The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site

Abstract

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

Graphical Abstract

Figure 1.

Cryo-EM structures of ex vivo New1-ribosome complexes. (A–F) cryo-EM maps of New1p (blue) in complex with (A–E) elongating (states 1–6) and (F) termination (State 7) state ribosomes. The 40S and 60S subunits are colored yellow and grey, respectively and the uL1 protein (pink) of the L1 stalk is indicated in the ‘in’, ‘out’ or intermediate (‘inter’) conformations. A-tRNA (red), P-tRNA (orange), E-tRNA (cyan), A/P-tRNA (dark blue), P/E-tRNA (green), P/E*-tRNA (dark blue), eIF-5A (lime) and eRF1 (magenta) are colored. The schematics below the maps indicate the conformation of the tRNAs in the (A–C) pre-translocational states (states 1–3) and (D–F) post-translocational states (states 4–7). As the ribosomes are shown in the same pose on panels A–F, the pictogram view provided on panel A is applicable to all of the panels.

Figure 2.

New1 samples the ribosome throughout the translation cycle. (A, B) 5PSeq metagene analysis. The 5′ read ends for of pulldown (blue) and lysate (black) samples aligned to start (A) and stop codons (B). Stalls represent the 5′ boundary of the ribosome-protected 5′-phosphorylated mRNA fragment. The 5′ position is offset by −14 nt from the fist nucleotide in the P site and −17 nt from the first nucleotide in the ribosomal A site. (C) Distributions of ribosome polarity scores across transcriptome for immunoprecipitated New1-HTF ribosome complexes (pulldown, blue) and the input lysate (black). Positive and negative scores reflect enrichment of reads towards either 3′ and 5′ end of CDS, respectively. (D) Ribosomal queuing scores (QS) calculated for ORFs parsed by the codon preceding the stop codon. The red dashed line is a guide for the expected position of data points in the absence of the systematic difference between in QS between the pulldown and lysate samples. All analyses were performed on pooled lysate and New1 pulldown datasets from four biological replicates each.

Figure 3.

New1 loss is associated with ribosomal queuing upstream of stop codons proceeded by C-terminal arginine, lysine and asparagine. (A) Metagene analysis of the 5′ read ends for the 5PSeq libraries generated from wild-type (WT MYJ1171, black trace) and New1-deficient (new1Δ MYJ1173, red trace) yeast strains grown at 20°C. Stalls represent the 5′ boundary of the ribosome-protected 5′-phosphorylated mRNA fragment. The 5′ position is offset by -17 nt from the first nucleotide in the ribosomal A site. With an 80S ribosome covering 30 mRNA bases, the 30-nt periodicity of the in the new1Δ metagene is indicative of multiple ribosomes queued at the stop codon. (B) An example of a heavily ‘queued’ ORF YGR195W encoding a C-terminal AGG arginine codon. Normalised read coverage around the stop codon is shown. (C) Ribosomal queuing scores (QS) calculated for ORFs parsed by the codon preceding the stop codon. The red dashed line is a guide for the expected position of data points in the absence of the systematic difference between in QS between the wild-type and new1Δ samples. All analyses were performed on pooled wild-type and new1Δ 5PSeq datasets from three biological replicates each.

Figure 4.

The absence of New1 specifically increases ribosomal readthrough when the weak stop codon UGA is preceded by C-terminal lysine. (A–C) Binned distributions of ribosome queuing scores (QS) for ORFs terminating in either UAA (A), UAG (B) or UGA (C) stop codons. (D–F) Same as (A–C), but with for ORFs encoding C-terminal lysine residues. Geomeans of QS distributions for wild type is black and for new1Δ is in red, the QS fold change is in green. Numbers of ORFs included in the analyses are shown on the figure, e.g. 1830 instances of ORFs with UAA stop codon. All analyses were performed on pooled datasets from three replicates collected at 20°C. (G, H) Same as (D-F), but with for ORFs encoding C-terminal aspartic acid. (J, K) Stop codon readthrough efficiencies in wild-type and new1Δ strains measured with dual-luciferase reporters harbouring UAA, UAG and UGA stop codons in combination with either a C-terminal AAA lysine codon (J) or GAU aspartic acid codon (K). (L) Readthrough efficiency of UAG G, UAG A, UAG U and UAG C extended stop codons in combination with a C-terminal AAA lysine codon in both wild-type and new1Δ strains. Note that the same AAA UGA C results are shown on both panels (J) and (L) in order to make the data comparison easier. Error bars represent standard deviations. All experiments were performed at 20°C.

Figure 5.

Synonymous codons encoding the C-terminal lysine and arginine residues have dramatically different effects on translation termination defects in new1Δ yeast. (A) Wild-type and new1Δ QS scores for ORFs parsed by the codon preceding the stop codon. (B–E) QS distributions with ORFs containing C-terminal lysine encoded by either AAG (B) or AAA (C), as well as arginine encoded by either AGA (D) or AGG (E). Wild-type: solid line, darker shading. Δnew1: dashed line, lighter shading. Geomeans of QS distributions for wild-type is black and for new1Δ is in red, the QS fold change is in green. All analyses were performed on pooled dataset from three replicates collected at 20°C. (F) Stop codon readthrough efficiencies of UGA stop codon combined with penultimate codons as indicated on the figure. Error bars represent standard deviations, all experiments were performed at 20°C.

Figure 6.

Termination defect in new1Δ yeast is dependent on the nature of the tRNA isoacceptor species present in the P-site of the pre-termination complex. (A) Decoding abilities of tRNA isoacceptors, adapted from Johansson and colleagues (89). Codons read by individual tRNA species are indicated by circles connected by lines. The gene copy number for each tRNA species is indicated and the position of the number denotes the cognate codon. Empty circles indicate that the tRNA is less likely to read the codon. The empty dashed circles indicate that the tRNA does not efficiently read the codon. C-terminal codons that display increased QS in the new1Δ strain are highlighted with green shading. (B, C) Queuing score distributions for NEW1 (solid outline) and new1Δ (dashed) strains either expressing (B) or lacking (C) . (D) Penultimate AGG codons are not associated with readthrough in new1Δ yeast when decoded by the near-cognate species. Readthrough efficiency for UGA stop codon preceded by penultimate AGG codon in strains either expressing or lacking . All experiments were performed at 20°C.

Figure 7.

Ribosomal queuing and termination defects of new1Δ strain are uncoupled from the growth defect at low temperature. (A, B) Polysome profile analysis of wild-type (MYJ1171) and new1Δ (MYJ1173) yeast cells grown exponentially at 20°C (F) or 30°C (G) in SC media. Polysomes were resolved on 7–45% sucrose gradients. All experiments performed at 30°C with the exception of (F), 5PSeq data analysis was performed on pooled data sets from three biological replicates. (C) Metagene analysis of the 5′ read ends for the 5PSeq libraries generated from wild-type (WT MYJ1171, black trace) and New1-deficient (new1Δ MYJ1173, red trace) yeast strains grown at 30°C. (D, E) QS distributions sorted by AAG lysine (D) or AAA lysine C-terminal codons (E), geomean QS values in the wild-type and new1Δ strains are given in black and red respectively and fold change in QS in green. (F) Readthrough of C-terminal AAA and AAG lysine codons in combination with a UGA stop codon. Error bars represent standard deviations. (G) Readthrough values of UAA, UAG and UGA stop codons in combination with a C-terminal AAA lysine codon. Error bars represent standard deviations. To make the data comparison easier, the same AAA UGA C results are shown in panel (F) and (G).