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THOC5/FMIP, an mRNA export TREX complex protein, is essential for hematopoietic primitive cell survival in vivo.

Mancini A, Niemann-Seyde SC, Pankow R, El Bounkari O, Klebba-Färber S, Koch A, Jaworska E, Spooncer E, Gruber AD, Whetton AD, Tamura T - BMC Biol. (2010)

Bottom Line: Thus we considered the hematopoietic system and found that seven days after poly injection, the number of blood cells in peripheral blood decreased drastically.THOC5/Fms interacting protein is an essential element in the maintenance of hematopoiesis.Furthermore, mechanistically depletion of THOC5/Fms interacting protein causes the down-regulation of its direct interacting partner, THOC1 which may contribute to altered THO complex function and cell death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Carl-Neuberg-Str, 1, D-30623 Hannover, Germany. annalisa_mancini@hotmail.de

ABSTRACT

Background: The transcription/export complex is evolutionarily conserved from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. FMIP(Fms interacting protein) is a member of the THO (suppressors of the transcriptional defects of hpr1delta by overexpression) complex which is a subcomplex of the transcription/export complex. THO complex (THOC) components are not essential for bulk poly (A)+ RNA export in higher eukaryotes, but for the nuclear export of subset of mRNAs, however, their exact role is still unclear.

Results: To study the role of THOC5/Fms interacting protein in vivo, we generated THOC5/Fms interacting protein knockout mice. Since these mice are embryonic lethal, we then generated interferon inducible conditional THOC5/Fms interacting protein knockout mice. After three poly injections all of the mice died within 14 days. No pathological alterations, however, were observed in liver, kidney or heart. Thus we considered the hematopoietic system and found that seven days after poly injection, the number of blood cells in peripheral blood decreased drastically. Investigation of bone marrow cells showed that these became apoptotic within seven days after poly injection. Committed myeloid progenitor cells and cells with long term reconstituting potential were lost from bone marrow within four days after poly injection. Furthermore, infusion of normal bone marrow cells rescued mice from death induced by loss of THOC5/Fms interacting protein.

Conclusion: THOC5/Fms interacting protein is an essential element in the maintenance of hematopoiesis. Furthermore, mechanistically depletion of THOC5/Fms interacting protein causes the down-regulation of its direct interacting partner, THOC1 which may contribute to altered THO complex function and cell death.

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Deletion of THOC5/FMIP causes apoptosis of leukocytes in bone marrow. Six-week-old Mx-cre THOC5/FMIP (flox/flox) mice (n = 9) and THOC5/FMIP (flox/flox) control mice (n = 6) were injected with poly (I:C) (250 μg each) and the femora were then isolated. (A): Bone marrow cells were spun down onto glass slides and then stained with May-Grunwald Giemsa and hematoxylin. (B): Aliquots of same preparation were stained with TUNEL and DAPI. Results are the mean +/- SEM of %TUNEL positive/DAPI positive cells (n >2000 cells). Original magnification: ×200 for all panels. (C): Aliquots of 2-3 μg of DNA from liver and bone marrow of poly (I:C) treated (+) and untreated (-) Mx-cre THOC5/FMIP (flox/flox) mice were separated on 1.5% (w/v) agarose gel, stained with ethidium bromide (2 μg/ml) and photographed under UV light. M: base pair Marker.
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Figure 6: Deletion of THOC5/FMIP causes apoptosis of leukocytes in bone marrow. Six-week-old Mx-cre THOC5/FMIP (flox/flox) mice (n = 9) and THOC5/FMIP (flox/flox) control mice (n = 6) were injected with poly (I:C) (250 μg each) and the femora were then isolated. (A): Bone marrow cells were spun down onto glass slides and then stained with May-Grunwald Giemsa and hematoxylin. (B): Aliquots of same preparation were stained with TUNEL and DAPI. Results are the mean +/- SEM of %TUNEL positive/DAPI positive cells (n >2000 cells). Original magnification: ×200 for all panels. (C): Aliquots of 2-3 μg of DNA from liver and bone marrow of poly (I:C) treated (+) and untreated (-) Mx-cre THOC5/FMIP (flox/flox) mice were separated on 1.5% (w/v) agarose gel, stained with ethidium bromide (2 μg/ml) and photographed under UV light. M: base pair Marker.

