PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif

Abstract

PAPD5 is one of the seven members of the family of noncanonical poly(A) polymerases in human cells. PAPD5 was shown to polyadenylate aberrant pre-ribosomal RNAs in vivo, similar to degradation-mediating polyadenylation by the noncanonical poly(A) polymerase Trf4p in yeast. PAPD5 has been reported to be also involved in the uridylation-dependent degradation of histone mRNAs. To test whether PAPD5 indeed catalyzes adenylation as well as uridylation of RNA substrates, we analyzed the in vitro properties of recombinant PAPD5 expressed in mammalian cells as well as in bacteria. Our results show that PAPD5 catalyzes the polyadenylation of different types of RNA substrates in vitro. Interestingly, PAPD5 is active without a protein cofactor, whereas its yeast homolog Trf4p is the catalytic subunit of a bipartite poly(A) polymerase in which a separate RNA-binding subunit is needed for activity. In contrast to the yeast protein, the C terminus of PAPD5 contains a stretch of basic amino acids that is involved in binding the RNA substrate.

FIGURE 1.

PAPD5 preparations used in the analysis. (A) PAPD5 was expressed as a fusion protein with N-terminal His₈ and Flag tag in HEK293 cells and purified via anti-Flag agarose (Flag), followed by metal chelating affinity chromatography (Flag + NiNTA). PAPD5 is marked by an arrowhead. (B) Bacterially expressed PAPD5 after metal chelating affinity chromatography (for details, see Materials and Methods).

FIGURE 2.

PAPD5 preferentially adds AMP residues to RNA substrates. (A) PAPD5 preparations from HEK293 cells (HEK293) or recombinant protein expressed in E. coli (rec.) were incubated with ribo-oligonucleotide A₁₅ in the presence of ATP (A), CTP (C), GTP (G), UTP (U), or a mixture of all four dNTPs (dN), respectively. (B) PAPD5 wild-type protein (wt) or catalytic site mutant (DADA) expressed in E. coli were incubated with ribo-oligonucleotide A₁₅ in the presence of ATP. (C) PAPD5 expressed in E. coli was incubated with ribo-oligonucleotide G₂U₁₅ in the presence of ATP (A), CTP (C), GTP (G), UTP (U), or a mixture of all four dNTPs (dN), respectively. Lane 1 always shows the RNA substrate incubated in the absence of protein.

FIGURE 3.

The C terminus of PAPD5 is involved in RNA binding. (A) Schematic representation of the truncation variants of PAPD5 tested for RNA binding. (B) Electrophoretic mobility shift assay of PAPD5 variants. Increasing amounts of PAPD5 full-length protein or fragments comprising the N terminus, central part, or C terminus of the protein were incubated with radioactively labeled in vitro–synthesized human tRNA_i^Met, and the reactions were resolved on a native polyacrylamide gel. Protein concentrations between 0.5 and 5 nM were tested for the full-length protein and the protein PAPD5 C2, 1–20 nM for the other variants. Lane 1 shows the tRNA after incubation in the absence of protein.

FIGURE 4.

The basic motif in the C terminus of PAPD5 is conserved among higher eukaryotes. Amino acid sequences of PAPD5 homologs (Ensembl) were aligned with ClustalW (Chenna et al. 2003) and refined manually (upper panel). The amino acid sequence of the yeast protein Trf4p is shown for comparison (lower panel). Basic amino acids are labeled in blue, acidic amino acids in red.

FIGURE 5.

Mutations in the basic motif of PAPD5 lead to loss of activity. PAPD5 wild-type protein (wt), C-terminal deletion mutant (ΔC, amino acids 1–551), or a point mutant in the basic motif (KE, position 560) expressed in E. coli were incubated with ribo-oligonucleotide A₁₅ in the presence of ATP.

FIGURE 6.

PAPD5 adds poly(A) tails to the unmodified in vitro transcript of S. cerevisiae tRNA_i^Met, but not to the native tRNA isolated from yeast. PAPD5 protein expressed in E. coli (PAPD5 rec.) or in HEK293 cells was incubated with the different RNA substrates in the presence of ATP, and the reactions were stopped after the reaction times indicated (0, 10, 20, 40, 60 min).

FIGURE 7.

PAPD5 is located in the nucleus. (A) PAPD5 was expressed as a fusion protein with N-terminal His₈-Flag tag in HEK293 cells. The protein was detected with anti-Flag antibody and Cy3-conjugated secondary antibody. Nuclei stained with DAPI (left panel); the tagged protein signal (middle); the merged picture (right panel). (B,C) PAPD5 was expressed as a fusion protein with green fluorescent protein fused to the N terminus (B) or the C terminus (C). Panels as in A. (D) PAPD5ΔC (amino acids 1–551) was expressed as a fusion protein with N-terminal His₈-Flag tag in HEK293 cells. The protein was detected with anti-Flag antibody and Cy3-conjugated secondary antibody. Panels as in A. DAPI stain of the nucleus (blue), Cy3 staining (red), and GFP signal (green).

FIGURE 8.

Distribution of pairwise Z-scores computed for 100-nt-long windows in pairs of samples. The reads obtained in two separate PAPD5 PAR-CLIP experiments as well as those previously obtained for the insulin growth factor 2 binding protein 1 (IGF2BP1) (Hafner et al. 2010) were mapped to the genome. After normalization, the Z-scores, giving a measure of the differential expression of individual 100-nt-long windows in each pair of experiments, were calculated.