Biochimie 106 (2014) 10e16

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Research paper

A functional involvement of ABCE1, eukaryotic ribosome recycling factor, in nonstop mRNA decay in Drosophila melanogaster cells Isao Kashima a, b, Masaki Takahashi a, Yoshifumi Hashimoto a, Eri Sakota a, Yoshikazu Nakamura a, *, Toshifumu Inada b, ** a b

The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 July 2014 Accepted 2 August 2014 Available online 14 August 2014

When ribosomes encounter mRNAs lacking stop codons, two quality-control machineries, NSD for nonstop mRNA decay and ribosome quality control (RQC) for co-translational degradation of the nonstop protein by the proteasome, are triggered to eliminate aberrant molecules. In yeast, it is known that Dom34 (a homolog of eRF1) and Ltn1 (an E3 ubiquitin ligase) play crucial roles in NSD and RQC, respectively, by triggering ribosome rescue at the 3' end of nonstop mRNAs and proteasome-dependent polypeptide degradation. Here we confirmed the essential role of Ltn1 in RQC for nonstop products in Drosophila cells, and further uncovered a functional role of ABCE1, a eukaryotic ribosome recycling factor, in NSD in Drosophila cells.  te  française de biochimie et biologie Mole culaire (SFBBM). All rights © 2014 Elsevier B.V. and Socie reserved.

Keywords: mRNA surveillance Protein surveillance Nonstop decay (NSD) Translation termination ABCE1

1. Introduction Nonstop mRNA decay (NSD) is an RNA surveillance mechanism that detects and degrades mRNAs lacking stop codons [1,2]. Nonstop mRNAs can be generated by alternative premature polyadenylation or endonucleolytic cleavage of transcripts within open reading frames [1,3e5]. NSD protects cells from the accumulation of aberrant polypeptides encoded by nonstop mRNAs [3]. It is known that the nonstop mRNAs are degraded mainly by 3'e5' decay pathway, in addition to 5'e3' decay pathway, composed of the Ski:Exosome complex and Xrn1, respectively, [6,7]. In yeast, the degradation of nonstop mRNA from 5' end is significantly accelerated in yeast, probably due to the removal of poly(A)-binding protein from poly(A) tail by translating ribosomes [6,7]. The trans-acting factors required for NSD involve Pelota/Dom34 and Hbs1, which are homologs of eRF1 and eRF3 in eukaryotes,

Abbreviations: NSD, nonstop mRNA decay; RQC, ribosome quality control; dsRNA, double strand RNA; cDNA, complementary DNA; RT-PCR, reverse transcription-polymerase chain reaction; RNAi, RNA interference; PAGE, polyacrylamide gel electrophoresis. * Corresponding author. Tel.: þ81 3 5449 5307; fax: þ81 3 5449 5415. ** Corresponding author. Tel.: þ81 22 795 6874; fax: þ81 22 795 6873. E-mail addresses: [email protected] (Y. Nakamura), [email protected] (T. Inada).

respectively [8]. Dom34/Pelota is structurally similar to eRF1 (the class-I eukaryotic release factor), but does not contain the GGQ motif, which catalyzes peptidyletRNA cleavage in the peptidyl transferase center [9]. Hbs1 is a GTPase that is a homolog of eRF3 (the class-II eukaryotic release factor) and eEF1 (an elongation factor) that enter into A site together with eRF1 and aminoacyltRNA, respectively [9,10]. Dom34/Pelota and Hbs1 form a stable complex and GTP binds to Hbs1, thus resembling eRF1 and eRF3 [11]. The resulting ternary complex Dom34/Pelota:Hbs1:GTP [12] gives rise to a conformational change enabling Pelota/Dom34 to adapt a tRNA-like structure [9,12e14]. The current model for NSD suggests that the Dom34/Pelota:Hbs1 complex enters the empty A site of the ribosome that is stalled at the 3' end of nonstop mRNA, promotes the ribosome dissociation, enabling access of the Ski:Exosome exonuclease complex from the 3' end of nonstop mRNAs [5,15,16]. Several lines of in vivo and in vitro observations support this view. First, Dom34/ Pelota:Hbs1 facilitates ribosome dissociation and peptidyl-tRNA drop-off in a codon independent-manner in the in vitro reconstitution systems of yeast and human [17,18]. Second, Pelota/Dom34 enhances nonstop protein production from nonstop mRNA when the 3'e5' exosome activity is hampered [9]. Third, accumulation of peptidyl-tRNAs derived from nonstop mRNA is triggered by a disability of Dom34 and Hbs1 in yeast [19]. NSD has also been observed in human tissue culture cells [1] and a recent study in

http://dx.doi.org/10.1016/j.biochi.2014.08.001  te  française de biochimie et biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2014 Elsevier B.V. and Socie

