Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways
2019; Springer Nature; Volume: 38; Issue: 5 Linguagem: Inglês
10.15252/embj.2018100276
ISSN1460-2075
AutoresKen Ikeuchi, Petr Těšina, Yoshitaka Matsuo, Takato Sugiyama, Jingdong Cheng, Yasushi Saeki, Keiji Tanaka, Thomas Becker, Roland Beckmann, Toshifumi Inada,
Tópico(s)Biochemical and Molecular Research
ResumoArticle4 January 2019free access Transparent process Collided ribosomes form a unique structural interface to induce Hel2-driven quality control pathways Ken Ikeuchi orcid.org/0000-0002-9578-7920 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Petr Tesina orcid.org/0000-0002-5227-1799 Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Yoshitaka Matsuo Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Takato Sugiyama orcid.org/0000-0001-8364-7050 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Jingdong Cheng Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Yasushi Saeki orcid.org/0000-0002-9202-5453 Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan Search for more papers by this author Keiji Tanaka Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan Search for more papers by this author Thomas Becker orcid.org/0000-0001-8458-2738 Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Roland Beckmann Corresponding Author [email protected] orcid.org/0000-0003-4291-3898 Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Toshifumi Inada Corresponding Author [email protected] orcid.org/0000-0002-2695-588X Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Ken Ikeuchi orcid.org/0000-0002-9578-7920 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Petr Tesina orcid.org/0000-0002-5227-1799 Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Yoshitaka Matsuo Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Takato Sugiyama orcid.org/0000-0001-8364-7050 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Jingdong Cheng Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Yasushi Saeki orcid.org/0000-0002-9202-5453 Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan Search for more papers by this author Keiji Tanaka Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan Search for more papers by this author Thomas Becker orcid.org/0000-0001-8458-2738 Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Roland Beckmann Corresponding Author [email protected] orcid.org/0000-0003-4291-3898 Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany Search for more papers by this author Toshifumi Inada Corresponding Author [email protected] orcid.org/0000-0002-2695-588X Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan Search for more papers by this author Author Information Ken Ikeuchi1,‡, Petr Tesina2,‡, Yoshitaka Matsuo1, Takato Sugiyama1, Jingdong Cheng2, Yasushi Saeki3, Keiji Tanaka3, Thomas Becker2, Roland Beckmann *,2 and Toshifumi Inada *,1 1Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2Department of Biochemistry, Gene Center and Center for Integrated Protein Science Munich, University of Munich, Munich, Germany 3Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +49 89 218076900; Fax: +49 89 218076945; E-mail: [email protected] *Corresponding author. Tel: +81 22 795 6874; Fax: +81 22 795 6873; E-mail: [email protected] EMBO J (2019)38:e100276https://doi.org/10.15252/embj.2018100276 See also: LL Yan & HS Zaher (March 2019) PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no-go decay) and the nascent polypeptide (RQC: ribosome-associated quality control). RQC requires Hel2-dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2-dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an in vitro translation reaction. Cryo-EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2-mediated cleavages upstream of the disome, governed by initial Not4-mediated monoubiquitination of eS7 and followed by Hel2-mediated K63-linked polyubiquitination. We propose that Hel2-mediated ribosome