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THOC5, a member of the mRNA export complex, contributes to processing of a subset of wingless/integrated (Wnt) target mRNAs and integrity of the gut epithelial barrier.

Saran S, Tran DD, Klebba-Färber S, Moran-Losada P, Wiehlmann L, Koch A, Chopra H, Pabst O, Hoffmann A, Klopfleisch R, Tamura T - BMC Cell Biol. (2013)

Bottom Line: Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney.Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist.These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5), an mRNA export protein, is involved in the expression of only 1% of all genes. Using an interferon inducible knockout mouse system, we have previously shown that THOC5 is an essential element in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis in adult mice. Here we interrogate THOC5 function in cell differentiation beyond the hematopoietic system and study pathological changes caused by THOC5 deficiency.

Results: To examine whether THOC5 plays a role in general differentiation processes, we generated tamoxifen inducible THOC5 knockout mice. We show here that the depletion of THOC5 impaired not only hematopoietic differentiation, but also differentiation and self renewal of the gut epithelium. Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney. We further show that THOC5 is indispensable for processing of mRNAs induced by Wnt (wingless/integrated) signaling which play key roles in epithelial cell differentiation/proliferation. A subset of Wnt target mRNAs, SRY-box containing gene 9 (Sox9), and achaete-scute complex homolog 2 (Ascl2), but not Fibronectin 1 (Fn1), were down-regulated in THOC5 knockout intestinal cells. The down-regulated Wnt target mRNAs were able to bind to THOC5. Furthermore, pathological alterations in the gastrointestinal tract induced translocation of intestinal bacteria and caused sepsis in mice. The bacteria translocation may cause Toll-like receptor activation. We identified one of the Toll-like receptor inducible genes, prostaglandin-endoperoxidase synthase 2 (Ptgs2 or COX2) transcript as THOC5 target mRNA.

Conclusion: THOC5 is indispensable for processing of only a subset of mRNAs, but plays a key role in processing of mRNAs inducible by Wnt signals. Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist. These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

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Depletion of the THOC5 gene did not cause pathological alteration to liver or kidney. (A) Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and six days after the first tamoxifen injection, kidney and liver were fixed in formalin. Paraffin sections were supplied for immunohistochemical staining using cleaved caspase 3 antibody. Bars represent 100 μm. (B) Protein extracts from liver obtained from the same mice as in (A) were supplied for THOC5, cleaved (cl.) caspase 3, caspase 3 and actin specific immunoblot. (C) Nuclear (Nuc) or cytoplasmic (Cyt) RNA from 20 μg of liver tissues before or after the tamoxifen treatment were supplied for the THOC5 specific RT-PCR or nuclear (Nuc) or cytoplasmic (Cyt) protein extracts from same tissues (200 μg of liver tissue) were supplied for GAPDH and Histone H3 specific immunoblot (Blot). (D) Aliquots of the same RNAs in (C) were supplied for Albumin, Transferrin and beta actin specific semi-quantitative RT-PCR. Cycles: Number of amplification cycles.
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Figure 4: Depletion of the THOC5 gene did not cause pathological alteration to liver or kidney. (A) Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and six days after the first tamoxifen injection, kidney and liver were fixed in formalin. Paraffin sections were supplied for immunohistochemical staining using cleaved caspase 3 antibody. Bars represent 100 μm. (B) Protein extracts from liver obtained from the same mice as in (A) were supplied for THOC5, cleaved (cl.) caspase 3, caspase 3 and actin specific immunoblot. (C) Nuclear (Nuc) or cytoplasmic (Cyt) RNA from 20 μg of liver tissues before or after the tamoxifen treatment were supplied for the THOC5 specific RT-PCR or nuclear (Nuc) or cytoplasmic (Cyt) protein extracts from same tissues (200 μg of liver tissue) were supplied for GAPDH and Histone H3 specific immunoblot (Blot). (D) Aliquots of the same RNAs in (C) were supplied for Albumin, Transferrin and beta actin specific semi-quantitative RT-PCR. Cycles: Number of amplification cycles.

Mentions: We next examined other organs, kidney and liver. In agreement with our previous data [23], we did not see any pathological alterations, such as inflammation, or any activated caspase 3 positive cells in kidney or liver (Figure 4A and B). Since hepatocytes produce a number of key molecules and enzymes such as albumin or transferrin for the maintenance of life, we examined whether albumin or transferrin mRNAs were exported into the cytoplasm in the THOC5 depleted liver. We isolated cytoplasmic and nuclear RNAs from 20 μg of liver tissue before and after THOC5 knockdown (Figure 4C,D), and then analyzed albumin, transferrin and actin expression by semi-quantitative RT-PCR. As control for fractionation, protein extracts of both fractions obtained from 200 μg liver tissues were supplied for Histone H3 (nuclear fraction), and GAPDH (cytoplasmic fraction) specific immunoblot (Figure 4C). In this experiment only spliced forms of mRNA can be detected since we have chosen the primer pair for RT-PCR which is located at two different exons (Table 1). As shown in Figure 4D, upon depletion of THOC5 the export of albumin as well as transferrin mRNAs were not altered.


THOC5, a member of the mRNA export complex, contributes to processing of a subset of wingless/integrated (Wnt) target mRNAs and integrity of the gut epithelial barrier.