Mentions: As severe leukocytopenia and anemia were observed in THOC5/FMIP depleted mice, we examined the bone marrow cells after induced depletion of THOC5/FMIP protein. Bone marrow cells from five to six-week-old mice were flushed from femora zero, four or seven days after the first 250 μg poly (I:C) injection (n = 9). Cytospin preparations were stained by May Grunwald solution and hematoxylin. Although after four days we did not detect any difference in morphology of bone marrow cells, after seven days it became apparent that few hematoxylin-stained cells were present in samples from Mx-cre THOC5/FMIP(flox/flox) mice (Figure 6A). A high proportion of those cells that were present showed dense chromatin staining, reminiscent of apoptotic cells. To examine whether the cells that survived were apoptotic more than 2,000 4', 6-Diamidino-2-phenyindole (DAPI) positive bone marrow cells were co-stained with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI for each preparation. Two and four percent respectively of DAPI positive bone marrow cells in control THOC5/FMIP(flox/flox) mice are stained with TUNEL in the presence or in the absence of poly (I:C) treatment (Figure 6B). In Mx-cre THOC5/FMIP(flox/flox) mice, 15% of bone marrow cells are stained without poly(I:C) injection, possibly as a result of the natural presence of interferons. After three poly (I:C) injections, only a few DAPI positive cells are left and all remaining cells were stained with TUNEL (Figure 6B), suggesting that deletion of THOC5/FMIP causes bone marrow cell apoptosis. Furthermore, no significant increase of TUNEL positive cells (10-20%) was observed, suggesting that apoptotic cells may fail to stay in bone marrow or be phagocytosed. DNA laddering, indicative of apoptosis, in bone marrow cells and hepatocytes was also examined. We detected DNA fragmentation associated with apoptosis from the poly (I:C) injected bone marrow cells, but not from the liver of the same mouse (Figure 6C).


THOC5/FMIP, an mRNA export TREX complex protein, is essential for hematopoietic primitive cell survival in vivo.

Mancini A, Niemann-Seyde SC, Pankow R, El Bounkari O, Klebba-Färber S, Koch A, Jaworska E, Spooncer E, Gruber AD, Whetton AD, Tamura T - BMC Biol. (2010)