ubiquitination is required both for canonical NGD (NGDRQC+) and RQC coupled to the disome and that RQC-uncoupled NGD outside the disome (NGDRQC−) can occur in a Not4-dependent manner. Synopsis Ribosome stalling triggers both ribosome-associated quality control (RQC) of nascent polypeptides and no-go decay (NGD) of mRNA. Structural and biochemical data show that collided ribosomes form a unique structural unit allowing the Hel2 ubiquitin ligase to operate in both pathways. Hel2-mediated uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di-ribosome (disome) unit. Cryo-EM reveals a unique composite interface between the small subunits of the stalled leading and the following colliding ribosome, which can serve as stalling-recognition pattern for Hel2. Hel2 preferentially ubiquitinates colliding ribosomes, where Hel2 ubiquitination targets on the respective small subunits congregate in close vicinity. Two distinct NGD branches act differentially on or near a disome unit, one coupled to and one uncoupled from RQC. RQC-uncoupled NGD is characterized by Not4-mediated mono-ubiquitination followed by Hel2-mediated polyubiquitination of ribosomal protein eS7, resulting in mRNA cleavage upstream of the disome unit. Introduction Cells have various quality control systems, which selectively degrade aberrant mRNA and defective proteins to ensure precise expression of genome-encoded information. Ribosome stalling during elongation results in degradation of both the mRNA and the arrested nascent protein by mRNA surveillance pathways and the ribosome-associated quality control (RQC) systems (Bengtson & Joazeiro, 2010; Becker et al, 2012; Brandman et al, 2012; Defenouillere et al, 2013). Specific mRNA decay pathways (non-stop decay, NSD; nonsense-mediated decay, NMD) are triggered by the lack of a stop codon and the presence of a premature a stop codon, respectively. In contrast, the no-go decay (NGD) mRNA surveillance pathway occurs when the ribosome is road blocked, for example, by stable RNA secondary structures, rare codons or stretches of consecutive positively charged amino acids in the nascent chain (Doma & Parker, 2006; Chen et al, 2010; van den Elzen et al, 2010; Kobayashi et al, 2010; Kuroha et al, 2010). NGD is initiated by endonucleolytic cleavage of mRNA proximal to the ribosomal stalling site (Doma & Parker, 2006). This cleavage results in the production of 5′ NGD and 3′ NGD-mRNA intermediates, which are further degraded by the Xrn1 exoribonuclease and the exosome, respectively (Doma & Parker, 2006). In this regard, certain combinations of rare codons coding for arginine (CGN; where N=G, C or A) were shown to be very potent for ribosomal stalling (Gamble et al, 2016). Notably, endonucleolytic cleavage events have been reported even in a more general context outside NGD in a study by Mourelatos and co-workers, showing that endogenous human mRNAs undergo repeated co-translational and ribosome-phased endonucleolytic cuts at the exit site of the mRNA ribosome channel (Ibrahim et al, 2018). Importantly, the potentially toxic protein products of stalled mRNAs have to be degraded. Via the ribosome-associated quality control (RQC) pathway, a truncated nascent protein is degraded and the stalled ribosome is recycled. Dissociation of the stalled 80S ribosome into 40S and 60S subunits is a crucial step in this process, with the resulting 60S subunit still bearing a peptidyl-tRNA. This complex is recognized by the Rqc2 and Ltn1 proteins: while Rqc2 stabilizes Ltn1 binding and adds a random sequence of alanines and threonines to the C-terminus of the nascent peptide (CAT-tails), the E3 ubiquitin ligase Ltn1 ubiquitinates the arrested peptide, thereby marking it for subsequent proteasomal degradation (Bengtson & Joazeiro, 2010; Brandman et al, 2012; Shao & Hegde, 2014; Hilal & Spahn, 2015; Shen et al, 2015). In yeast, the RQC pathway is triggered by ubiquitination of the ribosomal protein