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human cells indicates that the degradation of nonstop mRNA requires human Pelota and Hbs1 [20]. Since NSD has been investigated primarily in yeast, it is not yet certain whether the NSD system is conserved in other eukaryote cells. Protein synthesis is composed of four steps, initiation, elongation, termination and recycling. The factor responsible for the fourth step was first identified in bacteria and called RRF for ribosome recycling factor [14]. The factor equivalent to RRF was long unknown in eukaryotes. Finally, a mammalian in vitro reconstitution system has shown that ABCE1 (ATP-binding cassette subfamily E member 1) mediates ribosome recycling in concert with eRF1 in an A-site termination codon dependent manner [21]. Importantly, ABCE1 also induces the dissociation of the translating ribosome in concert with Dom34/Pelota and Hbs1 in an A site codon-independent manner [22]. These findings prompted us to speculate that ABCE1 might be involved in NSD. However, there is no literature reporting investigations of this possibility. Interestingly, it has been shown recently that the translational arrest of ribosomes triggers not only rapid mRNA degradation, but also the degradation of nascent polypeptide by the proteasome pathway in yeast [23]. Recent studies have clearly demonstrated that co-translational degradation of the aberrant products derived from aberrant mRNAs plays a crucial role in preventing aberrant protein expression, which is referred to as ribosome quality control, RQC [5,23,25,33,35]. An E3 ubiquitin ligase Ltn1 is specifically associated with the 60S ribosomal subunit, and involved in the ubiquitination of the peptidyl-tRNA on 60S subunit in vivo [25,26,37]. The co-translational quality control systems for both nonstop mRNA and nonstop polypeptide might well be conserved in eukaryotes because both the Pelota/Hbs1 complex and E3 ligase Ltn1 are highly conserved, although experimental evidence for this assumption is still largely missing. In this study, we addressed the possible involvement of ABCE1 and Ltn1 in NSD and RQC, respectively, in Drosophila cells by using cell lines constitutively expressing a stop-codon-less GFP mRNA (nonstop GFP mRNA) reporter system developed previously [9,19]. By knocking down relevant factors by double stranded RNA interference (RNAi), the functional role of ABCE1 and Ltn1 is shown in Drosophila cells. 2. Materials and methods

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penicillin and 50 mg/ml streptomycin. Stable Drosophila cell lines expressing the GFP-His (stop-codon-containing) and GFP-Rz-His (stop-codon-less) transcripts were established by co-transfecting the corresponding construct with pCoBlast (Invitrogen) that encodes the Streptomyces griseochromogenes bsd gene at a 19:1 ratio according to the manufacturer's instructions. S2 cells were transfected with HiglyMax (DOJINDO) according to the manufacturer's instructions and selected in medium containing 25 mg/ml blasticidin. 2.3. Double-strand RNA knock down RNAi was performed as described [24]; double-strand RNAs (dsRNAs) corresponding to Drosophila Ski8, Pelota, HBS1, ABCE1, Ltn1, and Xrn1 were transcribed in vitro by T7 RNA polymerase from ~500e700-bp DNA fragments amplified with primer sets shown in Supplementary data 3 from cDNAs carried on the relevant plasmids (Supplementary data 1). 20 mg of dsRNA were transfected per 2  106 cells in suspension culture at a density of 106 cells/ml. dsRNA interference described in this paper was carried out by transfecting cells with the corresponding dsRNAs on day 0 and day 4, and cells were harvested on day 7. 2.4. RNA isolation and northern blotting of steady-state mRNAs Total RNAs were isolated from relevant Drosophila cells using TRIzol Reagent (Life Technologies), separated in 1.4% agarose gels (3e6 mg/lane) using Glyoxal RNA loading buffer and blotted onto positively charged Hybond-H þ membranes (GE Healthcare). The mRNAs were detected by northern blotting with digoxigenin (DIG) reagents, and nonradioactive probes were prepared by PCR-based nucleic acid labeling with commercial kits (Roche). Bound probes were detected according to the procedure specified by the manufacturer (Roche). The DIG-labeled probes used in this study are listed in Supplementary data 4. The intensity of the bands on the blots was quantified with the LAS3000 mini or LAS4000 mini and Multi-Gauge ver 3.0 (Fuji Film) or imagequant (GE Healthcare). Relative mRNA levels were determined by comparison to a standard curve by using a series of dilutions of the corresponding samples as described in the figure legends. The intensities of bands from the diluted samples were compared with a standard curve, and the mRNA levels were normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32), which is a long-lived mRNA (half-life >8 h) [24].

