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A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor.

Addepalli B, Hunt AG - Nucleic Acids Res. (2007)

Bottom Line: In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding.The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein.These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546-0312, USA.

ABSTRACT
The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3' ends with hydroxyl groups, as they can be labeled by RNA ligase with [32P]-cytidine-3',5'-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.

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Mutational analysis of the nuclease activity. (A) Illustration of AtCPSF30 and the various mutants. For the wild-type protein (‘wt’), the three zinc fingers are represented as black bars. The parts of the protein present in the m4 and m9 deletion mutants (30) are shown. Mutated zinc fingers for each of the three zinc finger mutants are depicted as gray bars. (B) Stained gel showing the various purified protein preparations.Three microliter of each purified protein preparation was separated on a 10% gel, which was subsequently stained with Coomassie Brilliant Blue. The identity of each preparation is indicated above the scan of the gel; the full-sized MBP-AtCPSF30 is denoted with an arrow besides the gel, and the previously described breakdown product (30) noted with an *. The high molecular weight bands seen in the MBP and m4 lanes are not consistently seen in all preparations. s—SeeBlue2 (Invitrogen) pre-stained protein size standards. (C) Nuclease activity of the AtCPSF30 variants. Uniformly labeled RNA (2 pmol = 200 nM) was incubated with purified MBP-AtCPSF30 mutant proteins or with MBP for 30 min at 30°C and the remaining RNA recovered and analyzed on a sequencing gel. The identity of the AtCPSF30 variant is indicated above the gel image (‘-’: no added protein); the quantities of the enzyme preparations were: MBP—12 pmol; ‘wt’ (wild-type AtCPSF30)—6 pmol; m4—12 pmol; m9—10 pmol; ZF1—10 pmol; ZF2—10 pmol; ZF3—10 pmol.
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Figure 4: Mutational analysis of the nuclease activity. (A) Illustration of AtCPSF30 and the various mutants. For the wild-type protein (‘wt’), the three zinc fingers are represented as black bars. The parts of the protein present in the m4 and m9 deletion mutants (30) are shown. Mutated zinc fingers for each of the three zinc finger mutants are depicted as gray bars. (B) Stained gel showing the various purified protein preparations.Three microliter of each purified protein preparation was separated on a 10% gel, which was subsequently stained with Coomassie Brilliant Blue. The identity of each preparation is indicated above the scan of the gel; the full-sized MBP-AtCPSF30 is denoted with an arrow besides the gel, and the previously described breakdown product (30) noted with an *. The high molecular weight bands seen in the MBP and m4 lanes are not consistently seen in all preparations. s—SeeBlue2 (Invitrogen) pre-stained protein size standards. (C) Nuclease activity of the AtCPSF30 variants. Uniformly labeled RNA (2 pmol = 200 nM) was incubated with purified MBP-AtCPSF30 mutant proteins or with MBP for 30 min at 30°C and the remaining RNA recovered and analyzed on a sequencing gel. The identity of the AtCPSF30 variant is indicated above the gel image (‘-’: no added protein); the quantities of the enzyme preparations were: MBP—12 pmol; ‘wt’ (wild-type AtCPSF30)—6 pmol; m4—12 pmol; m9—10 pmol; ZF1—10 pmol; ZF2—10 pmol; ZF3—10 pmol.

Mentions: The purification of MBP-AtCPSF30, m4 and m9 proteins (Figure 4) has been described elsewhere (30). The zinc finger mutants of AtCPSF30 (Figure 4) were generated by using quick-change site-directed mutagenesis kit (Stratagene) using the pMALC2-AtCPSF30 plasmid (30) as template as per manufacturer's instructions. The results of mutagenesis were confirmed by DNA sequencing; accordingly, the C-terminus of the first zinc finger was changed from DACGFLHQF to DASTFLYQ, the second zinc finger from QDCVYKHTN to QDSTYKYTN and the third from PDCRYRHAK to PDSTYRYAK. The oligonucleotide primers that were used for the mutagenesis are given in Table 1. The cloning of coding sequence corresponding to the N-terminal 137 of the chromosome V-encoded Arabidopsis Fip1 protein into bacterial protein expression vectors pGEX-2T (Pharmacia) and pMAL-2C for making GST and MBP fusion proteins, respectively, was as described (24). The coding sequence corresponding to the N-terminal 483 amino acids was cloned into pDEST17 vector (Invitrogen) from the corresponding entry clone (24) to produce histidine-tagged fusion protein. Histidine-tagged and GST fusion proteins were purified as described by Forbes et al. (24) and MBP fusion proteins purified as described in Delaney et al. (30). It should be pointed out that the purification of MBP and GST fusion proteins included a wash of proteins bound to the affinity media with buffers containing 2 M NaCl, a step intended to remove almost all non-specifically bound bacterial proteins from the MBP fusion protein preparations. Control proteins (GST, MBP and histidine-tagged β-glucuronidase) were prepared as described (24,30). Protein concentrations were estimated by comparing the purified preparations with BSA standards by SDS–PAGE and staining of the gels with Coomassie Brilliant Blue.Table 1.


A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor.