uS10 at specific lysine residues by the E3 ubiquitin ligase Hel2 (also known as Rqt1, derived from RQC-trigger; ZNF598 in mammals) and the E2 enzyme Ubc4 (Matsuo et al, 2017). Moreover, initiating the RQC pathway requires an additional protein complex, the so-called RQC-trigger (RQT) complex (Matsuo et al, 2017). It is composed of the RNA helicase family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3 and yKR023W/Rqt4 (Matsuo et al, 2017; Sitron et al, 2017). The RQT complex was found to associate with the ribosome and Hel2, and the ubiquitin-binding activity of Cue3/Rqt3 and ATPase activity of Slh1/Rqt2 were shown to be crucial to trigger RQC (Matsuo et al, 2017). Recent studies in the mammalian system demonstrated that ZNF598 ubiquitinates the ribosomal proteins eS10 at lysines K138 and K139 and uS10 at K4 and K8. This ubiquitination triggered the RQC response on ribosomes stalled on a poly-lysine encoding mRNA reporter, indicating that the role of ribosome ubiquitination in quality control is conserved (Garzia et al, 2017; Juszkiewicz & Hegde, 2017; Sundaramoorthy et al, 2017). Both the NGD and the RQC require common factors and biochemical events suggesting a coupling of these two quality control pathways (Shoemaker & Green, 2012). Both are initiated by translation arrest and are dependent on the 40S subunit-associated Asc1 (RACK1) in yeast (Kuroha et al, 2010; Ikeuchi & Inada, 2016). Moreover, Hel2-mediated K63-linked polyubiquitination has been implicated in RQC after stalling at polybasic amino acid sequences and tandem CGA codons in yeast (Saito et al, 2015). Furthermore, Simms et al recently reported that ribosomal collisions represent the trigger for NGD-induced mRNA cleavage and proposed that colliding ribosomes induce robust ubiquitination of uS3 by Hel2 However, prior to our study, there was no evidence that K63-linked polyubiquitination of uS3 or other ribosomal proteins by Hel2 plays a role in NGD, and the exact biochemical and structural interdependencies between NGD and RQC induced by translation arrest remained unclear. In this study, we demonstrate that NGD and RQC are coupled and that both pathways respond to ribosome collision. We focused on a di-ribosome (disome) unit consisting of the stalled ribosome (here referred to as the leading ribosome) and the following colliding ribosome. This minimal ribosome collision unit is required to couple NGD and RQC via Hel2. We show that endonucleolytic cleavage of a NGD reporter mRNA occurs at sites within this disome unit. The mRNA cleavage is dependent on Hel2-mediated K63-linked polyubiquitination of uS10 as well as on the activity of the RQT component Slh1/Rqt2, showing that NGD and RQC are coupled via this ubiquitination event (here referred to as the NGDRQC+). Furthermore, we show that disomes are preferred as targets for Hel2-mediated uS10 ubiquitination over monosomes. In addition, we could dissect the NGD pathway into two interdependent branches. We identified a mutant of Hel2 that fails to trigger RQC and only initiates an alternative NGD. In this alternative NGD pathway, endonucleolytic mRNA cleavages occur upstream of the stalled disome (here referred to as the NGDRQC−). We further show that these cleavages require K63-linked polyubiquitination of ribosomal protein eS7. This polyubiquitination happens in a two-step mechanism, where the E3 ligase Not4 first monoubiquitinates eS7 which is followed by Hel2-mediated polyubiquitination. Taken together, we propose a dual role of Hel2 leading to two distinct NGD pathways, which require specific ubiquitination events on the stalled disome. To get a structural understanding of how ribosome collisions could provide a platform for signalling to mRNA and protein quality control pathways, we determined the Cryo-EM structure of a disome as an NGD substrate. This structure shows a defined disome arrangement in which the leading ribosome is stalled in the classical