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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.

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The ectopic expression of recombinant PARG-EGFP protein in Parg27.1 mutant animals rescues PARP1-DsRed localization to chromatin.The expression of UAS::PARG-EGFP transgene was induced in early second-instar larvae (A, B), or in early third-instar larvae (C), using hsp::Gal4 driver induction. Salivary glands were dissected and analyzed by confocal microscopy after 20 hrs following Gal4 induction (A, C) and after 30 hrs following Gal4 induction (B). PARG-EGFP protein is green; PARP1-DsRed is red, and DNA (Draq5) is blue. An arrow indicates colocalization of PARG-EGFP and PARP1-DsRed in the CB-like particle. D. The expression of recombinant catalytically inactive PARGAA-EGFP protein in Parg27.1 mutant animals does not rescue PARP1-DsRed protein localization to chromatin (after 30 hrs following Gal4 induction). E. Western blot hybridization was used to detect the accumulation of pADPr in Parg27.1 mutant and Parg27.1 mutant expressing PARGAA-EGFP protein, but not in wild-type and Parg27.1 mutant expressing PARG-EGFP protein.
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pgen-1000387-g005: The ectopic expression of recombinant PARG-EGFP protein in Parg27.1 mutant animals rescues PARP1-DsRed localization to chromatin.The expression of UAS::PARG-EGFP transgene was induced in early second-instar larvae (A, B), or in early third-instar larvae (C), using hsp::Gal4 driver induction. Salivary glands were dissected and analyzed by confocal microscopy after 20 hrs following Gal4 induction (A, C) and after 30 hrs following Gal4 induction (B). PARG-EGFP protein is green; PARP1-DsRed is red, and DNA (Draq5) is blue. An arrow indicates colocalization of PARG-EGFP and PARP1-DsRed in the CB-like particle. D. The expression of recombinant catalytically inactive PARGAA-EGFP protein in Parg27.1 mutant animals does not rescue PARP1-DsRed protein localization to chromatin (after 30 hrs following Gal4 induction). E. Western blot hybridization was used to detect the accumulation of pADPr in Parg27.1 mutant and Parg27.1 mutant expressing PARGAA-EGFP protein, but not in wild-type and Parg27.1 mutant expressing PARG-EGFP protein.

Mentions: In the following experiments, we examined if inducible expression of recombinant PARG protein could suppress accumulation of PARP1-DsRed protein in HLB/CBs and rescue the parg27.1 mutant phenotypes. To accomplish this, we expressed a previously constructed epitope-tagged version of UASt::PARG-EGFP transgenic construct [10] in parg27.1 mutant animals, along with PARP1-DsRed. When expressed using constitutive 69B Gal4-driver [25], PARG-EGFP rescues parg27.1 lethality. Using temperature-inducible hsp::Gal4 activator (gift of G. Cavalli lab), we were also able to suppress parg27.1 phenotypes. When expression of hsp::Gal4 was induced before third-instar larvae stage, the PARG-EGFP protein was strongly accumulated in nucleoplasm (Figure 5A, B). No ectopic PARP1-DsRed-containing bodies were observed in those animals. Moreover, 10–20 hours after Gal4 transgene induction, the PARP1-DsRed protein accumulation appeared in a subset of chromosomal loci (Figure 5A); after 30 hours, the PARP1-DsRed protein distribution in chromatin was similar to that of the wild-type (Figure 5B). Late expression of the PARG-EGFP (in third-instar larvae) did not rescue parg27.1 mutants. In this case, the accumulation of PARG-EGFP in nucleoplasm still occurs, but the amount of PARG protein is not sufficient to rescue mutant phenotypes. Therefore, the nucleoplasmic bodies persist and absorb the PARP1-DsRed (Figure 5C). It is also interesting to note that such conditions result in the presence of a detectable amount of PARG-EGFP protein in the HLB/CBs (Figure 5C). This last result suggests that the PARG protein, as well as PARP1, may be one of the HLB/CB components. Moreover, the same experiment performed with catalytically inactive PARGAA-EGFP proteins (see Material and Methods) does not rescue PARP1 protein localization to chromatin (Figure 5D) and does not suppress accumulation of pADPr (Figure 5E). This, in turn, implicates that the regulation of pADPr turnover may be one function of HLB/CB. If this is true, then poly(ADP-ribosyl)ated proteins necessarily pass through CB, giving further evidence that PARP1 is a key component of the CB transcription machinery, as proposed above. Considered together, these results 1) support the hypothesis that PARG enzyme cleaves pADPr in HLB/CBs and recycles proteins modified in chromatin and nucleoli, including PARP1 protein itself and 2) implicate these processes in maintaining control over the protein flow through Cajal bodies, a dynamic that we have proposed and demonstrated in this report.


