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

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.

Genome-Wide Control of Polyadenylation Site Choice by CPSF30 in Arabidopsis

“…Of these subunits, one of the more enigmatic is the 30-kD subunit of the Cleavage and Polyadenylation Stimulatory Factor (termed in this report as CPSF30). The Arabidopsis thaliana CPSF30 possesses three characteristic CCCH zinc finger motifs; these correspond to three of the five motifs found in the mammalian CPSF30 and its yeast counterpart, YTH1p (Delaney et al, 2006;Addepalli and Hunt, 2007). Together, these three motifs constitute the most highly conserved parts of the protein.…”

“…Together, these three motifs constitute the most highly conserved parts of the protein. Like its mammalian and yeast counterparts, the Arabidopsis CPSF30 is an RNA binding protein (Delaney et al, 2006;Addepalli and Hunt, 2007). The RNA binding activity of the Arabidopsis CPSF30 is largely determined by the N-terminal zinc finger motif (Addepalli and Hunt, 2007).…”

“…Like its mammalian and yeast counterparts, the Arabidopsis CPSF30 is an RNA binding protein (Delaney et al, 2006;Addepalli and Hunt, 2007). The RNA binding activity of the Arabidopsis CPSF30 is largely determined by the N-terminal zinc finger motif (Addepalli and Hunt, 2007). Arabidopsis CPSF30 is also an endonuclease, the action of which leaves a 39-OH group that is a suitable substrate for poly(A) polymerase (Addepalli and Hunt, 2007).…”

“…The RNA binding activity of the Arabidopsis CPSF30 is largely determined by the N-terminal zinc finger motif (Addepalli and Hunt, 2007). Arabidopsis CPSF30 is also an endonuclease, the action of which leaves a 39-OH group that is a suitable substrate for poly(A) polymerase (Addepalli and Hunt, 2007). The endonuclease activity is attributable to the C-terminal zinc finger motif.…”

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

“…The transcription template produced by PCR (using the primers Tyr-5Ј-T7 and Tyr-3Ј; supplemental Table S3) was in vitro transcribed, and the RNA was purified by an Ampliscribe kit (Epicenter Biotechnologies) and a MEGAclear kit (Ambion), respectively, as described (62).…”