POST-translocation state with an empty A-site and occupied P- and E-sites. The second ribosome is in a hybrid state with A/P and P/E tRNAs, apparently locked in an incomplete translocation step. The interface between the leading and the colliding ribosomes is mainly formed by the small 40S subunit and to lesser extent also the large 60S subunit. Importantly, the 40S inter-ribosomal contact interface brings all proteins targeted by Hel2 during quality control in close proximity. Moreover, both Asc1 (RACK1 in humans) molecules are in direct contact forming one of the inter-ribosomal interaction sites. We suggest that this defined interaction between a stalled leading ribosome and a colliding ribosome generates a unique composite surface for molecular recognition of translational stalling. It may represent the ideal substrate for Hel2, thereby specifically recognizing a prolonged translation stall to initiate RQC and NGDRQC+ by its E3 ubiquitin ligase activity. Results Hel2-mediated K63-linked polyubiquitination is required for both NGD and RQC In our previous study, we demonstrated that Hel2 is essential for ubiquitination of the small subunit ribosomal protein uS10, which leads to the degradation of the arrested peptide after translation arrest via RQC in yeast (Matsuo et al, 2017). To investigate the relation between the two quality control pathways induced by ribosome stalling, NGD and RQC, we now investigated the role of Hel2 in the endoribonucleolytic cleavage of mRNA, a triggering step of the NGD quality control using several stalling reporter mRNAs (Fig EV1A). The main reporter contains twelve consecutive CGN codons (N = A/G/C) coding for arginine (R(CGN)12). Notably, this sequence contains codon pairs (CGA-CGA) which have been described to efficiently cause ribosomal stalling in yeast (Gamble et al, 2016; Matsuo et al, 2017). To estimate the efficiency of NGD, we used the production of intermediate endonucleolytic mRNA cleavage products (Fig 1A). The 5′ NGD-mRNA intermediate (5′ NGD-IM) represents the primary product, which is rapidly degraded by the cytoplasmic exosome. Therefore, the 5′ NGD intermediates derived from various arrest-inducing sequences can be detected in mutants lacking the exosomal co-factor Ski2 (ski2∆). Click here to expand this figure. Figure EV1. Hel2 and Ubc4 were involved in NGD but not NSD and NMD Schematic drawing of reporter mRNAs containing various arrest-inducing sequences. The box indicates GFP and HIS3 open reading frames (ORFs), and the black line indicates the untranslated region (UTR). Ribosome stalling takes place during translation of the indicated arrest sequences (shown in red) and induces an endonucleolytic cleavage to produce two fragments, the 5′ NGD intermediate (5′ NGD-IM) and 3′ NGD intermediate (3′ NGD-IM). Northern blot analysis showing that Hel2 is required for endonucleolytic cleavages by R(CGN)12 arrest-inducing sequences. The GFP-R(CGN)12-HIS3 mRNA (FL) and the 5′ NGD-IM were detected in the indicated strains using a DIG-labelled GFP probe. SCR1 coding for RNA in the signal recognition particle (SRP) was used as a loading control. Northern blot analysis showing that Ubc4 but not Ubc5 is required for endonucleolytic cleavages by R(CGN)12 arrest-inducing sequences. The GFP-R(CGN)12-HIS3 mRNA (FL) and the 5′ NGD-IM were detected in the indicated strains with the DIG-labelled GFP probe. Northern blotting showing that Hel2 and Ubc4 are required for endonucleolytic cleavages by R(CGN)12 arrest-inducing sequences. The GFP-R(CGN)12-HIS3 mRNA (FL) and the 3′ NGD-IM were detected in the indicated strains with the DIG-labelled HIS3 probe. Northern blotting showing that Hel2 reduces the half-life of reporter mRNA containing R(CGN)12 or K(AAA)12 arrest sequences. The stability of GFP-K(AAA)12-FLAG-HIS3 and GFP-R(CGN)12-FLAG-HIS3 mRNAs was determined. The GFP-K(AAA)12-FLAG-HIS3 mRNA was stable in hel2 mutant cells than that in wild-type