2.1. DNA constructs 2.5. Reverse transcription-polymerase chain reaction (RT-PCR) The plasmids and oligonucleotides used for plasmid constructions are listed in Supplementary data 1 and 2. The plasmids expressing GFP-Rz-His3 or GFP-His3 were constructed by inserting the corresponding DNA fragments into the NotI and XbaI sites of pAc5.1A (Invitrogen). DNA fragments were amplified with primers listed in Supplementary data 2 using p416GPDp-GFP-HIS3 [33] or p416GPDp-GFP-Rz-HIS3 [23] as templates. Drosophila genes described in this paper correspond to those in FlyBase (http:// flybase.bio.indiana.edu): Ski8 (CG3039), Pelota (CG3959), Hbs1 (CG1898), ABCE1 (CG5651) Ltn1 (CG32210), Xrn1 (CG3291) and rp49 (CG7939). Plasmids used to prepare T7 templates, Ski8 (LD21537), Pelota (LD34262), Hbs1 (RE29053), ABCE1 (RE71924), Xrn1 (LD22664) and Ltn1 (SD01201) were obtained from The Drosophila Genomics Resource Center (DGRC, https://dgrc.cgb. indiana.edu/vectors/Collections).

Total RNAs were extracted from S2 cells using a TRIzol reagent (Life Technologies, Carlsbad, CA, USA), and treated with a DNase I (New England Biolabs, Ipswich, MA, USA) according to each manufacturer's instruction. Complementary DNA (cDNA) synthesis was performed with a Oligo(dT)20 primer (Life Technologies) and a ThermoScript reverse transcriptase (Life Technologies) according to the manufacturer's instructions. The synthesized cDNAs were subjected to real-time PCR using the Applied Biosystems 7500 Real Time PCR System (Life Technologies) with a Power SYBR Green PCR Master Mix (Life Technologies) according to the manufacturer's instructions. PCR primers for checking the knock-down efficacy with dsRNAs were designed to amplify 3' junctions (for Ski8, Pelota, HBS1, ABCE1, and Ltn1) or 5' junction (for Xrn1) processed by dsRNAs. Sequences and positions of the primers are indicated in Supplementary data 5.

2.2. Cell lines 2.6. Northern blotting with [32P]labeled probes Drosophila Schneider cells (S2 cells) were obtained from Invitrogen and maintained at 25  C in Schneider Medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum, 50 U/ml

DNA probes were amplified by PCR using primer sets shown in Supplementary data 6. These probes were labeled with [a-32P]

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dCTP (Perkin Elmer) by random primer DNA labeling procedure using BcaBEST Labeling kit (TAKARA) according to the manufacturer's instructions. Total RNAs isolated from S2 cells were blotted onto membranes as described in 2.4. [32P]labeled probes were hybridized with mRNAs in hybridization buffer (7% SDS, 250 mM Na-phosphate, 2 mM EDTA) and mRNAs were analyzed by autoradiography. The signals of the bands on the blots were detected by the FLA-5100 (Fuji Film).

2.7. Half-life experiments For the measurement of mRNA half-life, transfected cells were treated with actinomycin D (5 mg/ml final concentration), and harvested at the indicated time points. Total RNAs were isolated and analyzed by northern blotting using probes against GFP and rp49 mRNAs. The levels of GFP were quantified and normalized to the levels of rp49 mRNA.

2.8. Protein isolation and western blotting To prepare protein samples for SDS-PAGE, cells were lysed for 15 min on ice in lysis buffer (50 mM HEPES at pH 7.4, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2, 0.5 mM DTT) supplemented with protease inhibitor (Roche). Cell lysates were spun at 12,000  g for 15 min at 4  C. The cleared lysates were treated with sampling buffer [19]. The protein concentrations were determined and adjusted by protein assay kit (Biorad). The proteins were separated in 12% acrylamide gels (1e3 mg/lane) under neutral condition [19]. The proteins were detected by western blotting with anti-GFP monoclonal antibody (Santa Cruz). Secondary horseradish peroxidase-linked anti-mouse IgG antibody was purchased from GE Healthcare. Endogenous tubulin was detected by using anti-tubulin antibody (1:5000 dilution; DM1A, Millipore). All western blot experiments were developed with Immunostar (Wako) as recommended by the manufacturer and chemiluminescence detection was carried out by LAS-3000 mini or LAS-4000 mini.

