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. 2021 May 20;81(10):2112-2122.e7.
doi: 10.1016/j.molcel.2021.03.004. Epub 2021 Apr 27.

Convergence of mammalian RQC and C-end rule proteolytic pathways via alanine tailing

Anna Thrun  1 , Aitor Garzia  2 , Yu Kigoshi-Tansho  1 , Pratik R Patil  1 , Charles S Umbaugh  1 , Teresa Dallinger  1 , Jia Liu  1 , Sylvia Kreger  1 , Annarita Patrizi  3 , Gregory A Cox  4 , Thomas Tuschl  2 , Claudio A P Joazeiro  5
Affiliations

Convergence of mammalian RQC and C-end rule proteolytic pathways via alanine tailing

Anna Thrun et al. Mol Cell. .

Abstract

Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2KLHDC10 E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.

Keywords: Alanine-tail; C-end rule; KLHDC10; NEMF; Pirh2; RQC; Rchy1; Rqc2; RqcH; ribosome-associated quality control.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. NEMF promotes Listerin-independent degradation of non-stop proteins in mammalian cells
(A) Schematic diagram of mRNA encoded by bicistronic reporter constructs with DsRed and GFP being translated, and indicating a ribosome translating past the GFP coding sequence and into the poly(A) tail in the non-stop (NS) mRNA. (B) HeLa cells were transiently transfected with DsRed-IRES-GFP or -GFP NS and analyzed by flow cytometry. Stability of the reporter was assessed by normalizing GFP to DsRed fluorescence. Flow cytometry data was quantified from independent experiments (n = 3) and is represented as mean ± SEM. Ratios of mean GFP and DsRed fluorescence from cells expressing DsRed-IRES-GFP NS were normalized to mean GFP/DsRed ratios from cells expressing DsRed-IRES-GFP. Statistical analysis was performed using ordinary one-way ANOVA, P values are indicated. (C) Ubiquitylation analysis of GFP NS from indicated cell lysates by semi-denaturing GFP IP followed by the indicated immunoblot. Quantification of independent experiments (n = 4) shows a 75% reduction in the GFP NS ubiquitylation signal from LNKO cells compared to LKO. IRES, internal ribosomal entry site; WT, wild type; KO, knockout; NKO, NEMF KO; LKO, LTN1 KO; LNKO, LTN1/NEMF KO; NS, non-stop; WCL, whole cell lysate; IP, immunoprecipitation. See also Figure S1.
Figure 2.
Figure 2.. C-terminal Ala tails trigger ubiquitylation and proteasomal degradation
(A) Selective tRNA-Ala(IGC) enrichment by NEMF PAR-CLIP. x-axis: log2 average tRNA abundance in the cell (HydroSeq); y-axis: relative fold-changes in tRNA abundance in NEMF crosslinked reads (PAR-CLIP) compared to abundance in cell (HydroSeq). tRNA-Ala variants are colored in red, tRNA-Thr in blue, all other tRNAs in black. (B) Schematic diagrams of tRNA secondary structure (ASL, anticodon stem-loop). To identify NEMF crosslinking sites on tRNAs, the efficiency (%) of T-to-C conversion resulting from PAR-CLIP crosslinking was calculated for individual positions of tRNA-Ala (left) and all other tRNAs together except tRNA-Ala (right). Conserved nucleotides across tRNAs are indicated by letters or symbols (R=A/G; Y=C/T; Ψ=pseudouridine), non-conserved nucleotides are depicted as circles. Filled circles represent nucleotides covered with high frequency by NEMF PAR-CLIP sequence reads. (C) Flow cytometry analysis of HeLa cells transiently transfected with DsRed-IRES-GFP reporter constructs C-terminally fused to the indicated sequences. (D) RPE1 cells stably expressing DsRed-IRES-GFP or -GFP-Ala6 were treated or not with the proteasome inhibitor Bortezomib (20 μM for 6 h) and analyzed using flow cytometry. DMSO was used as a control vehicle. (E) HeLa cells expressing GFP or GFP-Ala6 were treated with the proteasome inhibitor MG132 (10 μM for 4 h) and subjected to GFP IP followed by immunoblot analysis to monitor ubiquitylation. Ub, ubiquitin. See also Figure S2.
Figure 3.
Figure 3.. C-terminal Ala tails are recognized by Pirh2 and KLHDC10