cells (t1/2 = 11.2 min for hel2Δ versus t1/2 = 4.3 min for wild type). The GFP-R(CGN) 12-FLAG-HIS3 mRNA was also stable in hel2 mutant cells than that in wild-type cells (t1/2 = 7.6 min for hel2 versus t1/2 = 4.9 min for wild type). In contrast, the stability of GFP-K(AAA)12-FLAG-HIS3 mRNA was essentially the same in hel2 mutant cells (t1/2 = 6.0 min) and in wild-type cells (t1/2 = 5.8 min). This stabilization of the reporter mRNAs that are substrate for NGD in hel2 mutant cells strongly supports the crucial role of Hel2 in NGD. On the other hand, mRNA stability did not change in slh1∆ and uS10-K6/8R mutant cells. Hel2 is dispensable for NSD and NMD. The GFP-Rz mRNA is a truncated poly(A) tail-less non-stop mRNA that is produced by self-cleavage of hammerhead ribozyme (Rz) and efficiently degraded in the NSD pathway (Tsuboi et al, 2012). The FLAG-HIS3-NS mRNA is a poly(A) tail containing nonstop mRNA and subjected to NSD (van Hoof et al, 2002). The FLAG-his3-100 mRNA contains premature termination codon and degraded by NMD quality control (Kuroha et al, 2009). The relative levels of reporter mRNAs in the indicated mutant cells were determined using a DIG-labeled GFP (GFP-Rz) or 5′ DIG-labeled-LNA-FLAG (FLAG-HIS3-NS or FLAG-his3-100) probes. The relative levels of each mRNA were normalized to the mRNA level in time 0, which was assigned a value of 100, and SCR RNA levels were used as a loading control for the RNA samples. Western blot analysis to check the expression levels of HA-tagged Hel2 deletion mutant proteins as schematically displayed in Fig 1B using an anti-HA antibody. Download figure Download PowerPoint Figure 1. Hel2-mediated K63-linked polyubiquitination is crucial for NGD and RQC Schematic drawing of the R(CGN)12 reporter mRNA including the two quality control pathways induced by the R(CGN)12 translation arrest sequence. Ribosome stalling occurs during translation of the R(CGN)12 arrest sequence (shown in orange) and induces RQC and NGD. In the RQC pathway, the stalled ribosome is dissociated into subunits, and peptidyl-tRNA remaining on the 60S subunit is ubiquitinated by Ltn1 (shown in pink) and degraded by the proteasome. In the NGD pathway, an endonucleolytic cleavage produces two fragments, the 5′ NGD intermediate (5′ NGD-IM) and 3′ NGD intermediate (3′ NGD-IM). The green and thin grey lines indicate GFP and HIS3 open reading frames (ORFs), and the black line indicates an untranslated region (UTR). Schematic drawing of the truncated Hel2 mutant proteins. Activities in RQC or NGD induced by the R(CGN)12 sequence are indicated. Western blot showing that Hel2(1–315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN)12 reporter in ltn1Δ cells expressing truncated Hel2 mutant protein were detected with an anti-GFP antibody. Northern blot showing the 5′ NGD-IM derived from the R(CGN)12 reporter in ski2Δ cells expressing the indicated Hel2 mutant proteins. 5′ NGD-IMs were detected with a DIG-labelled GFP probe. Primer extension mapping of 5′ ends of 3′ NGD intermediates in Hel2-WT or Hel2(1–315) mutant cells at nucleotide resolution. The primer extension samples were analysed using 5% TBE-Urea-PAGE and detected by fluorescence. Non-specific reverse transcription (ReTr) products are indicated by asterisks. Dissection of NGDRQC+ and NGDRQC−: the carboxyl-terminal region of Hel2 is required for both NGD and RQC which is likely triggered on a disome unit (pale yellow). It contains the primarily stalled, leading ribosome followed the colliding ribosome. For NGDRQC+, cleavages occur on mRNA covered by the disome, whereby the leading ribosome undergoes RQC. In the mutant Hel2 lacking the C-terminus, an alternative NGD pathway takes place (NGDRQC−). Here, cleavages occur on mRNA covered by the ribosomes following the disome unit (blue) and the leading ribosome is not affected by RQC (grey). Download figure Download PowerPoint While the 5′ NGD-IM in ski2∆ mutant cells could