Limits...
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.

Show MeSH

Related in: MedlinePlus

Cleavage by AtCPSF30 generates 3′OH groups. Unlabeled RNA (200 nM) was ligated with [32P]-pCp before pre-treatment with PAP + 3′-dATP (lane 1), after treatment with PAP + 3′-dATP (lane 2) and after treatment with PAP + 3′-dATP and subsequent incubation with AtCPSF30 (300 nM) for 10 min at 30°C (Lane 3). The positions of RNA size standards are indicated to the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1935010&req=5

Figure 2: Cleavage by AtCPSF30 generates 3′OH groups. Unlabeled RNA (200 nM) was ligated with [32P]-pCp before pre-treatment with PAP + 3′-dATP (lane 1), after treatment with PAP + 3′-dATP (lane 2) and after treatment with PAP + 3′-dATP and subsequent incubation with AtCPSF30 (300 nM) for 10 min at 30°C (Lane 3). The positions of RNA size standards are indicated to the right.

Mentions: One possible function for the endonuclease activity of AtCPSF30 is the processing of the pre-mRNA as a prelude to the polyadenylation. Such a function would require that the nuclease action leaves a 3′-hydroxyl group at the end of the RNA; alternative modes of nuclease action, in contrast, might leave 3′-phosphates or 2′-3′ cyclic phosphates. To examine this, the products of AtCPSF30 endonucleolytic action were end-labeled with RNA ligase + [32P] cytidine 3′,5′-bisphosphate ([32P]-pCp); such a treatment is expected to label RNAs bearing 3′-hydroxyl groups, but not 3′- or 2′,3′-cyclic phosphates. As shown in Figure 2 (lane 1), the end-labeling treatment of the input (unlabeled) RNA yielded a single discrete species. Pre-treatment of the input RNA with 3′-dATP + PAP (Figure 2, lane 2) eliminated this labeling, indicating a requirement for a free 3′-hydroxyl for the labeling with [32P]-pCp. Subsequent treatment of the 3′-blocked input RNA with AtCPSF30 yielded an array of breakdown products that were readily labeled with ([32P]-pCp (Figure 2, lane 3). This result indicates that the nuclease action of AtCPSF30 yields 3′-hydroxyl groups. It should be noted that the RNAs labeled by [32P]-pCp in lane 3 of Figure 2 do not correspond in size to those expected if cleavage was occurring at the three ‘natural’ polyadenylation sites in the substrate RNA (the latter would be expected to yield 5′ cleavage products of 125, 145 and 175 nt) (32,35). Thus, while purified AtCPSF30 processes the substrate at distinct sites, by itself it does not recapitulate the exact 3′-end profile seen in vivo.Figure 2.


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)

Cleavage by AtCPSF30 generates 3′OH groups. Unlabeled RNA (200 nM) was ligated with [32P]-pCp before pre-treatment with PAP + 3′-dATP (lane 1), after treatment with PAP + 3′-dATP (lane 2) and after treatment with PAP + 3′-dATP and subsequent incubation with AtCPSF30 (300 nM) for 10 min at 30°C (Lane 3). The positions of RNA size standards are indicated to the right.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935010&req=5

Figure 2: Cleavage by AtCPSF30 generates 3′OH groups. Unlabeled RNA (200 nM) was ligated with [32P]-pCp before pre-treatment with PAP + 3′-dATP (lane 1), after treatment with PAP + 3′-dATP (lane 2) and after treatment with PAP + 3′-dATP and subsequent incubation with AtCPSF30 (300 nM) for 10 min at 30°C (Lane 3). The positions of RNA size standards are indicated to the right.
Mentions: One possible function for the endonuclease activity of AtCPSF30 is the processing of the pre-mRNA as a prelude to the polyadenylation. Such a function would require that the nuclease action leaves a 3′-hydroxyl group at the end of the RNA; alternative modes of nuclease action, in contrast, might leave 3′-phosphates or 2′-3′ cyclic phosphates. To examine this, the products of AtCPSF30 endonucleolytic action were end-labeled with RNA ligase + [32P] cytidine 3′,5′-bisphosphate ([32P]-pCp); such a treatment is expected to label RNAs bearing 3′-hydroxyl groups, but not 3′- or 2′,3′-cyclic phosphates. As shown in Figure 2 (lane 1), the end-labeling treatment of the input (unlabeled) RNA yielded a single discrete species. Pre-treatment of the input RNA with 3′-dATP + PAP (Figure 2, lane 2) eliminated this labeling, indicating a requirement for a free 3′-hydroxyl for the labeling with [32P]-pCp. Subsequent treatment of the 3′-blocked input RNA with AtCPSF30 yielded an array of breakdown products that were readily labeled with ([32P]-pCp (Figure 2, lane 3). This result indicates that the nuclease action of AtCPSF30 yields 3′-hydroxyl groups. It should be noted that the RNAs labeled by [32P]-pCp in lane 3 of Figure 2 do not correspond in size to those expected if cleavage was occurring at the three ‘natural’ polyadenylation sites in the substrate RNA (the latter would be expected to yield 5′ cleavage products of 125, 145 and 175 nt) (32,35). Thus, while purified AtCPSF30 processes the substrate at distinct sites, by itself it does not recapitulate the exact 3′-end profile seen in vivo.Figure 2.

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