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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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Mouse DND1 and APOBEC3 co-localize to peri-nuclear sites in the cell cytoplasm.(a) COS7 cells were transfected with expression constructs for GFP-Dnd1 for visualization of green fluorescence due to GFP-DND1. Cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by confocal microscopy. (d) COS7 cells were transfected with expression constructs for mCherry-Apobec3 for visualization of red fluorescence due to mCherry-Apobec3 by confocal microscopy. (b,c,e and f) COS7 cells were co-transfected with expression constructs for GFP-DND1 and mCherry-Apobec3. (b) Red fluorescence due to mCherry-APOBEC3 was imaged using red filter (excitation at 548 nm). (c) Green fluorescence due to GFP-DND1 was imaged using green filter (excitation at 488 nm). (f) The merged image of cells co-transfected with GFP-Dnd1 and mCherry-Apobec3, which shows GFP and mCherry signal co-localized at the peri-nuclear regions of the cells. (e) The DIC (differential interference contrast) image of the cells.
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pone-0002315-g005: Mouse DND1 and APOBEC3 co-localize to peri-nuclear sites in the cell cytoplasm.(a) COS7 cells were transfected with expression constructs for GFP-Dnd1 for visualization of green fluorescence due to GFP-DND1. Cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by confocal microscopy. (d) COS7 cells were transfected with expression constructs for mCherry-Apobec3 for visualization of red fluorescence due to mCherry-Apobec3 by confocal microscopy. (b,c,e and f) COS7 cells were co-transfected with expression constructs for GFP-DND1 and mCherry-Apobec3. (b) Red fluorescence due to mCherry-APOBEC3 was imaged using red filter (excitation at 548 nm). (c) Green fluorescence due to GFP-DND1 was imaged using green filter (excitation at 488 nm). (f) The merged image of cells co-transfected with GFP-Dnd1 and mCherry-Apobec3, which shows GFP and mCherry signal co-localized at the peri-nuclear regions of the cells. (e) The DIC (differential interference contrast) image of the cells.

Mentions: To visualize the cellular localization of the DND1 and APOBEC3, we transfected expression plasmids encoding GFP-Dnd1 and mCherry-Apobec3 into different mammalian cells such as COS7, NIH3T3 and 293T cells. The transfected cells were observed using a Zeiss LSM 510 confocal microscope. Overall, when either GFP-DND1 or mCherry-APOBEC3 were transfected in cells, the expression of the fluorescent-tagged proteins was predominantly in the cytoplasm (Fig. 5a and d). However, when both GFP-DND1 and mCherry-APOBEC3 were co-transfected in COS7 cells, we observed a sharp overlap of the green and red signals to form a ring-like zone surrounding the nucleus indicating co-localization of the GFP-DND1 and mCherry-APOBEC3 fluorescent signals to perinuclear sites (Fig. 5b, c and f). The co-localization suggests that DND1 and APOBEC3 likely sequester each other.


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)

Mouse DND1 and APOBEC3 co-localize to peri-nuclear sites in the cell cytoplasm.(a) COS7 cells were transfected with expression constructs for GFP-Dnd1 for visualization of green fluorescence due to GFP-DND1. Cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by confocal microscopy. (d) COS7 cells were transfected with expression constructs for mCherry-Apobec3 for visualization of red fluorescence due to mCherry-Apobec3 by confocal microscopy. (b,c,e and f) COS7 cells were co-transfected with expression constructs for GFP-DND1 and mCherry-Apobec3. (b) Red fluorescence due to mCherry-APOBEC3 was imaged using red filter (excitation at 548 nm). (c) Green fluorescence due to GFP-DND1 was imaged using green filter (excitation at 488 nm). (f) The merged image of cells co-transfected with GFP-Dnd1 and mCherry-Apobec3, which shows GFP and mCherry signal co-localized at the peri-nuclear regions of the cells. (e) The DIC (differential interference contrast) image of the cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0002315-g005: Mouse DND1 and APOBEC3 co-localize to peri-nuclear sites in the cell cytoplasm.(a) COS7 cells were transfected with expression constructs for GFP-Dnd1 for visualization of green fluorescence due to GFP-DND1. Cells were fixed with 2% paraformaldehyde before visualization of fluorescent signal by confocal microscopy. (d) COS7 cells were transfected with expression constructs for mCherry-Apobec3 for visualization of red fluorescence due to mCherry-Apobec3 by confocal microscopy. (b,c,e and f) COS7 cells were co-transfected with expression constructs for GFP-DND1 and mCherry-Apobec3. (b) Red fluorescence due to mCherry-APOBEC3 was imaged using red filter (excitation at 548 nm). (c) Green fluorescence due to GFP-DND1 was imaged using green filter (excitation at 488 nm). (f) The merged image of cells co-transfected with GFP-Dnd1 and mCherry-Apobec3, which shows GFP and mCherry signal co-localized at the peri-nuclear regions of the cells. (e) The DIC (differential interference contrast) image of the cells.
Mentions: To visualize the cellular localization of the DND1 and APOBEC3, we transfected expression plasmids encoding GFP-Dnd1 and mCherry-Apobec3 into different mammalian cells such as COS7, NIH3T3 and 293T cells. The transfected cells were observed using a Zeiss LSM 510 confocal microscope. Overall, when either GFP-DND1 or mCherry-APOBEC3 were transfected in cells, the expression of the fluorescent-tagged proteins was predominantly in the cytoplasm (Fig. 5a and d). However, when both GFP-DND1 and mCherry-APOBEC3 were co-transfected in COS7 cells, we observed a sharp overlap of the green and red signals to form a ring-like zone surrounding the nucleus indicating co-localization of the GFP-DND1 and mCherry-APOBEC3 fluorescent signals to perinuclear sites (Fig. 5b, c and f). The co-localization suggests that DND1 and APOBEC3 likely sequester each other.

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