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. 2017 Jul 7:8:16056.
doi: 10.1038/ncomms16056.

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

Aitor Garzia  1 , Seyed Mehdi Jafarnejad  2 , Cindy Meyer  1 , Clément Chapat  2 , Tasos Gogakos  1 , Pavel Morozov  1 , Mehdi Amiri  2 , Maayan Shapiro  2 , Henrik Molina  3 , Thomas Tuschl  1 , Nahum Sonenberg  2
Affiliations

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

Aitor Garzia et al. Nat Commun. .

Abstract

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

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Conflict of interest statement

T.T. is cofounder and advisor to Alnylam Pharmaceuticals. The remaining authors declare no competing financial interests.

Figures

Figure 1
Figure 1. ZNF598 is a translation repressor that sediments with polysomes.
(a) Domain organization of the human ZNF598 protein and its yeast orthologue Hel2 as determined by Interpro. The protein length in amino acids (aa) is indicated. (b) Polysome profiles of empty vector (EV) and ZNF598-OE HEK293 cells. (c) Polysome profiles of parental (CTR) and ZNF598-KO HEK293 cells. (d) Western blot analysis of ZNF598 expression and EIF2S1 phosphorylation (p-EIF2S1) to probe for proteotoxic stress in ZNF598-OE and ZNF598-KO cells and controls. * Indicates a non-specific band recognized by the anti-ZNF598 antibody (GeneTex). Numbers indicate the ratio of ZNF598 expression relative to CTR cells. (e) Western blot analysis with the indicated antibodies of fractions of the ZNF598-OE HEK293 cell lysates after separation over a 10–50% sucrose gradient. The position of 80S ribosomes and polysomes in the gradient is indicated.
Figure 2
Figure 2. PAR-CLIP RNA targets of ZNF598 in HEK293 cells.
(a) Relative composition of ZNF598 PAR-CLIP sequence reads mapping to each RNA category with up to two mismatches. The reads mapped to nuclear encoded mRNAs are further subdivided into functional regions. (b) Meta-gene plot of PAR-CLIP reads mapping to mRNA defined by at least one read with T-to-C conversion. Each row in the matrix represents the relative coverage over each mRNA. mRNAs are ranked by the number of mapped T-to-C reads for the 3,000 most abundant mRNAs. The upper panel depicts the average coverage over the top 3,000 mRNAs. (c) Bin-normalized distribution of ZNF598 PAR-CLIP T-to-C reads mapping to tRNAs. (d) Schematic diagram of the secondary structure of tRNAs. Conserved nucleotides across cytosolic tRNAs are spelled out in letters, while non-conserved nucleotides are depicted by circles. The colour-code indicates the T-to-C conversion ratio. Filled circles at the 5′ end represent nucleotides covered by ZNF598 PAR-CLIP sequence reads (32 nt of the 5′ end). (e) Relative changes in tRNA abundance in ZNF598 PAR-CLIP versus HydroSeq (total cellular tRNA). All tRNALys(UUU) sequence variants are coloured in red, tRNALys(CUU) variants are coloured in orange, and all tRNAArg variants are coloured in blue. tRNAs, which are over-represented in PAR-CLIP with a false discovery rate (FDR) of <5% are labelled by their corresponding gene names. tRNAs collecting the top 85% of sequencing reads are to the right and residual tRNAs are to the left of the dotted vertical line. (f) Average read composition of two replicates of ZNF598 PAR-CLIP experiments for the rRNA category. Reads were assigned as d0 (dark grey), d1 T-to-C (red), d1 other than T-to-C and (light grey).
Figure 3
Figure 3. The RING domain of ZNF598 is essential for ribosome stalling at polyA residing within coding sequences.
(a) Schematic diagram of the reporter constructs sandwiching a polybasic oligopeptide track between the fluorescent GFP and mCherry (mCh) fusion protein. (ACT AGC)6 [(ThrSer)6] encoded a neutrally charged amino-acid tract that served as a control. (b) Detection of GFP and mCherry fluorescent signals by FACS analyses in samples from (a) shown as relative cell numbers. Each experiment was performed in triplicates. (c) Domain structures of ZNF598 full-length and truncation mutants, with numbers referring to the position of amino acids. (d) Detection of GFP and mCherry fluorescent signals by FACS analyses in samples expressing full-length or truncated versions of ZNF598 and transiently transfected with GFP-mCherry reporter with (ACT AGC)6 and (AAA)12 linkers. Each experiment was performed in triplicate. (e) Upper panel: autoradiograph of cross-linked, 32P-labelled, RNA- Flag/HA-ZNF598 immunoprecipitate. Flag/HA-tagged full-length ZNF598 or truncated versions were separated by SDS-PAGE after 4SU PAR-CLIP. Lower panel: Anti-HA Western blot analyses of the cross-linked RNA-protein immunoprecipitates; HC, antibody heavy chain.
Figure 4
Figure 4. Ribosome stalling at coding polyA sequences requires the E3 ubiquitin ligase activity of ZNF598 and the E2 ubiquitin ligase UBE2D3.
(a) Identification of differentially ubiquitinated proteins by ubiquitin remnant immuno-affinity profiling. Log2 ratios of the enrichment of the quantified diGly-containing peptides are shown. Only peptides with an average log2 ≥2 upregulation for ZNF598OE/CTR and downregulation for ZNF598KO/CTR are shown. Error bars represent the s.d. (n=2). See Supplementary Data 5 for a complete list of all detected peptides. (b) Volcano plots of the quantitative proteomic analysis of the ZNF598 interactome. The t-test difference based on label free quantitation for each detected protein is plotted against the negative logarithmic P value of a Welch’s t-test. The intensity based absolute quantitation (iBAQ) values correspond to the sum of all the peptide intensities divided by the number of observable peptides of a protein and are represented by point size. Proteins with a permutation-based FDR-value of <5% and t-test difference >0 are labelled in red and represent putative ZNF598 interactors (see also Supplementary Fig. 15 and Supplementary Data 6). (c) Analysis of siRNA-mediated knockdown of UBE2D2, UBE2D3 or both UBE2D2 and UBE2D3 in HEK293 cells by western blot. C indicates mock transfection. (d) Detection of GFP and mCherry fluorescent signals by FACS analyses in samples from (c), for reporter constructs containing (ACT AGC)6 and (AAA)12 linkers. Each experiment was performed in triplicate. (e) Model for ZNF598-dependent ribosome stalling and RQC at cryptic polyadenylated protein-coding mRNAs.
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