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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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Related in: MedlinePlus

DND1 interacts with APOBEC3 in vitro.(a) Experimental design to test interaction of DND1 to the APOBEC proteins in vitro. All reactions contained [35S]methionine-labeled APOBEC proteins (blue circles). The labeled proteins were incubated with purified GST-DND1 (indicated by+in panels c–e). Glutathione Sepharose 4B beads (beads, white circles) were added to the reactions. The beads are able to bind to the GST moiety of DND1 and should also “pull-down” proteins that associate with GST-DND1. Control incubations used beads alone incubated with labeled APOBEC proteins (indicated by–in panels c–e). (b) In vitro transcribed/translated [35S]methionine-labeled APOBEC proteins. 5 μL was loaded from each translation reaction. A1 (APOBEC1), A2 (APOBEC2), A3 (8) (8-exon isoform APOBEC3) and A3 (9) (9-exon isoform of APOBEC3). The translated proteins used as input are marked with red bars (in panels b, d–f). (c) Binding of APOBEC proteins to GST-DND1α. 40 μL of [35S]methionine-labeled APOBEC proteins was incubated with GST beads and either with (+) or without (−) GST-DND1α. Lanes 5 and 7 show binding of APOBEC3 to GST-DND1α. Lanes 6 and 8 show no binding of APOBEC3 to the beads. (d) Binding of APOBEC proteins to GST-DND1β. 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST-DND1β. Lanes 3 and 5 show binding of APOBEC3 to GST-DND1β. Lanes 4 and 6 show no binding of APOBEC3 to the beads. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (e) Testing interaction of human APOBEC1 and ACF to GST-DND1α. 40 μL of [35S]methionine-labeled human APOBEC1 (hA1), mouse APOBEC2 (A2) and human ACF were incubated with GST beads and either with (+) or without (−) GST-DND1α. Comparison of lanes 1 and 2 indicate weak binding of human APOBEC1 (hA1) to GST-DND1α. Comparison of lanes 5 and 6 indicate no specific binding of ACF to GST-DND1α. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (f) Binding of APOBEC proteins to GST (second control). 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (g) Effect of RNase (left) and DNase (right) on APOBEC3 and GST-DND1 interaction. [35S]methionine-labeled APOBEC proteins incubated together with GST-DND1α̃ and GST beads were divided into two. Each aliquot was treated with RNase (+) or DNAse (+) or not treated (−) prior to electrophoresis.
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pone-0002315-g002: DND1 interacts with APOBEC3 in vitro.(a) Experimental design to test interaction of DND1 to the APOBEC proteins in vitro. All reactions contained [35S]methionine-labeled APOBEC proteins (blue circles). The labeled proteins were incubated with purified GST-DND1 (indicated by+in panels c–e). Glutathione Sepharose 4B beads (beads, white circles) were added to the reactions. The beads are able to bind to the GST moiety of DND1 and should also “pull-down” proteins that associate with GST-DND1. Control incubations used beads alone incubated with labeled APOBEC proteins (indicated by–in panels c–e). (b) In vitro transcribed/translated [35S]methionine-labeled APOBEC proteins. 5 μL was loaded from each translation reaction. A1 (APOBEC1), A2 (APOBEC2), A3 (8) (8-exon isoform APOBEC3) and A3 (9) (9-exon isoform of APOBEC3). The translated proteins used as input are marked with red bars (in panels b, d–f). (c) Binding of APOBEC proteins to GST-DND1α. 40 μL of [35S]methionine-labeled APOBEC proteins was incubated with GST beads and either with (+) or without (−) GST-DND1α. Lanes 5 and 7 show binding of APOBEC3 to GST-DND1α. Lanes 6 and 8 show no binding of APOBEC3 to the beads. (d) Binding of APOBEC proteins to GST-DND1β. 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST-DND1β. Lanes 3 and 5 show binding of APOBEC3 to GST-DND1β. Lanes 4 and 6 show no binding of APOBEC3 to the beads. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (e) Testing interaction of human APOBEC1 and ACF to GST-DND1α. 40 μL of [35S]methionine-labeled human APOBEC1 (hA1), mouse APOBEC2 (A2) and human ACF were incubated with GST beads and either with (+) or without (−) GST-DND1α. Comparison of lanes 1 and 2 indicate weak binding of human APOBEC1 (hA1) to GST-DND1α. Comparison of lanes 5 and 6 indicate no specific binding of ACF to GST-DND1α. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (f) Binding of APOBEC proteins to GST (second control). 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (g) Effect of RNase (left) and DNase (right) on APOBEC3 and GST-DND1 interaction. [35S]methionine-labeled APOBEC proteins incubated together with GST-DND1α̃ and GST beads were divided into two. Each aliquot was treated with RNase (+) or DNAse (+) or not treated (−) prior to electrophoresis.

Mentions: The TnT Coupled Reticulocyte Lysate Systems (Promega) was used to generate [35S]methionine-labeled APOBEC proteins in in vitro transcription and coupled translation reactions (see Experimental Procedures section) (Fig. 2b). For controls, human APOBEC1 (hA1) and human ACF were also generated by in vitro transcription/translation of cloned expression constructs (Fig. 2e). The sizes and amount of translated [35S]methionine-labeled APOBEC protein products were determined by electrophoreses.


