAX expression leading to reduced cell proliferation Previous studies with mice lacking TB-RBP [2] and MEFs (MEFs) derived from TB-RBP null mice revealed that the loss of TB-RBP caused an equivalent loss of its partner protein, TRAX, and the MEFs from null mice showed a slower growth rate [11]. Having established that our intronbased vector works with the exogenous GFP gene, we targeted TRAX in Hela cells with a similar vector expressing TRAX shRNAs to determine whether TRAX deficiency affected cell proliferation.…”