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. 2000 Nov 15;19(22):6230-9.
doi: 10.1093/emboj/19.22.6230.

Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting

S Aigner  1 , J Lingner, K J Goodrich, C A Grosshans, A Shevchenko, M Mann, T R Cech
Affiliations

Euplotes telomerase contains an La motif protein produced by apparent translational frameshifting

S Aigner et al. EMBO J. .

Abstract

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNA-binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

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Figures

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Fig. 1. Sequencing of p43 by nanoelectrospray tandem mass spectrometry. m/z, mass-to-charge ratio. (A) Mass spectrum of the unfractionated digest of the p43 doublet band. Trypsin autolysis products are marked with asterisks. Tryptic peptides whose entire sequences were determined are designated with T. Tryptic peptides sequenced partially and retrospectively matched to the full-length sequence are designated with t. (B) Mass spectrum of product ions resulting from fragmentation of peptide precursor ion T42+ from (A). Interpretation of these mass spectra relative to those obtained from esterified peptides allowed assignment of y-ions and sequence determination.
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Fig. 2. p43 gene and protein. (A) Structure of the macronuclear chromosome containing the p43 gene. Protein-coding regions are depicted by solid black boxes, introns are numbered and shown as lines. 5′ and 3′ non-coding regions are shown as empty boxes proximal to the telomeres, which are represented with their 3′-terminal single-stranded extensions. The location of the putative translational frameshift site is marked by an arrow. (B) Predicted amino acid sequence of the p43 gene product. Peptides sequenced de novo from purified protein and assigned to p43 are shown underlined and are labeled as in Figure 1A. Peptide T6 could not be assigned to p43. Amino acids 86–88, whose identity could not be determined unambiguously due to the putative frameshift, are in lower case letters. Shown here is the sequence assuming the frameshift event at site 3 (see Figure 3). The La motif and the putative RRM are shaded and boxed, respectively.
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Fig. 3. Close-up of the frameshift region. The mRNA sequence as deduced from cDNA is shown in bold at the top. The four theoretically possible +1 frameshift events are illustrated by curved arrows above the ‘skipped’ nucleotides, which are underlined. Conceptual translations in the reading frame of peptide t1 (designated frame 0) and peptide t3 (designated frame +1) are aligned below the mRNA sequence using the one-letter amino acid code and asterisks to denote stop codons. The translation products originating from each of the four possible +1 frameshifts are given below the vertical arrow. Bold letters signify amino acids whose identity depends on the actual site of frameshifting.
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Fig. 4. Relationship of p43 to La proteins. Parts of the predicted protein sequence of p43 (E.a.) are aligned with the respective regions of La proteins from human (H.s.), D.melanogaster (D.m.), the fission yeast S.pombe (S.p.) and S.cerevisiae (S.c.) (Chambers et al., 1988; Yoo and Wolin, 1994; van Horn et al., 1997). Amino acids in black shading are similar or identical in the majority of the five proteins (see Materials and methods). Numbers to the left and right of each of the sequences refer to their start and end amino acid positions, respectively. Top: alignment of the La motif regions. The three amino acids in p43 whose identity is uncertain due to lack of information on the exact location of the frameshift are underlined. The sequence shown here assumes the frameshift event at site 3 (see Figure 3). The phenylalanine residue conserved in La proteins is marked by an asterisk. Bottom: alignment of parts of the putative RRM region. The RNP2 and RNP1 submotifs are overlined.
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Fig. 5. Affinity-purified α-p43 antibodies against recombinant p43 recognize a ∼51 kDa protein in E.aediculatus nuclear extract but a ∼43 kDa protein in purified telomerase. (A) Purified recombinant His6-tagged p43 (lane 1) and 0.4 pmol of telomerase purified by heparin chromatography and affinity chromatography (lane 2) were denatured and separated on a 4–20% polyacrylamide gel, followed by immunoblot analysis with α-p43 antibodies. (B) Recombinant p43 (lanes 1 and 2) and nuclear extract (NXT, lane 3) were electrophoresed as in (A), followed by either Coomassie Blue staining (left) or immunoblot analysis (right). Recombinant p43 migrates with slightly decreased mobility relative to endogenous p43 due to its His6 tag, which adds ∼2 kDa (data not shown). (C) Recombinant p43 (lanes 1 and 2), nuclear extract that was stored at 4°C for 1 day (lane 3), 2 days (lane 4) or 5 days (lane 5), or 0.04 pmol of purified telomerase (lane 6) were denatured and electrophoresed on an 8% polyacrylamide gel and subjected to immunoblot analysis. p51 and p43 designate the proteins migrating at apparent mol. wts of 51 and 43 kDa, respectively.
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Fig. 6. α-p43 antibody efficiently co-immunoprecipitates telomerase RNA from nuclear extract. Euplotes aediculatus nuclear extract was subjected to three successive immunodepletion reactions with either α-p43 antibody beads or control beads. After each round of immunodepletion, supernatants were separated from the beads and added to fresh beads. After addition of a 110mer RNA as a recovery control, samples containing equal amounts of input nuclear extract (In, lane 1), the three α-p43 bead fractions (Bound, lanes 2–4) and control bead fractions (Bound, lanes 5–7) as well as the final flowthroughs (F.t.) of the α-p43 beads (lane 8) and control beads (lane 9) were treated with protease, phenol extracted and analyzed by northern blot hybridization with probes specific for the 189 nt E.aediculatus telomerase RNA and the control RNA. Lanes M, uniformly labeled RNAs as size markers.
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Fig. 7. The vast majority of telomerase activity is associated with p43. Euplotes aediculatus nuclear extract was partially purified by glycerol gradient centrifugation and subjected to three consecutive rounds of immunodepletion with α-p43 antibody beads or control antibody beads, as described in the legend to Figure 6. Input, bound and flowthrough fractions were assayed for telomerase activity by measuring extension of a telomeric primer in the presence of a radiolabeled substrate nucleotide, followed by phenol/chloroform extraction and separation of telomerase products in a denaturing polyacrylamide gel. Five percent of input nuclear extract before (lane 1) or after (lane 2) RNase A treatment, 50% of α-p43 antibody beads (lanes 3–5) or control antibody beads (lanes 6–8), as well as 0.25, 0.5, 2.5 and 5% of the final flowthroughs from the α-p43 antibody beads (lanes 9–12) or control antibody beads (lanes 13–16) were assayed. Equal amounts of an end-labeled 23mer oligonucleotide were added to the samples prior to phenol/chloroform extraction to control for recovery and loading.
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Fig. 8. Purified recombinant p43 binds in vitro transcribed E.aediculatus telomerase RNA in vitro. 32P-labeled telomerase RNA (∼6.6 nM) carrying the photoaffinity chromophore 5-iodo-U at all U positions was incubated on ice with 330 nM p43 (lanes 1 and 3–20), without p43 (lane 2), or with p43 pre-treated with protease (lane 3), followed by UV irradiation to induce cross-linking (except lane 4). Samples were denatured and analyzed on 4–20% acrylamide gels. Samples run in lanes 5, 7, 9, 11 and 6, 8, 10, 12 included 50-fold and 500-fold molar excess, respectively, of the indicated unlabeled competitor RNAs over labeled E.aediculatus telomerase RNA. Samples run in lanes 13, 15, 17, 19 and 14, 16, 18, 20 included 5- and 50-fold excess by mass, respectively, of the indicated homopolymers over labeled E.aediculatus telomerase RNA. Protein molecular weight markers are indicated on the right. XL, cross-linked RNA–protein complexes.
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