We have previously shown that depletion of THOC5/FMIP by siRNA or ectopic expression causes abnormal hematopoiesis and abnormal adipocyte differentiation in myloid progenitor or mesenchymal progenitor cell lines [14
]. Furthermore, although THO complex does not play a role in nuclear export of bulk mRNA, THOC1 deletion causes embryonic lethality [13
]. This led us to investigate the potential roles for THOC5/FMIP in vivo
. To do this we first generated conventional knockout mice, deleting THOC5/FMIP. These mice died at an early embryonic stage. Furthermore, although heterozygotic THOC5/FMIP deletion mutant mice were fertile and did not display any phenotypic or histological abnormalities after birth, 50% of THOC5/FMIP heterozygotes died during embryogenesis (El Bounkari and Tamura, unpublished data), suggesting that the level of expression of THOC5/FMIP plays a key role in embryo development.
When an inducible system was developed a key developmental system, hematopoiesis, was found to be sensitive to depletion of THOC5/FMIP. Our data showed that bone marrow cells and spleen cells, but no other organs, such as liver, heart, kidney, intestine, testicle, or lung, became apoptotic in THOC5/FMIP deficient adult mice, suggesting hematopoietic required THOC5/FMIP. In addition, transplantation with bone marrow cells derived from cre minus THOC5/FMIP(flox/flox) mice rescues poly (I:C) injected Mx-cre THOC5/FMIP(flox/flox) mice from death. This extends back to the primitive cells in the hematopoietic system, since THOC5/FMIP depleted mice showed a major decrease in progenitor cells (GEMM-CFU, GM-CFU, Figure ) and also a fall in the slowly cycling bone marrow LSK cells (Figure ). The effects of an RNA processing protein on stem cells were also profound and this we argue in part explains the massive effect on the committed progenitor cell compartment compared to LSK cells. Nonetheless, the rapid onset of the effect also argues that the progenitor cell compartment is sensitive to loss of FMIP/THOC5 expression.
Mx-cre directed depletion by poly I:C injection was observed in bone marrow cells, heart, kidney, liver and spleen [21
]. Indeed, THOC5/FMIP specific PCR analyses using genomic DNA as a template revealed that exons IV/V were deleted from these organs, including spleen after only one × times poly (I:C) injection. We did not observe the depletion of THOC5/FMIP protein in spleen for any time period however, the size of spleen is drastically reduced after seven days poly (I:C) injection, suggesting that apoptotic cells are released from spleen and that the THOC5/FMIP protein half life in certain cells was longer than in other cells.
The issue remains how and why THOC5/FMIP is required for primitive hematopoietic as well as embryo stem cell survival and/or proliferation. Our data suggest that the THO complex is required for mRNA export of particular genes that play a key role in hematopoietic primitive (multipotent) and committed progenitor cell survival and/or proliferation. Alternatively, the THO complex may be essential for bulk poly (A)+ RNA export in primitive cells. The yeast TREX complex is composed of the THO transcription elongation complex (Hpr1 (the THOC1 orthologue), Tho2 (the THOC2 orthologue), Mht1 and Thp2), Tex1 (the THOC3 orthologue), Sub2 (the UAP56 orthologue) and Yra1 (the Aly/THOC4 orthologue) [30
]. Yeast TREX mutants show a nuclear export defect for bulk poly A+ RNAs and are synthetically lethal with many mutants of the mRNA machinery [30
]. In Drosophila loss of the THO complex function results in only minor differences in transcription profiles as revealed by the whole genome array [5
]. The analysis of the cytoplasmic mRNA from bone marrow cells was not successful, because cytoplasmic mRNA from bone marrow cells was not stable enough for microarray analysis. Therefore, we analyzed the expression level of cytoplasmic mRNA in fibroblasts. In the presence or absence of THOC5/FMIP, we found that less than 100 genes were down-regulated more than three-fold using the mouse whole genome array (Hauser and Tamura, unpublished data). In the fibroblast system, however, the depletion of THOC5/FMIP did not cause apoptosis, but reduced cell growth (Tran and Tamura, unpublished data), suggesting that THOC5/FMIP has a specific function in proliferating cells. It has been shown recently that in response to treatment of mice with interferon-alpha hematopoietic stem cells efficiently exit G0
and enter an active cell cycle [31
], suggesting that poly (I:C) treatment may exacerbate the phenotype which is observed in THOC5/FMIP deletion mice. Further studies will be required to examine whether the mice would present the same phenotype if other methods of conditional deletion of THOC5/FMIP in the relevant cell types were used.
We show here that the THOC5/FMIP binding partner, THOC1 was down-regulated via THOC5/FMIP knockdown. Furthermore, we have shown that the expression of mutant THOC5/FMIP lacking the THOC1 binding site degraded faster than the wild-type and down-regulated the level of endogenous THOC1 [19
], suggesting that these two molecules may stabilize each other forming a functional unit. It indicates that the phenotype we observed might be the result of both THOC1 and THOC5/FMIP depletion. It has been shown that over-expression of THOC1 causes apoptosis in several cells [12
]. The deletion of THOC1 also causes apoptosis in cancer cells, but not in normal fibroblasts [12
]. Furthermore, the embryonic development of conventional THOC1 knockout mice is arrested around the time of implantation [13
]. Interestingly, THOC1 possesses the death domain and interacts with Rb protein, suggesting that THOC1 may involve the apoptosis signal directly [32
Our analysis demonstrates a link between THOC5/FMIP protein function and the normal developmental processes seen in adult hematopoiesis. We now have a suitable model system to determine how THO complex proteins act, enabling primitive cell survival and proliferation.