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 vitro.(a) Experimental design to test interaction of DND1 to the APOBEC proteins in vitro. All reactions contained [35S]methionine-labeled APOBEC proteins (blue circles). The labeled proteins were incubated with purified GST-DND1 (indicated by+in panels c–e). Glutathione Sepharose 4B beads (beads, white circles) were added to the reactions. The beads are able to bind to the GST moiety of DND1 and should also “pull-down” proteins that associate with GST-DND1. Control incubations used beads alone incubated with labeled APOBEC proteins (indicated by–in panels c–e). (b) In vitro transcribed/translated [35S]methionine-labeled APOBEC proteins. 5 μL was loaded from each translation reaction. A1 (APOBEC1), A2 (APOBEC2), A3 (8) (8-exon isoform APOBEC3) and A3 (9) (9-exon isoform of APOBEC3). The translated proteins used as input are marked with red bars (in panels b, d–f). (c) Binding of APOBEC proteins to GST-DND1α. 40 μL of [35S]methionine-labeled APOBEC proteins was incubated with GST beads and either with (+) or without (−) GST-DND1α. Lanes 5 and 7 show binding of APOBEC3 to GST-DND1α. Lanes 6 and 8 show no binding of APOBEC3 to the beads. (d) Binding of APOBEC proteins to GST-DND1β. 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST-DND1β. Lanes 3 and 5 show binding of APOBEC3 to GST-DND1β. Lanes 4 and 6 show no binding of APOBEC3 to the beads. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (e) Testing interaction of human APOBEC1 and ACF to GST-DND1α. 40 μL of [35S]methionine-labeled human APOBEC1 (hA1), mouse APOBEC2 (A2) and human ACF were incubated with GST beads and either with (+) or without (−) GST-DND1α. Comparison of lanes 1 and 2 indicate weak binding of human APOBEC1 (hA1) to GST-DND1α. Comparison of lanes 5 and 6 indicate no specific binding of ACF to GST-DND1α. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (f) Binding of APOBEC proteins to GST (second control). 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (g) Effect of RNase (left) and DNase (right) on APOBEC3 and GST-DND1 interaction. [35S]methionine-labeled APOBEC proteins incubated together with GST-DND1α̃ and GST beads were divided into two. Each aliquot was treated with RNase (+) or DNAse (+) or not treated (−) prior to electrophoresis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002315-g002: DND1 interacts with APOBEC3 in vitro.(a) Experimental design to test interaction of DND1 to the APOBEC proteins in vitro. All reactions contained [35S]methionine-labeled APOBEC proteins (blue circles). The labeled proteins were incubated with purified GST-DND1 (indicated by+in panels c–e). Glutathione Sepharose 4B beads (beads, white circles) were added to the reactions. The beads are able to bind to the GST moiety of DND1 and should also “pull-down” proteins that associate with GST-DND1. Control incubations used beads alone incubated with labeled APOBEC proteins (indicated by–in panels c–e). (b) In vitro transcribed/translated [35S]methionine-labeled APOBEC proteins. 5 μL was loaded from each translation reaction. A1 (APOBEC1), A2 (APOBEC2), A3 (8) (8-exon isoform APOBEC3) and A3 (9) (9-exon isoform of APOBEC3). The translated proteins used as input are marked with red bars (in panels b, d–f). (c) Binding of APOBEC proteins to GST-DND1α. 40 μL of [35S]methionine-labeled APOBEC proteins was incubated with GST beads and either with (+) or without (−) GST-DND1α. Lanes 5 and 7 show binding of APOBEC3 to GST-DND1α. Lanes 6 and 8 show no binding of APOBEC3 to the beads. (d) Binding of APOBEC proteins to GST-DND1β. 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST-DND1β. Lanes 3 and 5 show binding of APOBEC3 to GST-DND1β. Lanes 4 and 6 show no binding of APOBEC3 to the beads. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (e) Testing interaction of human APOBEC1 and ACF to GST-DND1α. 40 μL of [35S]methionine-labeled human APOBEC1 (hA1), mouse APOBEC2 (A2) and human ACF were incubated with GST beads and either with (+) or without (−) GST-DND1α. Comparison of lanes 1 and 2 indicate weak binding of human APOBEC1 (hA1) to GST-DND1α. Comparison of lanes 5 and 6 indicate no specific binding of ACF to GST-DND1α. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (f) Binding of APOBEC proteins to GST (second control). 40 μL of [35S]methionine-labeled APOBEC proteins (APOBEC1 and both isoforms of APOBEC3) were incubated with GST beads and either with (+) or without (−) GST. Lanes, 7, 8 and 9 are the translated proteins used as input (5 μL). (g) Effect of RNase (left) and DNase (right) on APOBEC3 and GST-DND1 interaction. [35S]methionine-labeled APOBEC proteins incubated together with GST-DND1α̃ and GST beads were divided into two. Each aliquot was treated with RNase (+) or DNAse (+) or not treated (−) prior to electrophoresis.
Mentions: The TnT Coupled Reticulocyte Lysate Systems (Promega) was used to generate [35S]methionine-labeled APOBEC proteins in in vitro transcription and coupled translation reactions (see Experimental Procedures section) (Fig. 2b). For controls, human APOBEC1 (hA1) and human ACF were also generated by in vitro transcription/translation of cloned expression constructs (Fig. 2e). The sizes and amount of translated [35S]methionine-labeled APOBEC protein products were determined by electrophoreses.

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