3. Results 3.1. dsRNA-mediated knock down of factors involved in non-stop mRNA decay in Drosophila cells The potential role of Drosophila factors, whose homologs are known to participate in nonstop mRNA and protein decay in yeast, was investigated in Drosophila cells by the dsRNA-mediated gene silencing procedure. mRNAs encoding ABCE1, Xrn1, Ski8, Pelota, HBS1 and Ltn1 were depleted by RNAi upon transfection of S2 cells with ~500e700-bp dsRNAs. Knock down of each mRNA was examined by RT-PCR and northern blot procedures. RNAs prepared from dsRNA-treated S2 cells were first subjected to RT-PCR using primers designed to amplify 3' or 5' proximal segments truncated by dsRNA cleavage. As shown in Fig. 1A, RT-PCR amplification of each targeted sequence was markedly hampered, while that of non-targeted sequences was unaffected (data not shown). The same RNA preparations were then subjected to northern blotting using [a-32P]labeled oligonucleotide probes for ABCE1, Xrn1, Ski8, Pelota, HBS1 and Ltn1. The data confirmed depletion of each targeted RNA segment, while non-targeted sequences remained unchanged (Fig. 1B). 3.2. Generation and characterization of an NSD reporter in Drosophila cells We had developed previously an in vivo reporter for NSD in yeast using artificial NSD transcripts that are generated by the hammer-head ribozyme (Rz) sequence inserted in the GFP-His reporter gene [19]. This provides a self-cleaved transcript of 5'-capped stop-codon-less mRNA. The degradation of this artificial mRNA is known to be stimulated by Dom34:Hbs1-mediated ribosome dissociation at the 3' end of stop-codon-less mRNAs in yeast [19] and the product derived from 5'-capped stop-codon-less mRNA is degraded by the proteasome in a Ltn1-dependent manner [25,26]. We transplanted this reporter system (GFP-Rz-His) into Drosophila cells by establishing Drosophila cell lines that expressed stably the 5'-capped stop-codon-less transcript. Briefly, the reporter constructs were co-transfected with pCoBlast into S2 cells to

Fig. 1. dsRNA-mediated knock down of Drosophila factors. Total RNAs from S2 cells treated with dsRNAs for N-terminal ~166e233 amino acids of ABCE1, HBS1, Ltn1, Pelota, Ski8 and Xrn1 were examined for depletion of targeted RNAs. (A) RT-PCR analysis. Experimental procedures and conditions are as described in Materials and methods. Delta Ct method [39] was employed using the level of Rp49 as a control. The data of each gene were further normalized to the expression level obtained from control sample without dsRNA treatment. PCR amplifications were performed in triplicate wells using the same cDNA templates. Data are shown as mean ± S.D. (B) Northern blot analysis. RNA blots were hybridized to [32P] DNA probes against ABCE1, HBS1, Ltn1, Pelota, Ski8 and Xrn1, and subjected to autoradiography.

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Fig. 2. Generation of an NSD reporter in Drosophila cells. (A) Schematic representation of the GFP-Rz-His reporter used in this study. Abbreviations: Ac5.1, Drosophila actin promoter; GFP, green fluorescent protein; Rz, hammer-head ribozyme; His, imidazoleglycerol-phosphate dehydratase. The GFP-Rz-His reporter generates the capped nonstop mRNA transcript. (B) The steady state levels of nonstop mRNA. S2 cells constitutively expressing GFP-Rz-His were treated with the indicated dsRNAs for RNA interference. Total RNA samples were isolated and analyzed by northern blot using probes against GFP and rp49 mRNAs. In lanes 1e5, dilutions of RNA samples isolated from Ski8-knocked down cells were loaded for the quantifications. In lanes 6e15, the equal amounts of RNA samples were loaded. The levels of the reporters were quantitated in three independent experiments and normalized to those of rp49 mRNA. The values are presented relative to those from non-treated (control) cells. Mean values ± SD are shown. (C) The steady state levels of nonstop protein. S2 cells constitutively expressing GFP-Rz-His were treated with the indicated dsRNAs. Total proteins were isolated and analyzed by western blot using antibodies against GFP and tubulin. In lanes 1e5, dilutions of protein samples isolated from Ski8-knocked down cells were loaded for quantification. In lanes 6e15, the equal amounts of protein samples were loaded. The levels of the reporters were quantitated in three independent experiments and normalized to those of tubulin. The values are presented relative to those from non-treated control cells.

