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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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Two-hybrid analysis of the interaction between AtCPSF30 and AtFip1(V). The battery of mutants tested in the assay is shown on the left; as in Figure 4A, the three zinc fingers are represented as black bars, and mutated zinc fingers depicted as gray bars. The extents of each protein present in the two-hybrid construct are shown for each variant; the representation is not drawn to scale. The results of the tests are summarized on the right; ‘yes’ indicates that different independently isolated dual transformants were able to grow on the selective media (and therefore denotes an interaction in yeast cells), whereas ‘no’ indicates an inability to grow on such media.
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Figure 6: Two-hybrid analysis of the interaction between AtCPSF30 and AtFip1(V). The battery of mutants tested in the assay is shown on the left; as in Figure 4A, the three zinc fingers are represented as black bars, and mutated zinc fingers depicted as gray bars. The extents of each protein present in the two-hybrid construct are shown for each variant; the representation is not drawn to scale. The results of the tests are summarized on the right; ‘yes’ indicates that different independently isolated dual transformants were able to grow on the selective media (and therefore denotes an interaction in yeast cells), whereas ‘no’ indicates an inability to grow on such media.

Mentions: The third zinc finger of AtCPSF30 corresponds to the motif of Yth1 that is involved in its interaction with Fip1, an interaction that affects the binding of Yth1 to RNA (14). Since this is also the motif that is needed for endonuclease activity (Figure 4), it seemed possible that plant Fip1 orthologs might have effects on the nuclease activity of AtCPSF30. Previously, it was reported that an Arabidopsis Fip1 ortholog, encoded by At5g58040, was able to interact with AtCPSF30, and that the N-terminal 137 amino acids of the 1196 amino acid Fip1 ortholog [AtFip1(V)] contained the domain responsible for this interaction (24). This interaction was further dissected using a standard yeast two-hybrid assay. For this, the battery of AtCPSF30 mutants illustrated in Figure 4A, along with others described previously (30), were tested for interactions with the N-terminus of AtFip1(V). As summarized in Figure 6, all of the variants that possessed the third zinc finger retained the ability to interact with AtFip1(V); importantly, alteration of just the third zinc finger eliminated the interaction, indicating that this interaction requires just the third zinc finger motif of AtCPSF30.Figure 6.


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)

Two-hybrid analysis of the interaction between AtCPSF30 and AtFip1(V). The battery of mutants tested in the assay is shown on the left; as in Figure 4A, the three zinc fingers are represented as black bars, and mutated zinc fingers depicted as gray bars. The extents of each protein present in the two-hybrid construct are shown for each variant; the representation is not drawn to scale. The results of the tests are summarized on the right; ‘yes’ indicates that different independently isolated dual transformants were able to grow on the selective media (and therefore denotes an interaction in yeast cells), whereas ‘no’ indicates an inability to grow on such media.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Two-hybrid analysis of the interaction between AtCPSF30 and AtFip1(V). The battery of mutants tested in the assay is shown on the left; as in Figure 4A, the three zinc fingers are represented as black bars, and mutated zinc fingers depicted as gray bars. The extents of each protein present in the two-hybrid construct are shown for each variant; the representation is not drawn to scale. The results of the tests are summarized on the right; ‘yes’ indicates that different independently isolated dual transformants were able to grow on the selective media (and therefore denotes an interaction in yeast cells), whereas ‘no’ indicates an inability to grow on such media.
Mentions: The third zinc finger of AtCPSF30 corresponds to the motif of Yth1 that is involved in its interaction with Fip1, an interaction that affects the binding of Yth1 to RNA (14). Since this is also the motif that is needed for endonuclease activity (Figure 4), it seemed possible that plant Fip1 orthologs might have effects on the nuclease activity of AtCPSF30. Previously, it was reported that an Arabidopsis Fip1 ortholog, encoded by At5g58040, was able to interact with AtCPSF30, and that the N-terminal 137 amino acids of the 1196 amino acid Fip1 ortholog [AtFip1(V)] contained the domain responsible for this interaction (24). This interaction was further dissected using a standard yeast two-hybrid assay. For this, the battery of AtCPSF30 mutants illustrated in Figure 4A, along with others described previously (30), were tested for interactions with the N-terminus of AtFip1(V). As summarized in Figure 6, all of the variants that possessed the third zinc finger retained the ability to interact with AtFip1(V); importantly, alteration of just the third zinc finger eliminated the interaction, indicating that this interaction requires just the third zinc finger motif of AtCPSF30.Figure 6.

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