(A) Top: schematic and cartoon diagrams of the domain arrangement of Pirh2; bottom: schematic diagram of the domain arrangement of KLHDC10 and cartoon representation of the subunit organization of the CRL2KLHDC10 complex (Sekine et al., 2012). The E3 ligases are shown bound to Ub-conjugated E2, and KLHDC10 is also shown bound to an Ala-tailed (orange) substrate. (B) The interaction of GFP, GFP-Ala6, GFP-Ala4 or GFP-Thr2-Ala2 with FLAG-tagged Pirh2 WT or the catalytically-inactive mutant M176E in MG132-treated HeLa cells was analyzed by GFP IP followed by anti-FLAG immunoblot. (C) The interaction of GFP, GFP-Ala6, GFP-Ala4 or GFP-Thr2-Ala2 and FLAG-tagged KLHDC10 was analyzed as in “B”, but in the absence of MG132. (D) Interaction of GFP-Ala6 or GFP-SelK (a substrate of KLHDC2 ending in -GlyGly (Rusnac et al., 2018)) and FLAG-tagged KLHDC10 or KLHDC2 analyzed as in “C”. (E) HeLa cells transiently transfected with GFP reporters were treated with Pirh2 and/or KLHDC10 siRNA and MG132. Ubiquitylation of GFP-Ala6 was analyzed by GFP IP followed by immunoblot. (F) RPE1 cells stably expressing GFP fusion reporters were treated with siRNAs targeting Pirh2 and/or KLHDC10 and with the Neddylation inhibitor MLN4924 (see “A”; 1 μM for 5 h) as indicated. NTD, N-terminal domain; CTD, C-terminal domain; K1-6, Kelch repeats 1-6; CRL, Cullin-RING ligase; SelK, Selenoprotein K. See also Figures S3, S4, S7 and Table S1.
Figure 4.
Figure 4.. Pirh2 and KLHDC10 promote degradation of non-stop proteins in a NEMF-dependent manner
(A) HeLa LKO or LNKO cells expressing GFP or GFP NS and FLAG-tagged Pirh2 or KLHDC10 were treated with MG132 (10 μM for 4 h). Interaction of GFP NS with the E3 ligases was analyzed by GFP IP followed by anti-FLAG immunoblot. (B) Interaction between GFP NS and KLHDC10 was analyzed in the presence or absence of NEMF WT or the catalytically inactive mutant D96A/R97A (DR) as described in “A”, but in the absence of MG132. (C) Different cell lines expressing GFP, GFP-Ala6 or GFP-NS were treated with control, Pirh2 and/or KLHDC10 siRNA, as indicated. Ubiquitylation levels of GFP NS were assessed by semi-denaturing GFP IP and anti-ubiquitin immunoblot. (D) Effect of Pirh2 and KLHDC10 depletion on GFP NS stability in HeLa WT, NKO, LKO, and LNKO cell lines. Cells were knocked down with control or Pirh2 and KLHDC10 siRNA followed by transfection with DsRed-IRES-GFP or -GFP NS. GFP and DsRed fluorescence was measured by flow cytometry. Ratios of median GFP and DsRed fluorescence from cells expressing DsRed-IRES-GFP NS were normalized to median GFP/DsRed ratios from cells expressing DsRed-IRES-GFP. Data was quantified from independent experiments (n = 3) and is represented as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA; E3 depletion only had a large and statistically significant effect on GFP NS stability in LKO cells (P < 0.0001). (E) Parental and KO HeLa (top panel) and RPE1 (bottom panel) cells were treated with control or Pirh2 and KLHDC10 siRNA and transiently transfected with GFP NS and mCherry. Expression and gel mobility of GFP NS were analyzed by immunoblot. mCherry serves as a transfection control. See also Figures S5 and S6.
Figure 5.
Figure 5.. Model of the actions of Pirh2 and KLHDC10 in mammalian RQC
Exposure of 60S subunit-associated peptidyl-tRNA in response to ribosome stalling and splitting is the key trigger for NEMF recruitment, the first step in mammalian RQC. Subsequent ubiquitylation of the nascent-chain by Listerin promotes extraction and proteasomal degradation (RQC-L; L for Listerin). The extent to which NEMF elongates nascent-chains with Ala tails prior to Listerin-mediated ubiquitylation likely varies according to the Lys accessibility of each substrate, with the dashed arrow indicating situations in which C-terminal tailing is not required for Listerin to act. When Listerin action becomes limiting, NEMF-mediated Ala tails serve as a proteolytic signal for the E3 ligases Pirh2 and KLHDC10, which bind to and ubiquitylate the released nascent-chains (RQC-C; C for C-end rule pathway). Represented are: ribosomal subunits (grey), tRNA-Lys and Lys (blue), NEMF (green), tRNA-Ala and Ala (orange), Listerin (yellow), Ubiquitin (red), E2 (navy) and Pirh2 or KLHDC10 (magenta).
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