Limits...
Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Kotova E, Jarnik M, Tulin AV - PLoS Genet. (2009)

Bottom Line: At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear.Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo.We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

Show MeSH

Related in: MedlinePlus

The components of Cajal body are targets for pADPr.(A) The immunostaining of thick sections prepared from Parg27.1 mutant salivary gland is shown. “Free” nucleoplasmic bodies (arrow) and “chromatin-embedded” CBs (arrowhead) are shown. Specific antibodies were used: rabbit anti-pADPr (green) and mouse anti-histone H1 (blue). PARP1-DsRed (red) was visualized by DsRed autofluorescence. Inset. The single CB-like particle is magnified. Arrow indicates accumulation of pADPr in the CB cavity. Arrowhead shows enrichment of PARP1 protein in the CB matrix. (B, C) Immunostaining of Parg27.1 mutant salivary gland is shown. Specific antibodies were used: mouse (10H) anti-pADPr (green) (B–C); Guinea Pig anti-Coilin (blue) (B) and rabbit anti-Fibrillarin (C). PARP1-DsRed (red) was visualized by DsRed autofluorescence. (B) The single chromatin-embedded CB-like particle is presented. Arrow indicates the colocalization of Coilin and pADPr on periphery of CB. (C) Three CBs are shown. Arrows show the colocalization of Fibrillarin and pADPr on periphery of CBs. (D) Immunoprecipitation assays using mouse and rabbit antibody against pADPr. Wild-type Drosophila stock was used to prepare protein extracts. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin, mouse anti-pADPr, rabbit anti-Fibrillarin, rabbit anti-LSM11 and mouse anti-Lamin C. Arrow indicates hypermodified isoform of Coilin. (E) Western blot analysis of total protein extracts from wild-type (WT), Parp1 (PARP−) and Parg (PARG−) mutant flies was performed. Rabbit anti-Coilin antibody was used. Arrow indicates hypermodified isoform of Coilin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2637609&req=5

pgen-1000387-g006: The components of Cajal body are targets for pADPr.(A) The immunostaining of thick sections prepared from Parg27.1 mutant salivary gland is shown. “Free” nucleoplasmic bodies (arrow) and “chromatin-embedded” CBs (arrowhead) are shown. Specific antibodies were used: rabbit anti-pADPr (green) and mouse anti-histone H1 (blue). PARP1-DsRed (red) was visualized by DsRed autofluorescence. Inset. The single CB-like particle is magnified. Arrow indicates accumulation of pADPr in the CB cavity. Arrowhead shows enrichment of PARP1 protein in the CB matrix. (B, C) Immunostaining of Parg27.1 mutant salivary gland is shown. Specific antibodies were used: mouse (10H) anti-pADPr (green) (B–C); Guinea Pig anti-Coilin (blue) (B) and rabbit anti-Fibrillarin (C). PARP1-DsRed (red) was visualized by DsRed autofluorescence. (B) The single chromatin-embedded CB-like particle is presented. Arrow indicates the colocalization of Coilin and pADPr on periphery of CB. (C) Three CBs are shown. Arrows show the colocalization of Fibrillarin and pADPr on periphery of CBs. (D) Immunoprecipitation assays using mouse and rabbit antibody against pADPr. Wild-type Drosophila stock was used to prepare protein extracts. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin, mouse anti-pADPr, rabbit anti-Fibrillarin, rabbit anti-LSM11 and mouse anti-Lamin C. Arrow indicates hypermodified isoform of Coilin. (E) Western blot analysis of total protein extracts from wild-type (WT), Parp1 (PARP−) and Parg (PARG−) mutant flies was performed. Rabbit anti-Coilin antibody was used. Arrow indicates hypermodified isoform of Coilin.

Mentions: Multiple in vivo and in vitro observations (reviewed in [2]) report that the main, if not the exclusive, target for pADPr reaction is the PARP1 protein itself. Therefore, we investigated whether most pADPr molecules co-migrate into the same nucleoplasmic domains where PARP1 is localized. In order to test this in respect to the ultrastructure of ectopic HLB/CBs, we performed immunostaining of thick sections prepared from the salivary glands of parg27.1 mutant third-instar larvae. As the PARP1-DsRed protein relocated from chromatin, we observed two types of nucleoplasmic PARP1-containing particles: “embedded” in chromatin (one or four per nucleus) (Figure 6A; Figure S7, arrowhead) and “free” nucleoplasmic (five to forty per nucleus) (Figure 6A; Figure S7, arrow). The position of “embedded” bodies is similar to that of normal CBs [22], and these “embedded” bodies also often demonstrate structural properties similar to those of classical CBs [20] by showing PARP1-positive CB matrix and pADPr-positive CB cavity (Figure 6A, Inset; Fig S7A, Inset). As expected [10], a significant amount of pADPr is associated with these PARP1-containing bodies (Figure 6A). However, the “chromatin-embedded” particles demonstrated separation of PARP1-DsRed from the main pool of pADPr (Figure 6A, Inset). To confirm this segregation, we performed immuno-gold staining of thin sections prepared from the same materials, followed by electron microscopy.


Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Kotova E, Jarnik M, Tulin AV - PLoS Genet. (2009)

The components of Cajal body are targets for pADPr.(A) The immunostaining of thick sections prepared from Parg27.1 mutant salivary gland is shown. “Free” nucleoplasmic bodies (arrow) and “chromatin-embedded” CBs (arrowhead) are shown. Specific antibodies were used: rabbit anti-pADPr (green) and mouse anti-histone H1 (blue). PARP1-DsRed (red) was visualized by DsRed autofluorescence. Inset. The single CB-like particle is magnified. Arrow indicates accumulation of pADPr in the CB cavity. Arrowhead shows enrichment of PARP1 protein in the CB matrix. (B, C) Immunostaining of Parg27.1 mutant salivary gland is shown. Specific antibodies were used: mouse (10H) anti-pADPr (green) (B–C); Guinea Pig anti-Coilin (blue) (B) and rabbit anti-Fibrillarin (C). PARP1-DsRed (red) was visualized by DsRed autofluorescence. (B) The single chromatin-embedded CB-like particle is presented. Arrow indicates the colocalization of Coilin and pADPr on periphery of CB. (C) Three CBs are shown. Arrows show the colocalization of Fibrillarin and pADPr on periphery of CBs. (D) Immunoprecipitation assays using mouse and rabbit antibody against pADPr. Wild-type Drosophila stock was used to prepare protein extracts. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin, mouse anti-pADPr, rabbit anti-Fibrillarin, rabbit anti-LSM11 and mouse anti-Lamin C. Arrow indicates hypermodified isoform of Coilin. (E) Western blot analysis of total protein extracts from wild-type (WT), Parp1 (PARP−) and Parg (PARG−) mutant flies was performed. Rabbit anti-Coilin antibody was used. Arrow indicates hypermodified isoform of Coilin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637609&req=5

pgen-1000387-g006: The components of Cajal body are targets for pADPr.(A) The immunostaining of thick sections prepared from Parg27.1 mutant salivary gland is shown. “Free” nucleoplasmic bodies (arrow) and “chromatin-embedded” CBs (arrowhead) are shown. Specific antibodies were used: rabbit anti-pADPr (green) and mouse anti-histone H1 (blue). PARP1-DsRed (red) was visualized by DsRed autofluorescence. Inset. The single CB-like particle is magnified. Arrow indicates accumulation of pADPr in the CB cavity. Arrowhead shows enrichment of PARP1 protein in the CB matrix. (B, C) Immunostaining of Parg27.1 mutant salivary gland is shown. Specific antibodies were used: mouse (10H) anti-pADPr (green) (B–C); Guinea Pig anti-Coilin (blue) (B) and rabbit anti-Fibrillarin (C). PARP1-DsRed (red) was visualized by DsRed autofluorescence. (B) The single chromatin-embedded CB-like particle is presented. Arrow indicates the colocalization of Coilin and pADPr on periphery of CB. (C) Three CBs are shown. Arrows show the colocalization of Fibrillarin and pADPr on periphery of CBs. (D) Immunoprecipitation assays using mouse and rabbit antibody against pADPr. Wild-type Drosophila stock was used to prepare protein extracts. To detect protein on Western blots, the following antibodies were used: Guinea Pig anti-Coilin, mouse anti-pADPr, rabbit anti-Fibrillarin, rabbit anti-LSM11 and mouse anti-Lamin C. Arrow indicates hypermodified isoform of Coilin. (E) Western blot analysis of total protein extracts from wild-type (WT), Parp1 (PARP−) and Parg (PARG−) mutant flies was performed. Rabbit anti-Coilin antibody was used. Arrow indicates hypermodified isoform of Coilin.
Mentions: Multiple in vivo and in vitro observations (reviewed in [2]) report that the main, if not the exclusive, target for pADPr reaction is the PARP1 protein itself. Therefore, we investigated whether most pADPr molecules co-migrate into the same nucleoplasmic domains where PARP1 is localized. In order to test this in respect to the ultrastructure of ectopic HLB/CBs, we performed immunostaining of thick sections prepared from the salivary glands of parg27.1 mutant third-instar larvae. As the PARP1-DsRed protein relocated from chromatin, we observed two types of nucleoplasmic PARP1-containing particles: “embedded” in chromatin (one or four per nucleus) (Figure 6A; Figure S7, arrowhead) and “free” nucleoplasmic (five to forty per nucleus) (Figure 6A; Figure S7, arrow). The position of “embedded” bodies is similar to that of normal CBs [22], and these “embedded” bodies also often demonstrate structural properties similar to those of classical CBs [20] by showing PARP1-positive CB matrix and pADPr-positive CB cavity (Figure 6A, Inset; Fig S7A, Inset). As expected [10], a significant amount of pADPr is associated with these PARP1-containing bodies (Figure 6A). However, the “chromatin-embedded” particles demonstrated separation of PARP1-DsRed from the main pool of pADPr (Figure 6A, Inset). To confirm this segregation, we performed immuno-gold staining of thin sections prepared from the same materials, followed by electron microscopy.

Bottom Line: At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear.Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo.We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

Show MeSH
Related in: MedlinePlus