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

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

The ectopic expression of recombinant PARG-EGFP protein in Parg27.1 mutant animals rescues PARP1-DsRed localization to chromatin.The expression of UAS::PARG-EGFP transgene was induced in early second-instar larvae (A, B), or in early third-instar larvae (C), using hsp::Gal4 driver induction. Salivary glands were dissected and analyzed by confocal microscopy after 20 hrs following Gal4 induction (A, C) and after 30 hrs following Gal4 induction (B). PARG-EGFP protein is green; PARP1-DsRed is red, and DNA (Draq5) is blue. An arrow indicates colocalization of PARG-EGFP and PARP1-DsRed in the CB-like particle. D. The expression of recombinant catalytically inactive PARGAA-EGFP protein in Parg27.1 mutant animals does not rescue PARP1-DsRed protein localization to chromatin (after 30 hrs following Gal4 induction). E. Western blot hybridization was used to detect the accumulation of pADPr in Parg27.1 mutant and Parg27.1 mutant expressing PARGAA-EGFP protein, but not in wild-type and Parg27.1 mutant expressing PARG-EGFP protein.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000387-g005: The ectopic expression of recombinant PARG-EGFP protein in Parg27.1 mutant animals rescues PARP1-DsRed localization to chromatin.The expression of UAS::PARG-EGFP transgene was induced in early second-instar larvae (A, B), or in early third-instar larvae (C), using hsp::Gal4 driver induction. Salivary glands were dissected and analyzed by confocal microscopy after 20 hrs following Gal4 induction (A, C) and after 30 hrs following Gal4 induction (B). PARG-EGFP protein is green; PARP1-DsRed is red, and DNA (Draq5) is blue. An arrow indicates colocalization of PARG-EGFP and PARP1-DsRed in the CB-like particle. D. The expression of recombinant catalytically inactive PARGAA-EGFP protein in Parg27.1 mutant animals does not rescue PARP1-DsRed protein localization to chromatin (after 30 hrs following Gal4 induction). E. Western blot hybridization was used to detect the accumulation of pADPr in Parg27.1 mutant and Parg27.1 mutant expressing PARGAA-EGFP protein, but not in wild-type and Parg27.1 mutant expressing PARG-EGFP protein.
Mentions: In the following experiments, we examined if inducible expression of recombinant PARG protein could suppress accumulation of PARP1-DsRed protein in HLB/CBs and rescue the parg27.1 mutant phenotypes. To accomplish this, we expressed a previously constructed epitope-tagged version of UASt::PARG-EGFP transgenic construct [10] in parg27.1 mutant animals, along with PARP1-DsRed. When expressed using constitutive 69B Gal4-driver [25], PARG-EGFP rescues parg27.1 lethality. Using temperature-inducible hsp::Gal4 activator (gift of G. Cavalli lab), we were also able to suppress parg27.1 phenotypes. When expression of hsp::Gal4 was induced before third-instar larvae stage, the PARG-EGFP protein was strongly accumulated in nucleoplasm (Figure 5A, B). No ectopic PARP1-DsRed-containing bodies were observed in those animals. Moreover, 10–20 hours after Gal4 transgene induction, the PARP1-DsRed protein accumulation appeared in a subset of chromosomal loci (Figure 5A); after 30 hours, the PARP1-DsRed protein distribution in chromatin was similar to that of the wild-type (Figure 5B). Late expression of the PARG-EGFP (in third-instar larvae) did not rescue parg27.1 mutants. In this case, the accumulation of PARG-EGFP in nucleoplasm still occurs, but the amount of PARG protein is not sufficient to rescue mutant phenotypes. Therefore, the nucleoplasmic bodies persist and absorb the PARP1-DsRed (Figure 5C). It is also interesting to note that such conditions result in the presence of a detectable amount of PARG-EGFP protein in the HLB/CBs (Figure 5C). This last result suggests that the PARG protein, as well as PARP1, may be one of the HLB/CB components. Moreover, the same experiment performed with catalytically inactive PARGAA-EGFP proteins (see Material and Methods) does not rescue PARP1 protein localization to chromatin (Figure 5D) and does not suppress accumulation of pADPr (Figure 5E). This, in turn, implicates that the regulation of pADPr turnover may be one function of HLB/CB. If this is true, then poly(ADP-ribosyl)ated proteins necessarily pass through CB, giving further evidence that PARP1 is a key component of the CB transcription machinery, as proposed above. Considered together, these results 1) support the hypothesis that PARG enzyme cleaves pADPr in HLB/CBs and recycles proteins modified in chromatin and nucleoli, including PARP1 protein itself and 2) implicate these processes in maintaining control over the protein flow through Cajal bodies, a dynamic that we have proposed and demonstrated in this report.

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