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Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1).

Bhattacharya C, Aggarwal S, Kumar M, Ali A, Matin A - PLoS ONE (2008)

Bottom Line: The 3'-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins.In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression.The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, University of Texas, MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT

Background: The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3'-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.

Methodology/principal findings: Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.

Conclusions/significance: The 3'-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

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DND1 interacts with APOBEC3 in mammalian cells.(a) Expression of APOBEC proteins and DND1 in 293T transfected cells. Cells were co-transfected with expression vectors encoding HA-tagged Dnd1 (lanes 1, 2, 3 & 4) and myc-tagged Apobec1, Apobec2 and Apobec3 (lanes 2, 3 & 4, respectively). After 48 h, transfected cells were lysed. An aliquot (10 μg) was electrophoresed for western blotting with anti-myc (top panel) and anti-DND1 antibody (bottom panel) to detect expression of proteins. (b) HA-DND1 pulls-down APOBEC3 from cells. Lysates from transfected cells in (a) were used. Equal aliquots (50 μg) were used for immunoprecipitation (IP) using anti-HA antibody (+) to pull down HA-DND1 (lanes 6, 7, 9, 11 & 13). Control lanes had no antibody (−). Following IP, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.
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pone-0002315-g003: DND1 interacts with APOBEC3 in mammalian cells.(a) Expression of APOBEC proteins and DND1 in 293T transfected cells. Cells were co-transfected with expression vectors encoding HA-tagged Dnd1 (lanes 1, 2, 3 & 4) and myc-tagged Apobec1, Apobec2 and Apobec3 (lanes 2, 3 & 4, respectively). After 48 h, transfected cells were lysed. An aliquot (10 μg) was electrophoresed for western blotting with anti-myc (top panel) and anti-DND1 antibody (bottom panel) to detect expression of proteins. (b) HA-DND1 pulls-down APOBEC3 from cells. Lysates from transfected cells in (a) were used. Equal aliquots (50 μg) were used for immunoprecipitation (IP) using anti-HA antibody (+) to pull down HA-DND1 (lanes 6, 7, 9, 11 & 13). Control lanes had no antibody (−). Following IP, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.

Mentions: Next, we tested whether DND1 can interact with APOBEC proteins in mammalian cells. HA-tagged Dnd1 and myc-tagged Apobec plasmid constructs were co-transfected into 293T cells. myc-APOBEC1, 2, and 3 expression in transfected 293T cells were detected by immunoblotting with anti-myc (Fig. 3a, top panel). Expression of HA-tagged DND1 was also detected using anti-DND1 antibody, antibody C [32] (Fig. 3a, lower panel). The lysates were used for immunoprecipitation with anti-HA antibody to “pull-down” HA-DND1 and associated proteins. No antibody was added to controls. After immunoprecipitation with anti-HA antibody, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.


Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1).

Bhattacharya C, Aggarwal S, Kumar M, Ali A, Matin A - PLoS ONE (2008)

DND1 interacts with APOBEC3 in mammalian cells.(a) Expression of APOBEC proteins and DND1 in 293T transfected cells. Cells were co-transfected with expression vectors encoding HA-tagged Dnd1 (lanes 1, 2, 3 & 4) and myc-tagged Apobec1, Apobec2 and Apobec3 (lanes 2, 3 & 4, respectively). After 48 h, transfected cells were lysed. An aliquot (10 μg) was electrophoresed for western blotting with anti-myc (top panel) and anti-DND1 antibody (bottom panel) to detect expression of proteins. (b) HA-DND1 pulls-down APOBEC3 from cells. Lysates from transfected cells in (a) were used. Equal aliquots (50 μg) were used for immunoprecipitation (IP) using anti-HA antibody (+) to pull down HA-DND1 (lanes 6, 7, 9, 11 & 13). Control lanes had no antibody (−). Following IP, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2384002&req=5

pone-0002315-g003: DND1 interacts with APOBEC3 in mammalian cells.(a) Expression of APOBEC proteins and DND1 in 293T transfected cells. Cells were co-transfected with expression vectors encoding HA-tagged Dnd1 (lanes 1, 2, 3 & 4) and myc-tagged Apobec1, Apobec2 and Apobec3 (lanes 2, 3 & 4, respectively). After 48 h, transfected cells were lysed. An aliquot (10 μg) was electrophoresed for western blotting with anti-myc (top panel) and anti-DND1 antibody (bottom panel) to detect expression of proteins. (b) HA-DND1 pulls-down APOBEC3 from cells. Lysates from transfected cells in (a) were used. Equal aliquots (50 μg) were used for immunoprecipitation (IP) using anti-HA antibody (+) to pull down HA-DND1 (lanes 6, 7, 9, 11 & 13). Control lanes had no antibody (−). Following IP, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.
Mentions: Next, we tested whether DND1 can interact with APOBEC proteins in mammalian cells. HA-tagged Dnd1 and myc-tagged Apobec plasmid constructs were co-transfected into 293T cells. myc-APOBEC1, 2, and 3 expression in transfected 293T cells were detected by immunoblotting with anti-myc (Fig. 3a, top panel). Expression of HA-tagged DND1 was also detected using anti-DND1 antibody, antibody C [32] (Fig. 3a, lower panel). The lysates were used for immunoprecipitation with anti-HA antibody to “pull-down” HA-DND1 and associated proteins. No antibody was added to controls. After immunoprecipitation with anti-HA antibody, electrophoresis and transfer to membranes, western blotting was performed using anti-myc antibody.

Bottom Line: The 3'-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins.In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression.The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, University of Texas, MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT

Background: The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3'-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.

Methodology/principal findings: Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.

Conclusions/significance: The 3'-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

Show MeSH
Related in: MedlinePlus