Limits...
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.

Show MeSH

Related in: MedlinePlus

DND1 and APOBEC3 in mouse tissues.(a) DND1 interacts with endogenous APOBEC3. Mouse testis lysates were incubated with GST-DND1α (lane 1), GST-DND1β⪽ (lane 2) or GST protein (lane 3) and incubated with Sepharose 4B beads. The beads were washed and the contents eluted into loading dye prior to electrophoresis and immunoblotting with rabbit anti-mouse APOBEC3 antibody. Control lanes on the left are cell lysates from untransfected cells (lane 4) and lysates from cells transfected with myc-APOBEC3 expression constructs (lane 5). (b) Dnd1 and Apobec3 are expressed in embryonic germ (EG) cells and genital ridges. RT-PCR for Apobec3, Dnd1 and HPRT (hypoxanthine-guanine phosphoribosyltransferase) on total RNA from EG cells, mouse embryo fibroblasts (MEF), and E13.5 genital ridges (GR). (c) EG cells in culture, stained with alkaline phosphatase (orange clusters), growing on MEF cell layer.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2384002&req=5

pone-0002315-g004: DND1 and APOBEC3 in mouse tissues.(a) DND1 interacts with endogenous APOBEC3. Mouse testis lysates were incubated with GST-DND1α (lane 1), GST-DND1β⪽ (lane 2) or GST protein (lane 3) and incubated with Sepharose 4B beads. The beads were washed and the contents eluted into loading dye prior to electrophoresis and immunoblotting with rabbit anti-mouse APOBEC3 antibody. Control lanes on the left are cell lysates from untransfected cells (lane 4) and lysates from cells transfected with myc-APOBEC3 expression constructs (lane 5). (b) Dnd1 and Apobec3 are expressed in embryonic germ (EG) cells and genital ridges. RT-PCR for Apobec3, Dnd1 and HPRT (hypoxanthine-guanine phosphoribosyltransferase) on total RNA from EG cells, mouse embryo fibroblasts (MEF), and E13.5 genital ridges (GR). (c) EG cells in culture, stained with alkaline phosphatase (orange clusters), growing on MEF cell layer.

Mentions: Our results showed that GST-DND1α was able to “pull-down” APOBEC3 from mouse testes (Fig. 4a). APOBEC3 from mouse testis was the same size as myc-APOBEC3 expressed in cells transfected with myc-Apobec3 expression vector (Fig. 4a). In this experiment, GST-DND1β was not able to efficiently “pull-down” APOBEC3 from mouse testes and this suggests that DND1α isoform interacts more efficiently with endogenous, testes APOBEC3. DND1α isoform is expressed in germ cells during embryogenesis as well as in adult stages. In contrast, DND1β is expressed in meiotic and post-meiotic germ cells in adult mice [32].


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 and APOBEC3 in mouse tissues.(a) DND1 interacts with endogenous APOBEC3. Mouse testis lysates were incubated with GST-DND1α (lane 1), GST-DND1β⪽ (lane 2) or GST protein (lane 3) and incubated with Sepharose 4B beads. The beads were washed and the contents eluted into loading dye prior to electrophoresis and immunoblotting with rabbit anti-mouse APOBEC3 antibody. Control lanes on the left are cell lysates from untransfected cells (lane 4) and lysates from cells transfected with myc-APOBEC3 expression constructs (lane 5). (b) Dnd1 and Apobec3 are expressed in embryonic germ (EG) cells and genital ridges. RT-PCR for Apobec3, Dnd1 and HPRT (hypoxanthine-guanine phosphoribosyltransferase) on total RNA from EG cells, mouse embryo fibroblasts (MEF), and E13.5 genital ridges (GR). (c) EG cells in culture, stained with alkaline phosphatase (orange clusters), growing on MEF cell layer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2384002&req=5

pone-0002315-g004: DND1 and APOBEC3 in mouse tissues.(a) DND1 interacts with endogenous APOBEC3. Mouse testis lysates were incubated with GST-DND1α (lane 1), GST-DND1β⪽ (lane 2) or GST protein (lane 3) and incubated with Sepharose 4B beads. The beads were washed and the contents eluted into loading dye prior to electrophoresis and immunoblotting with rabbit anti-mouse APOBEC3 antibody. Control lanes on the left are cell lysates from untransfected cells (lane 4) and lysates from cells transfected with myc-APOBEC3 expression constructs (lane 5). (b) Dnd1 and Apobec3 are expressed in embryonic germ (EG) cells and genital ridges. RT-PCR for Apobec3, Dnd1 and HPRT (hypoxanthine-guanine phosphoribosyltransferase) on total RNA from EG cells, mouse embryo fibroblasts (MEF), and E13.5 genital ridges (GR). (c) EG cells in culture, stained with alkaline phosphatase (orange clusters), growing on MEF cell layer.
Mentions: Our results showed that GST-DND1α was able to “pull-down” APOBEC3 from mouse testes (Fig. 4a). APOBEC3 from mouse testis was the same size as myc-APOBEC3 expressed in cells transfected with myc-Apobec3 expression vector (Fig. 4a). In this experiment, GST-DND1β was not able to efficiently “pull-down” APOBEC3 from mouse testes and this suggests that DND1α isoform interacts more efficiently with endogenous, testes APOBEC3. DND1α isoform is expressed in germ cells during embryogenesis as well as in adult stages. In contrast, DND1β is expressed in meiotic and post-meiotic germ cells in adult mice [32].

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