Addepalli B, Hunt AG - Nucleic Acids Res. (2007)

Mutational analysis of the nuclease activity. (A) Illustration of AtCPSF30 and the various mutants. For the wild-type protein (‘wt’), the three zinc fingers are represented as black bars. The parts of the protein present in the m4 and m9 deletion mutants (30) are shown. Mutated zinc fingers for each of the three zinc finger mutants are depicted as gray bars. (B) Stained gel showing the various purified protein preparations.Three microliter of each purified protein preparation was separated on a 10% gel, which was subsequently stained with Coomassie Brilliant Blue. The identity of each preparation is indicated above the scan of the gel; the full-sized MBP-AtCPSF30 is denoted with an arrow besides the gel, and the previously described breakdown product (30) noted with an *. The high molecular weight bands seen in the MBP and m4 lanes are not consistently seen in all preparations. s—SeeBlue2 (Invitrogen) pre-stained protein size standards. (C) Nuclease activity of the AtCPSF30 variants. Uniformly labeled RNA (2 pmol = 200 nM) was incubated with purified MBP-AtCPSF30 mutant proteins or with MBP for 30 min at 30°C and the remaining RNA recovered and analyzed on a sequencing gel. The identity of the AtCPSF30 variant is indicated above the gel image (‘-’: no added protein); the quantities of the enzyme preparations were: MBP—12 pmol; ‘wt’ (wild-type AtCPSF30)—6 pmol; m4—12 pmol; m9—10 pmol; ZF1—10 pmol; ZF2—10 pmol; ZF3—10 pmol.
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Figure 4: Mutational analysis of the nuclease activity. (A) Illustration of AtCPSF30 and the various mutants. For the wild-type protein (‘wt’), the three zinc fingers are represented as black bars. The parts of the protein present in the m4 and m9 deletion mutants (30) are shown. Mutated zinc fingers for each of the three zinc finger mutants are depicted as gray bars. (B) Stained gel showing the various purified protein preparations.Three microliter of each purified protein preparation was separated on a 10% gel, which was subsequently stained with Coomassie Brilliant Blue. The identity of each preparation is indicated above the scan of the gel; the full-sized MBP-AtCPSF30 is denoted with an arrow besides the gel, and the previously described breakdown product (30) noted with an *. The high molecular weight bands seen in the MBP and m4 lanes are not consistently seen in all preparations. s—SeeBlue2 (Invitrogen) pre-stained protein size standards. (C) Nuclease activity of the AtCPSF30 variants. Uniformly labeled RNA (2 pmol = 200 nM) was incubated with purified MBP-AtCPSF30 mutant proteins or with MBP for 30 min at 30°C and the remaining RNA recovered and analyzed on a sequencing gel. The identity of the AtCPSF30 variant is indicated above the gel image (‘-’: no added protein); the quantities of the enzyme preparations were: MBP—12 pmol; ‘wt’ (wild-type AtCPSF30)—6 pmol; m4—12 pmol; m9—10 pmol; ZF1—10 pmol; ZF2—10 pmol; ZF3—10 pmol.
Mentions: The purification of MBP-AtCPSF30, m4 and m9 proteins (Figure 4) has been described elsewhere (30). The zinc finger mutants of AtCPSF30 (Figure 4) were generated by using quick-change site-directed mutagenesis kit (Stratagene) using the pMALC2-AtCPSF30 plasmid (30) as template as per manufacturer's instructions. The results of mutagenesis were confirmed by DNA sequencing; accordingly, the C-terminus of the first zinc finger was changed from DACGFLHQF to DASTFLYQ, the second zinc finger from QDCVYKHTN to QDSTYKYTN and the third from PDCRYRHAK to PDSTYRYAK. The oligonucleotide primers that were used for the mutagenesis are given in Table 1. The cloning of coding sequence corresponding to the N-terminal 137 of the chromosome V-encoded Arabidopsis Fip1 protein into bacterial protein expression vectors pGEX-2T (Pharmacia) and pMAL-2C for making GST and MBP fusion proteins, respectively, was as described (24). The coding sequence corresponding to the N-terminal 483 amino acids was cloned into pDEST17 vector (Invitrogen) from the corresponding entry clone (24) to produce histidine-tagged fusion protein. Histidine-tagged and GST fusion proteins were purified as described by Forbes et al. (24) and MBP fusion proteins purified as described in Delaney et al. (30). It should be pointed out that the purification of MBP and GST fusion proteins included a wash of proteins bound to the affinity media with buffers containing 2 M NaCl, a step intended to remove almost all non-specifically bound bacterial proteins from the MBP fusion protein preparations. Control proteins (GST, MBP and histidine-tagged β-glucuronidase) were prepared as described (24,30). Protein concentrations were estimated by comparing the purified preparations with BSA standards by SDS–PAGE and staining of the gels with Coomassie Brilliant Blue.Table 1.

Bottom Line: In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding.The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein.These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546-0312, USA.

ABSTRACT
The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3' ends with hydroxyl groups, as they can be labeled by RNA ligase with [32P]-cytidine-3',5'-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.

Show MeSH
Related in: MedlinePlus