Deletion of THOC5/FMIP causes apoptosis of leukocytes in bone marrow. Six-week-old Mx-cre THOC5/FMIP (flox/flox) mice (n = 9) and THOC5/FMIP (flox/flox) control mice (n = 6) were injected with poly (I:C) (250 μg each) and the femora were then isolated. (A): Bone marrow cells were spun down onto glass slides and then stained with May-Grunwald Giemsa and hematoxylin. (B): Aliquots of same preparation were stained with TUNEL and DAPI. Results are the mean +/- SEM of %TUNEL positive/DAPI positive cells (n >2000 cells). Original magnification: ×200 for all panels. (C): Aliquots of 2-3 μg of DNA from liver and bone marrow of poly (I:C) treated (+) and untreated (-) Mx-cre THOC5/FMIP (flox/flox) mice were separated on 1.5% (w/v) agarose gel, stained with ethidium bromide (2 μg/ml) and photographed under UV light. M: base pair Marker.
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Figure 6: Deletion of THOC5/FMIP causes apoptosis of leukocytes in bone marrow. Six-week-old Mx-cre THOC5/FMIP (flox/flox) mice (n = 9) and THOC5/FMIP (flox/flox) control mice (n = 6) were injected with poly (I:C) (250 μg each) and the femora were then isolated. (A): Bone marrow cells were spun down onto glass slides and then stained with May-Grunwald Giemsa and hematoxylin. (B): Aliquots of same preparation were stained with TUNEL and DAPI. Results are the mean +/- SEM of %TUNEL positive/DAPI positive cells (n >2000 cells). Original magnification: ×200 for all panels. (C): Aliquots of 2-3 μg of DNA from liver and bone marrow of poly (I:C) treated (+) and untreated (-) Mx-cre THOC5/FMIP (flox/flox) mice were separated on 1.5% (w/v) agarose gel, stained with ethidium bromide (2 μg/ml) and photographed under UV light. M: base pair Marker.
Mentions: As severe leukocytopenia and anemia were observed in THOC5/FMIP depleted mice, we examined the bone marrow cells after induced depletion of THOC5/FMIP protein. Bone marrow cells from five to six-week-old mice were flushed from femora zero, four or seven days after the first 250 μg poly (I:C) injection (n = 9). Cytospin preparations were stained by May Grunwald solution and hematoxylin. Although after four days we did not detect any difference in morphology of bone marrow cells, after seven days it became apparent that few hematoxylin-stained cells were present in samples from Mx-cre THOC5/FMIP(flox/flox) mice (Figure 6A). A high proportion of those cells that were present showed dense chromatin staining, reminiscent of apoptotic cells. To examine whether the cells that survived were apoptotic more than 2,000 4', 6-Diamidino-2-phenyindole (DAPI) positive bone marrow cells were co-stained with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI for each preparation. Two and four percent respectively of DAPI positive bone marrow cells in control THOC5/FMIP(flox/flox) mice are stained with TUNEL in the presence or in the absence of poly (I:C) treatment (Figure 6B). In Mx-cre THOC5/FMIP(flox/flox) mice, 15% of bone marrow cells are stained without poly(I:C) injection, possibly as a result of the natural presence of interferons. After three poly (I:C) injections, only a few DAPI positive cells are left and all remaining cells were stained with TUNEL (Figure 6B), suggesting that deletion of THOC5/FMIP causes bone marrow cell apoptosis. Furthermore, no significant increase of TUNEL positive cells (10-20%) was observed, suggesting that apoptotic cells may fail to stay in bone marrow or be phagocytosed. DNA laddering, indicative of apoptosis, in bone marrow cells and hepatocytes was also examined. We detected DNA fragmentation associated with apoptosis from the poly (I:C) injected bone marrow cells, but not from the liver of the same mouse (Figure 6C).

Bottom Line: Thus we considered the hematopoietic system and found that seven days after poly injection, the number of blood cells in peripheral blood decreased drastically.THOC5/Fms interacting protein is an essential element in the maintenance of hematopoiesis.Furthermore, mechanistically depletion of THOC5/Fms interacting protein causes the down-regulation of its direct interacting partner, THOC1 which may contribute to altered THO complex function and cell death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Carl-Neuberg-Str, 1, D-30623 Hannover, Germany. annalisa_mancini@hotmail.de

ABSTRACT

Background: The transcription/export complex is evolutionarily conserved from yeast to man and is required for coupled transcription elongation and nuclear export of mRNAs. FMIP(Fms interacting protein) is a member of the THO (suppressors of the transcriptional defects of hpr1delta by overexpression) complex which is a subcomplex of the transcription/export complex. THO complex (THOC) components are not essential for bulk poly (A)+ RNA export in higher eukaryotes, but for the nuclear export of subset of mRNAs, however, their exact role is still unclear.

Results: To study the role of THOC5/Fms interacting protein in vivo, we generated THOC5/Fms interacting protein knockout mice. Since these mice are embryonic lethal, we then generated interferon inducible conditional THOC5/Fms interacting protein knockout mice. After three poly injections all of the mice died within 14 days. No pathological alterations, however, were observed in liver, kidney or heart. Thus we considered the hematopoietic system and found that seven days after poly injection, the number of blood cells in peripheral blood decreased drastically. Investigation of bone marrow cells showed that these became apoptotic within seven days after poly injection. Committed myeloid progenitor cells and cells with long term reconstituting potential were lost from bone marrow within four days after poly injection. Furthermore, infusion of normal bone marrow cells rescued mice from death induced by loss of THOC5/Fms interacting protein.

Conclusion: THOC5/Fms interacting protein is an essential element in the maintenance of hematopoiesis. Furthermore, mechanistically depletion of THOC5/Fms interacting protein causes the down-regulation of its direct interacting partner, THOC1 which may contribute to altered THO complex function and cell death.

Show MeSH