A family of poly(U) polymerases

Abstract

The GLD-2 family of poly(A) polymerases add successive AMP monomers to the 3' end of specific RNAs, forming a poly(A) tail. Here, we identify a new group of GLD-2-related nucleotidyl transferases from Arabidopsis, Schizosaccharomyces pombe, Caenorhabditis elegans, and humans. Like GLD-2, these enzymes are template independent and add nucleotides to the 3' end of an RNA substrate. However, these new enzymes, which we refer to as poly(U) polymerases, add poly(U) rather than poly(A) to their RNA substrates.

FIGURE 1.

Tethered GLD-2 family members. (A) Protocol. mRNA encoding an MS2 fusion protein with the candidate polypeptide was injected into oocytes. The fusion protein was expressed during a 6-h incubation, and then two reporter mRNAs were coinjected. The luciferase mRNA contained MS2 binding sites; the β-galactosidase lacked MS2 sites, and was used as a control. (B) Results. (Top) Relative stimulation of translation by each protein, expressed as the ratio of luciferase to β-galactosidase activities. Values were normalized to CeGLD-2 D608A, a catalytically inactive form of the enzyme (Kwak et al. 2004). Error bars were derived from more than three experiments. (Bottom) Chimeric fusion proteins detected by Western blotting using anti-HA tag antibody. Lysates equivalent to three oocytes were loaded into each lane.

FIGURE 2.

RNAs are elongated by proteins that do not stimulate translation. (A) Protocol. A radiolabeled RNA containing three MS2 binding sites was injected into oocytes that were expressing a fusion protein. (B) Results. The radiolabeled RNA substrates were analyzed by electrophoresis through urea–polyacrylamide gels. The products are grouped by what proved to be their enzymatic activities (PAPs [lanes 2–6], PUPs [lanes 7–13], or inactive [lanes 14–18]). RNAs from two oocytes were loaded on each lane. (Lane 1) An uninjected input RNA. Markers on left indicate approximate sizes of tails.

FIGURE 3.

Poly(A) analysis of the products. (A) RNaseH/oligo(dT) reactions. RNAs produced by each tethered protein, prepared as in Figure 2B, were treated with oligo(dT) and RNaseH and analyzed by electrophoresis and autoradiography. (−) No DNA oligo added; (+) reactions included oligo(dT)₁₈. (B) Binding to oligo(dT). RNAs were fractionated using biotinylated oligo(dT). (−) RNA that did not bind to biotinylated oligo(dT); (+) RNA that did bind.

FIGURE 4.

Tail sequencing. (A) Protocol. Luciferase reporter mRNA was injected and RNAs prepared after a 16-h incubation. Step (1): RNAs were ligated to the 3′-amino modified DNA primer P1. Step (2): Reverse transcriptase was used to synthesize DNAs complementary to the substrate RNAs. Oligonucloeotide P1′, which is complementary to primer P1, was used as a primer for this step. Step (3): cDNAs were amplified by PCR reactions using a substrate specific primer, P2, and P1′. PCR products contain tails of the ligated products. Step (4): PCR reaction products were cloned into vectors using blunt-end ligation and transformed in E. coli. DNA was prepared from independent E. coli transformants, and the sequence of the insert in the plasmid determined. (B) Results. The tethered enzymes add poly(U). Sequences obtained from the RNAs products of reactions using eight different tethered enzymes, as indicated in the figures. The sequences in the figure correspond to that present in the RNA. The number of clones obtained is given in the second column: thus, with At1, seven clones contained 13 T residues.

FIGURE 5.

SpCID1 activity in vitro. (A) Divalent metals. Purified recombinant SpCID1 was incubated with a ³²P-labeled RNA substrate and one of four nucleoside triphosphates. Reactions contain either MgCl₂ or MnCl₂, as indicated above the lanes. (B) Preference for UTP and poly(U) synthesis in vitro. (Lane 1) End-labeled RNA corresponding to the substrate of the reactions in lanes 2–7. (Lanes 2–5) SpCID1 was incubated with an unlabeled RNA substrate and all four nucleoside triphosphates. Reactions contained either ³²P-labeled ATP, UTP, GTP, or CTP, as indicated above each lane. Only UTP was significantly incorporated. (Lanes 6,7) Recombinant ScPAP1 was incubated with an unlabeled RNA substrate and the four nucleoside triphosphates plus either ³²P-labeled ATP or UTP, as indicated. (Lanes 8–11) Similar reactions using total RNA from S. pombe as a substrate. (C) Oligo(dA)/RNaseH treatment confirms that the products of SpCID1 are poly(U). In vitro reactions were performed using radiolabeled RNA substrates and four NTPs. The products were treated with RNase H and either oligo(dA) or oligo(dT), as indicated. (Lane 1) substrate RNA (no A or U added); (lanes 2,5) no oligonucleotides added; (lanes 3,6) oligo(dT) added; (lanes 4,7) oligo (dA) added.

FIGURE 6.

Rooted phylogenetic tree of PUPs and PAPs and related proteins. (Black boxes) Proteins that add poly(U); (white boxes) proteins that add poly(A); (boxes with crosses) no activity detected in the tethered protein assay.