be readily detected, it was absent in hel2∆ski2∆ cells (Fig EV1B) and ubc4∆ski2∆ cells (Fig EV1C). This indicates that Hel2 and its E2 ubiquitin-conjugating enzyme Ubc4 are indeed required for endonucleolytic mRNA cleavage in various arrest-inducing sequences and subsequent mRNA decay. Consistently, 3′ NGD-IMs, which are degraded by the 5′–3′ exonuclease Xrn1, were also not detected in either hel2∆xrn1∆ or ubc4∆xrn1∆ cells (Fig EV1D). Moreover, the full-length reporter mRNAs containing R(CGN)12 and K(AAA)12 arrest sequences were stabilized in hel2∆ mutant cells (Fig EV1E). Notably, we observed that the half-lives of the NGD reporters in the hel2Δ mutant cells were longer than in wild-type cells. This confirms the crucial role of Hel2 in NGD. In contrast to the observed Hel2 dependency of these analysed NGD scenarios, we further confirmed that Hel2 and Ubc4 were not involved in either NSD or NMD (Fig EV1F). Taken together, we show that Hel2 is not only required for degradation of the aberrant peptide by the RQC system, but also for the decay of aberrant mRNA by the NGD pathway. To obtain a more detailed insight into which parts of Hel2 are involved in NGD and RQC, we introduced a series of deletions in Hel2 (Fig 1B). Hel2 contains an N-terminal RING domain (61–109), three C2H2-type ZnF domains (222–307) and a proline-rich motif at the C-terminus (Fig 1B). Our deletions comprised N-terminal deletions including or lacking the RING domain (61–109), which is essential for target ubiquitination, and several C-terminal deletions of the presumably functionally important ZnF domains. After checking the expression levels of mutant Hel2 proteins (Fig EV1G), we determined the levels of 5′ NGD-IMs in the ski2∆ background and the protein arrest products in the ltn1∆ background to estimate the efficiencies of NGD and RQC. Both RQC and NGD were effective in the cells expressing the Hel2(1–539) and Hel2(61–539) mutant proteins (Fig 1C, lanes 9–10 and 17–18; Fig 1D, lanes 5 and 9) indicating that the ultimate N- and C-termini of Hel2 are not required for its function. However, neither the 5′ NGD-IM nor the peptide arrest products accumulated in the hel2 mutants carrying deletions of the RING domain (ΔRING) or alanine substitution of the conserved zinc finger (ZnF) cysteine residues (C64A, C67A) within the RING domain indicating a loss of function. Interestingly, we found two mutants, Hel2(1–315) and Hel2(61–315), which were only defective in inducing RQC but not NGD. Moreover, this singular function in NGD seemed to be inhibited by the Hel2 region comprising residues 316–439. In fact, these mutants enabled us to investigate a potential NGD-specific function of Hel2 (Fig 1C, lanes 13–14 and 21–22; Fig 1D, lanes 7 and 11). Intriguingly, the length of 5′ NGD intermediates produced by Hel2(1–315)-associated endoribonucleolytic cleavages was altered and the cleavage sites shifted upstream compared to the normal condition (Fig EV2). To determine the precise endonucleolytic mRNA cleavage sites induced by Hel2 and Hel2(1–315) mutant protein, we mapped the 5′ ends of 3′ NGD-IMs derived from the R(CGN)12 reporter mRNA in the xrn1∆ background by primer extension experiments. The 5′ ends of 3′ NGD-IMs were determined with a fluorescence-labelled primer that hybridized to the region of the FLAG-encoding mRNA sequence (Fig 1A and E). Thereby, we could map four Hel2-dependent cleavage sites (X1–X4; Fig 1E, lanes 6–7). Our previous study demonstrated that the ribosome is stalled mainly at positions R2(CGA) and R3(CGA) of the R(CGN)12 sequence in P- and A-sites, respectively, and subjected to RQC (Matsuo et al, 2017), indicating that the X1 cleavage site is located in the P-site of the stalled ribosome. The X2–X4 cleavage sites were approximately 26–36 nt upstream from the X1 cleavage site (X2–X4 in red arrowheads, in Fig 1E and F). Interestingly, in cells expressing the Hel2(1–315) mutant, X1 cleavage was minor