generate polyclonal cell lines constitutively expressing GFP-Rz-His, resulting in 5'-capped GFP-stop-codon-less mRNA (Fig. 2A). The steady-state levels of the corresponding transcripts were analyzed by northern blot. In all experiments, levels of the NSD reporters were normalized to those of the endogenous rp49 mRNA (encoding ribosomal protein L32). Following this normalization, the steadystate levels of nonstop mRNAs in cells depleted of Ski8 were increased approximately by two-fold compared to the non-treated cells (Fig. 2B, compare lanes 6 and 7) and further increased in cells doubly depleted of Ski8 and Pelota or Hbs1 (Fig. 2B, compare lane 6 and lanes 11 and 12). It was noteworthy that the nonstop reporter mRNA was also increased in Ski8/ABCE1 doubly depleted cells (Fig. 2B, lane 13). The nonstop protein product detected by anti-GFP antibody was markedly increased in cells depleted of Ski8 by about 9-fold compared to the non-treated cells (Fig. 2C, lane 7). However, this increase in nonstop protein production disappeared in cells doubly depleted of Ski8 and other factors involved in ribosome dissociation, regardless of the steady-state accumulation of the nonstop reporter mRNA (Fig. 2C). These results are interpreted as indicating a hampered translatability of the nonstop reporter mRNA, presumably due to stalling of ribosomes generated by depletion of ribosome disassembly factor(s) as previously found in yeast [9,19].

3.3. Half-life measurement of the NSD reporter in Drosophila cells The stability of 5'-capped GFP-stop-codon-less mRNAs was then measured at time intervals upon addition of actinomycin D that blocks transcription. Half-lives of nonstop mRNAs in cells depleted of Xrn1 (5'e3' decay pathway) or Ski8 (3'e5' decay pathway) were 6.8 and 20.3 min (Fig. 3C and E), respectively, while in non-depleted cells was 5.7 min (Fig. 3A). These results suggest that the nonstop mRNA generated by the Rz sequence in the GFP-His reporter was primarily degraded by the Ski:Exosome complex from the 3' end. Under these conditions, the depletion of ABCE1 significantly prolonged the half-life to 8.9 min (Fig. 3B); this prolongation was reproducibly longer than that by Xrn1 depletion (6.8 min). The prolongation of the half-life of the nonstop mRNA by the depletion of ABCE1 became even more evident in Xrn1-depleted cells; the half-life of 6.8 min in Xrn1depleted cells was prolonged to 29 min in Xrn1/ABCE1 doubly depleted cells (Fig. 3D). The depletion of Ltn1 did not affect the half-life appreciably (Fig. 3G). Then, the stability of stop-codon-containing GFP-His transcripts was examined in Xrn1/ABCE1 singly or doubly depleted cells in the same experimental conditions. The half-lives of GFP-His mRNAs in non-depleted or ABCE1-depleted cells were 21.4 and 26.0 min (Fig. 3H and I), respectively, while in Xrn1-depleted or Xrn1/

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Fig. 3. ABCE1 facilitates nonstop mRNA decay in Drosophila cells. S2 cells constitutively expressing GFP-Rz-His (AeG; stop-codon-less transcripts) or GFP-His (HeK; stop-codoncontaining transcripts) were treated with the indicated dsRNAs and cells were withdrawn at the indicated time intervals upon addition of actinomycin D (5 mg/ml). Total RNA samples were isolated and the same amounts of each RNA were analyzed by northern blotting using probes against GFP and rp49 mRNAs. In lanes 1e5, dilutions of RNA samples isolated from time 0 of each experiment were loaded for quantification. In lanes 5e10, the equal amounts of RNA samples were loaded. The levels of nonstop mRNAs with dsRNAs were quantitated in three independent experiments and normalized to those of rp49 mRNA. The half-life of each nonstop mRNA (AeG) or stop mRNA (HeK) is indicated with standard deviations. (A, H) non-treated cells (control). (B, I) ABCE1 depletion. (C, J) Xrn1 depletion. (D, K) Xrn1/ABCE1 double depletion. (E) Ski8 depletion. (F) Ski8/ABCE1 double depletion. (G) Ltn1 depletion.