Saran S, Tran DD, Klebba-Färber S, Moran-Losada P, Wiehlmann L, Koch A, Chopra H, Pabst O, Hoffmann A, Klopfleisch R, Tamura T - BMC Cell Biol. (2013)

Depletion of the THOC5 gene did not cause pathological alteration to liver or kidney. (A) Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and six days after the first tamoxifen injection, kidney and liver were fixed in formalin. Paraffin sections were supplied for immunohistochemical staining using cleaved caspase 3 antibody. Bars represent 100 μm. (B) Protein extracts from liver obtained from the same mice as in (A) were supplied for THOC5, cleaved (cl.) caspase 3, caspase 3 and actin specific immunoblot. (C) Nuclear (Nuc) or cytoplasmic (Cyt) RNA from 20 μg of liver tissues before or after the tamoxifen treatment were supplied for the THOC5 specific RT-PCR or nuclear (Nuc) or cytoplasmic (Cyt) protein extracts from same tissues (200 μg of liver tissue) were supplied for GAPDH and Histone H3 specific immunoblot (Blot). (D) Aliquots of the same RNAs in (C) were supplied for Albumin, Transferrin and beta actin specific semi-quantitative RT-PCR. Cycles: Number of amplification cycles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4222586&req=5

Figure 4: Depletion of the THOC5 gene did not cause pathological alteration to liver or kidney. (A) Rosa26-CreERT2:THOC5 (flox/flox) mice were treated with (+) or without (−) tamoxifen and six days after the first tamoxifen injection, kidney and liver were fixed in formalin. Paraffin sections were supplied for immunohistochemical staining using cleaved caspase 3 antibody. Bars represent 100 μm. (B) Protein extracts from liver obtained from the same mice as in (A) were supplied for THOC5, cleaved (cl.) caspase 3, caspase 3 and actin specific immunoblot. (C) Nuclear (Nuc) or cytoplasmic (Cyt) RNA from 20 μg of liver tissues before or after the tamoxifen treatment were supplied for the THOC5 specific RT-PCR or nuclear (Nuc) or cytoplasmic (Cyt) protein extracts from same tissues (200 μg of liver tissue) were supplied for GAPDH and Histone H3 specific immunoblot (Blot). (D) Aliquots of the same RNAs in (C) were supplied for Albumin, Transferrin and beta actin specific semi-quantitative RT-PCR. Cycles: Number of amplification cycles.
Mentions: We next examined other organs, kidney and liver. In agreement with our previous data [23], we did not see any pathological alterations, such as inflammation, or any activated caspase 3 positive cells in kidney or liver (Figure 4A and B). Since hepatocytes produce a number of key molecules and enzymes such as albumin or transferrin for the maintenance of life, we examined whether albumin or transferrin mRNAs were exported into the cytoplasm in the THOC5 depleted liver. We isolated cytoplasmic and nuclear RNAs from 20 μg of liver tissue before and after THOC5 knockdown (Figure 4C,D), and then analyzed albumin, transferrin and actin expression by semi-quantitative RT-PCR. As control for fractionation, protein extracts of both fractions obtained from 200 μg liver tissues were supplied for Histone H3 (nuclear fraction), and GAPDH (cytoplasmic fraction) specific immunoblot (Figure 4C). In this experiment only spliced forms of mRNA can be detected since we have chosen the primer pair for RT-PCR which is located at two different exons (Table 1). As shown in Figure 4D, upon depletion of THOC5 the export of albumin as well as transferrin mRNAs were not altered.

Bottom Line: Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney.Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist.These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5), an mRNA export protein, is involved in the expression of only 1% of all genes. Using an interferon inducible knockout mouse system, we have previously shown that THOC5 is an essential element in the maintenance of hematopoietic stem cells and cytokine-mediated hematopoiesis in adult mice. Here we interrogate THOC5 function in cell differentiation beyond the hematopoietic system and study pathological changes caused by THOC5 deficiency.

Results: To examine whether THOC5 plays a role in general differentiation processes, we generated tamoxifen inducible THOC5 knockout mice. We show here that the depletion of THOC5 impaired not only hematopoietic differentiation, but also differentiation and self renewal of the gut epithelium. Depletion of the THOC5 gene did not cause pathological alterations in liver or kidney. We further show that THOC5 is indispensable for processing of mRNAs induced by Wnt (wingless/integrated) signaling which play key roles in epithelial cell differentiation/proliferation. A subset of Wnt target mRNAs, SRY-box containing gene 9 (Sox9), and achaete-scute complex homolog 2 (Ascl2), but not Fibronectin 1 (Fn1), were down-regulated in THOC5 knockout intestinal cells. The down-regulated Wnt target mRNAs were able to bind to THOC5. Furthermore, pathological alterations in the gastrointestinal tract induced translocation of intestinal bacteria and caused sepsis in mice. The bacteria translocation may cause Toll-like receptor activation. We identified one of the Toll-like receptor inducible genes, prostaglandin-endoperoxidase synthase 2 (Ptgs2 or COX2) transcript as THOC5 target mRNA.

Conclusion: THOC5 is indispensable for processing of only a subset of mRNAs, but plays a key role in processing of mRNAs inducible by Wnt signals. Furthermore, THOC5 is dispensable for general mRNA export in terminally differentiated organs, indicating that multiple mRNA export pathways exist. These data imply that THOC5 may be a useful tool for studying intestinal stem cells, for modifying the differentiation processes and for cancer therapy.

Show MeSH
Related in: MedlinePlus