and X2–X4 cleavages were almost absent, suggesting that the 316–639 region of Hel2 is important for these cleavage events (Fig 1E, lane 9). To our surprise, this mutant produced alternative major endonucleolytic cleavage sites instead, which occurred more than 45-51 nt upstream of the X1 cleavage site (blue arrowheads, lane 9 in Fig 1E). This indicates that the X1–X4 cleavage sites are not preferentially used in the Hel2 knockout or the Hel2(1–315) mutant cells and that the cleavage sites could be protected by the stalled ribosomes in RQC-deficient cells. The leading ribosome would be stalled covering the X1 site, and the colliding ribosome would thereby cover X2–X4. Click here to expand this figure. Figure EV2. Alteration of mRNA cleavage sites in Hel2(1–315) expressing cells A, B. Northern blot showing that the length of 5′ NGD intermediate was altered in Hel2(1–315) expressing cells and slh1∆ cells. The full-length GFP-R(CGN)12-FLAG-HIS3 mRNA and 5′ NGD intermediates (5′ NGD-IM) or 3′ NGD intermediates (3′ NGD-IM) were detected in the indicated mutant cells with expression of Hel2 wild-type or 1–315 mutant from plasmid by Northern blotting with DIG-labelled probes. 5′ NGD intermediates were detected by DIG-labelled GFP probe in (A), and 3′ NGD intermediates were detected by the DIG-labelled HIS3 probe in (B). SCR1 is used as loading control. FL; full length. Note an upstream shift of NGD cleavage sites in lanes A3-4 and B3-4. Download figure Download PowerPoint We have previously described an interesting phenotype of sensitivity to the anisomycin translation elongation inhibitor in RQC-deficient cells (Matsuo et al, 2017). Intriguingly, Hel2 mutants that were unable to trigger RQC (1–315, 61–315, 1–439 and 61–439) remained sensitive to anisomycin, whereas RQC-competent Hel2 mutants (1–539, 61–539) were not susceptible to this drug (Appendix Fig S1). Anisomycin binds in the ribosomal A-site and most likely prevents tRNA accommodation, which leads to stalling and ribosome collisions. Thus, it could trap ribosomes stalled in a state which occurs during RQC (presumably a disome) and increase RQC turnover demand. Based on these results, we hypothesized that at least two adjacent ribosomes as a disome unit, rather than a single-stalled 80S ribosome, would serve as a minimal control hub on which RQC and NGD are induced after translation arrest. In this scenario, ribosomal collision and subsequent oligoribosome formation could be recognized as a signal of translational arrest, which is in complete agreement with earlier concepts and data by Simms and colleagues (Simms et al, 2017). Further supporting this idea, the observed cleavage pattern occurring in the presence of the RQC-deficient Hel2(1–315) mutant, with a putative disome unit protecting the canonical cleavage sites (X1–X4), precisely positioned the alternative cleavage sites between following colliding ribosomes (Fig 1F). Together, this allowed us to dissect NGD of mRNA occupied by ribosomes into two branches: the first branch occurs on the primary stalled disome unit with NGD coupled to the RQC response and is dependent on the Hel2 C-terminal and RING domains (NGDRQC+). The second branch affects the ribosomes colliding with the disome, with NGD uncoupled from RQC, and is only dependent on the Hel2 RING domain (NGDRQC−, Fig 1F). Both uS10 ubiquitination and Slh1/Rqt2 are required for NGDRQC+ We further characterized the role of Hel2 in ribosomal protein ubiquitination by first checking which mode of ubiquitination is employed for NGD. Notably, the role of K63-linked polyubiquitination has been implicated in translation arrest and RQC before (Saito et al, 2015). To examine whether K63-linked ubiquitin chains are also critical for NGD, we used ub-K63R mutant cell strain in which all endogenous ubiquitin-encoding genes are modified. As a result, only the K63R mutant ubiquit
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