ABCE1-doubly depleted cells were 32.4 and 52.0 min (Fig. 3J and K), respectively. These control experiments demonstrated that the stop-codon-less mRNAs in Xrn1-depleted cells was more significantly stabilized by ABCE1-depletion (4.3-fold) than stop-codoncontaining mRNAs (1.6-fold). This, in turn, suggests that the mammalian recycling factor ABCE1 functionally participates in the Ski:Exosome complex-mediated 3'e5' nonstop mRNA decay in Drosophila cells by facilitating the dissociation of stalled ribosome from nonstop mRNA.

3.4. The E3 ubiquitin ligase Ltn1 is responsible for RQC of nonstop products in Drosophila cells The E3 ubiquitin ligase Ltn1 involved in the degradation of the polypeptide produced from nonstop mRNA functions cotransnationally via the proteasome pathway in yeast [25]. To test whether such a crucial role exists for Ltn1 in the degradation of the polypeptide produced from nonstop mRNA in Drosophila cells, we examined the expression level of the nonstop protein upon

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Fig. 4. Ltn1 degrades the polypeptide produced from nonstop mRNA via the proteasome pathway in Drosophila cells. (A) S2 cells constitutively expressing GFP-Rz-His were treated with MG132 (50 mg/ml) for the indicated time periods. Total proteins were isolated and analyzed by western blot using antibodies against GFP and Tubulin. (B) S2 cells constitutively expressing GFP-Rz-His were treated with the indicated dsRNAs. Total proteins were isolated and analyzed by western blot using antibodies against GFP and tubulin. In lanes 1e5, dilutions of protein samples isolated from Ski8-knocked down cells were loaded for quantification. In lanes 8e14, the equal amounts of protein samples were loaded. The levels of the reporters were quantitated in three independent experiments and normalized to those of tubulin. The values are presented relative to those from non-treated control cells. Mean values ± SD are shown.

treatment of Ltn1-depleted cells with MG132 (a proteasome inhibitor). The nonstop protein level in cells treated with MG132 increased compared with non-treatment cells (Fig. 4A). The level of the nonstop protein in cells depleted of Ltn1 was increased by 4.9fold compared to the non-depletion level (Fig. 4B, lane 11). Under these conditions, the half-life of nonstop mRNAs in Ltn1-depleted cells was unchanged (Fig. 3G). We also confirmed that the stop codon-containing GFP protein level was not affected by Ltn1 depletion (data not shown). A more drastic effect of Ltn1 depletion on nonstop protein accumulation was observed in Ski8-depleted cells: the nonstop protein level increased by 13.6-fold in cells depleted of Ski8 alone, and further increased by 81.1-fold compared to the non-depleted level in cells doubly depleted of Ski8 and Ltn1 (Fig. 4C, lanes 9 and 13). It was noteworthy that depletion of Pelota significantly reduced the nonstop protein accumulation in Ski8-depleted cells (Fig. 4, lanes 10 and 12), presumably due to disabled ribosome dissociation from nonstop mRNA. These results indicate that Drosophila Ltn1 is responsible for the degradation of polypeptides derived from nonstop mRNA via the proteasome pathway. 4. Discussion It is crucial for cells to eliminate aberrant mRNAs such as mRNA containing premature termination codons or lacking termination codons to guarantee proper gene regulation and expression. These transcripts encoding aberrant polypeptides, regardless of harmful or non-harmful, are silenced by mRNA-surveillance pathways called nonsense-mediated mRNA decay (NMD) or nonstop mRNA decay (NSD) in eukaryote cells. These two pathways share the mechanism that recognizes the aberrant mRNAs by sensing an unusual translation termination process, showing that translation termination is a checkpoint of mRNA surveillance in eukaryotes [15,27]. NMD has been well characterized in several eukaryote cells and shown already that the fundamental molecular mechanism is widely conserved except for some diversities depending on the substrate mRNAs and organisms [27e29]. On the other hand, NSD has been studied primarily in yeast or in vitro, and the mechanism of NSD is not well understood in other eukaryotes [5,28]. Nonstop mRNAs lacking a termination codon and polyA tail are rapidly degraded from their 3' and 5' termini by the Ski:Exosome complex and Xrn1 in yeast [6]. Dom34 and Hbs1 stimulate nonstop mRNA degradation by mediating ribosome dissociation at the 3' end of the mRNA, allowing access of the Ski:Exosome exonuclease complex at the 3' end of nonstop mRNAs in yeast [19]. To elucidate the evolutionary conservation and molecular mechanism of the bona fide NSD pathway in vivo, we examined NSD in

