Evidence that polyadenylation factor CPSF-73 is the mRNA 3' processing endonuclease

Abstract

Generation of the polyadenylated 3' end of an mRNA requires an endonucleolytic cleavage followed by synthesis of the poly(A) tail. Despite the seeming simplicity of the reaction, more than a dozen polypeptides are required, and nearly all appear to be necessary for the cleavage reaction. Because of this complexity, the identity of the endonuclease has remained a mystery. Here we present evidence that a component of the cleavage-polyadenylation specificity factor CPSF-73 is the long-sought endonuclease. We first show, using site-specific labeling and UV-cross-linking, that a protein with properties of CPSF-73 is one of only two polypeptides in HeLa nuclear extract to contact the cleavage site in an AAUAAA-dependent manner. The recent identification of CPSF-73 as a possible member of the metallo-beta-lactamase family of Zn(2+)-dependent hydrolytic enzymes suggests that this contact may identify CPSF-73 as the nuclease. Supporting the significance of the putative hydrolytic lactamase domain in CPSF-73, we show that mutation of key residues predicted to be required for activity in the yeast CPSF-73 homolog result in lethality. Furthermore, in contrast to long held belief, but consistent with properties of metallo-beta-lactamases, we show that 3' cleavage is metal-dependent, likely reflecting a requirement for tightly protein-bound Zn(2+). Taken together, the available data provide strong evidence that CPSF-73 is the 3' processing endonuclease.

FIGURE 1.

Two Hela cell proteins photo cross-link to the SV40 L poly(A) site in vitro. (A) Diagram of the two SV40L pre-mRNA substrates used in this study. The asterisk in the site-specifically labeled (SSL) substrate denotes the natural SV40L cleavage site, the site-specific ³²P phosphate label, and the site of ligation between the 179-nucleotide upstream in vitro transcribed piece and the 30-nt downstream chemically synthesized piece. The uniformly labeled substrate (UL) was made by in vitro transcription of the DraI-digested pG3SVL-A plasmid using either [α-³²P]GTP or UTP. The underlined U was changed to A in the mutated poly(A) substrate (mutant). (B) UV cross-linking of both proteins is AAUAAA-dependent. Either the mutant or wild-type substrates, UL or SSL, were irradiated in nuclear extract, digested with RNAse A then immunoprecipitated with the anti-Cstf-64 monoclonal antibody 3A7. (CIP) calf intestinal alkaline phosphatase treatment just before IP; (H.D.) heat-denaturation just before IP. Input, I, either 7.5% or 10% of one 10-μL reaction. Prestained protein markers (M) are indicated with dashes. (C) As in B except: wild-type SV40L substrates and immunoprecipitated with the indicated antibody.

FIGURE 2.

Mutation of conserved residues in the metallo-β-lactamase and β-CASP consensus motifs of the yeast CPSF-73 homolog Ysh1p causes lethality. (A) Abbreviated sequence alignment between scYsh1p, hCPSF-73, and hArtemis showing the β-lactamase motifs 1–4 and β-CASP motifs A, B, and C (adapted from Callebaut et al. 2002). Residues substituted individually in the plasmid shuffle assay are shown directly below the native Ysh1p residue. (B) Plasmid shuffling was used to test whether E209 of the β-CASP consensus in Ysh1p is essential for cell viability. Chromosomal YSH1 was disrupted with HIS3 and replaced by YSH1 on a URA3 plasmid. Cells were transformed with a LEU plasmid bearing wild-type YSH1, ysh1-209, or no insert (vector). Individual colonies were isolated and streaked on plates with or without 5-FOA. (Inset) Western blot of cell extracts comparing flu epitope-tagged wild-type and mutant ysh1p. (C) Plasmid shuffling was done as in B with four additional ysh1 mutants that alter the residues indicated (see text).

FIGURE 3.

3′ Cleavage is divalent cation-dependent, and zinc supports both cleavage and polyadenylation in vitro. (A) ZnCl₂ partially restores cleavage activity lost during nuclear extract dialysis. (Lane 1) Cleavage of uniformly labeled SV40L pre-mRNA in the presence of 2 mM EDTA. Cleavage (lane 3) or cleavage and polyadenylation (lane 2) in the presence of added ZnCl₂. (Lane 4) Loss of cleavage activity upon dialysis (cf. lane 1). (Lanes 5,6) Partial restoration of cleavage activity by addition of increasing zinc concentration. (Lane 7) ZnCl₂ supports poly(A) addition in the absence of exogenous MgCl₂. (B) EDTA at high concentrations and the zinc-specific chelators TPEN and OP at low concentrations inhibit cleavage. In addition to the chelators listed above the gel, each sample contained 2 mM EDTA. (C) Inhibition of cleavage by EDTA and TPEN is concentration dependent.

FIGURE 4.

CPSF-73 as the 3′ processing nuclease. The core polyadenylation machinery (e.g., RNAP II is not included) is illustrated as it would assemble in a precleavage complex on an mRNA precursor, with CPSF-73 at the site of cleavage. The AAAUAA and GU-rich signal sequences are indicated, and the arrow denotes the cleavage site. The protein–protein and protein–RNA interactions depicted are consistent with available data. See text for details of protein factors.