Drosophila cells using ribozyme-mediated nonstop mRNA reporter systems. The present study revealed, for the first time, that ABCE1 functionally participates in nonstop mRNA decay in eukaryotes. ABCE1 (Rli1 in yeast) plays an essential role in translation termination and ribosome recycling together with eRF1. ABCE1 mediates ribosome dissociation when ribosomes encounter the stop codon [21,30]. ABCE1 also triggers the dissociation of translating ribosomes by the interaction with Dom34 in an A-site codonindependent-manner [18,22,31]. The prolonged half-life of nonstop mRNA in ABCE1-depleted cells, evidently in conjunction with Xrn1-depletion, strongly suggests that ribosomes stalled on the mRNA impede the access of nucleases, at most the Ski:Exosome complex. The polypeptide production from nonstop mRNA in ABCE1-depleted cells is reduced strongly even though nonstop mRNA is stabilized by ABCE1 depletion. Taken together, ABCE1 stimulates the degradation of nonstop mRNA from the 3' end by dissociating the stalled ribosome, and mediates the high nonstop protein production in Ski8-depleted cells. Ribosomes often stall during translation of mRNAs containing polybasic amino acid sequences, rare codons or premature stop codons, leading to an endonucleolytic cleavage of mRNA, a process called no-go decay (NGD) [5,28,32,33]. Recent studies have shown that ribosome stalling induces co-translational degradation of the nascent polypeptide via the proteasome pathway [23,34,35]. In yeast, Ltn1 is responsible for the degradation of the polypeptides derived from polyA minus or polyA plus nonstop mRNAs [25]. In this regard, a ribosome quality control (RQC) complex has been proposed, consisting of Ltn1 associated with Rqc1, Tae2 and the AAA ATPase Cdc48, which stimulates efficient polyubiqutination and initiates polypeptide destruction by the proteasome [5,25,36e38]. In this paper, we clearly demonstrate that Drosophila Ltn1 specifically targets the polypeptide derived from nonstop mRNAs. It remains to be determined whether Tae2 and Rqc1 might be involved in the clearance of nonstop products in Drosophila cells. Recently we found that the aberrant nonstop polypeptide derived from nonstop mRNA lacking a poly(A) tail were dramatically stabilized following Ltn1 deletion in the absence of the ribosome dissociation factor Dom34:Hbs1 in yeast [25]. We showed in this study that the level of nonstop product derived from GFP-Rz mRNA is significantly increased by the Ltn1 depletion in Pelota and Ski8-depleted cells (Fig. 4, lanes 12 and 14). These suggest that Ltn1 destabilizes aberrant nonstop polypeptides in the absence of Pelota:Hbs1 in Drosophila cells. One possibility is that a putative factor might be involved in the dissociation of ribosome that is stalled at the 3' end of mRNA for the Ltn1-dependent nonstop

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protein degradation in Drosophila cells. The levels of peptidyl-tRNA in stalled ribosomes derived from nonstop mRNA should be determined with cells extracts depleted of Pelota and/or Ltn1 as previously shown in yeast [25]. It could not be excluded at present that stalled ribosomes may be dissociated into subunits in a Pelota/ Hbs1-independent manner. In summary, the present studies show, for the first time, a functional role of ABCE1 in NSD as well as a conserved role of Ltn1 in RQC for nonstop products in Drosophila cells.

[14] [15] [16]

[17]

[18]

Conflict of interest [19]

The authors declare that there is no conflict of interest. [20]

Acknowledgments We are grateful to Michiru Ozawa for excellent technical assistance, Kazushige Kuroha and Toshinobu Fujiwara for helpful discussions, and John Hershey for his reading and editing of the manuscript. This work was supported in part by grants from The Ministry of Education, Sports, Culture, Science and Technology of Japan (MEXT) to I.K., Y.N and T.I., and from the Uehara Memorial Foundation to I.K. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2014.08.001.

[21]

[22]

[23]

[24]

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