Shifting frames to make more proteins
Severe acute respiratory syndrome coronavirus 2 critically depends on the ribosomal frameshifting that occurs between two large open reading frames in its genomic RNA for expression of viral replicase. Programmed frameshifting occurs during translation, when the ribosome encounters a stimulatory pseudoknot RNA fold. Using a combination of cryo–electron microscopy and biochemistry, Bhatt et al. revealed that the pseudoknot resists unfolding as it lodges at the entry of the ribosomal messenger RNA channel. This causes back slippage of the viral RNA, resulting in a minus-1 shift of the reading frame of translation. A partially folded nascent viral polyprotein forms specific interactions inside the ribosomal tunnel that can influence the efficiency of frameshifting.
Science, abf3546, this issue p. 1306
Abstract
Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo–electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.
Ribosomal frameshifting, a process during which the reading frame of translation is changed at the junction between open reading frames (ORFs) 1a and 1b, is one of the key events during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive-sense single-stranded RNA genome. This programmed −1 translational frameshifting is conserved in all coronaviruses and is necessary for the synthesis of viral RNA-dependent RNA polymerase (RdRp or Nsp12) and downstream viral nonstructural proteins that encode core enzymatic functions involved in capping of viral RNA, RNA modification and processing, and RNA proofreading (1). Although the translational machinery typically prevents frameshifting as a potential source of one of the most disruptive errors in translation (2, 3), many viruses rely on programmed ribosomal frameshifting to expand and fine-tune the repertoire and stoichiometry of expressed proteins (4).
Programmed −1 frameshifting in SARS-related coronaviruses occurs at the slippery sequence U_UUA_AAC in the context of a 3′ stimulatory RNA sequence that was predicted to form a three-stemmed pseudoknot structure (5) and, in parallel, was independently tested by our lab and others (6–8). The frameshifting occurs with high efficiency (25 to 75%), depending on the system used (6, 7, 9–11), and changes the reading frame to UUU_AAA_C (12) (Fig. 1A). Consequently, two viral polyproteins are synthesized: one encoded by ORF1a when frameshifting does not take place, and ORF1ab, which is expressed as a result of frameshifting. Translation of ORF1a produces polyprotein 1a, which ends with Nsp10 followed by the short Nsp11. Conversely, when the frameshift occurs, the polyprotein 1ab is generated, which contains almost 2700 additional amino acids and in which the viral RdRp, Nsp12, is produced after Nsp10 as a consequence of translation in the −1 frame. A putative secondary structure element in the viral RNA that forms a loop upstream of the shift site has been proposed to play an attenuating role in frameshifting and is referred to as the 5′ attenuator loop (8). Maintaining the precise level of coronavirus frameshifting efficiency is crucial for viral infectivity, as evidenced by the fact that mutation of a single nucleotide in the frameshifting region of the SARS-CoV-1 RNA results in a concomitant abrogation of viral replication (13). Therefore, the importance of three-stemmed pseudoknot-dependent −1 ribosomal frameshifting for the propagation of SARS-related coronaviruses, a process that has not been seen to occur on any endogenous human transcript in human cells, presents itself as an opportune drug target with minimal tolerance for drug-resistant mutations.
(A) Schematic of the SARS-CoV-2 main ORF. In the close-up view of the frameshift event, codons and corresponding amino acids are shown. During −1 frameshifting, the slippery site codons UUA (Leu) and AAC (Asn) are the last codons decoded in the 0 frame. Upon −1 frameshifting of the AAC codon to AAA, translation resumes at the CGG (Arg) triplet, where elongation proceeds uninterrupted to produce full-length Nsp12. (B) In vitro translation reaction depicting pausing at the frameshift site, as shown with Western blotting. Efficient frameshifting is observed for the WT template, consistent with our dual luciferase assays (see methods). Samples for cryo-EM originally intended to be trapped by dominant negative eRF1 (AAQ) show a tRNA-bound pause in proximity of the frameshift site. The tRNA-associated band is lost upon RNase treatment. Reactions without added eRF1 (AAQ) produce a similarly paused product. (C) Overview of the density low-pass filtered to 6 Å with the pseudoknot found close to the entry of the mRNA channel on the small subunit (SSU). The SSU proteins are colored in yellow, the large subunit (LSU) proteins in blue, and the rRNA in gray. The pseudoknot is colored according to its secondary structure as in (F), and the P-site tRNA is colored in green. (D) Close-up view of the pseudoknot from the solvent-exposed side of the SSU. Helix h16 of the 18S rRNA interacts with the base of Stem 1. Unpaired loop-forming nucleotides are colored in cyan. (E) P-site codon-anticodon interactions reveal a Phe (UUU) codon interacting with tRNA(Phe). yW37, wybutosine at position 37. (F) Schematic of the revised secondary structure elements in the pseudoknot necessary for −1 programmed ribosomal frameshifting, with different functional regions labeled and colored accordingly.
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(A) Schematic of the SARS-CoV-2 main ORF. In the close-up view of the frameshift event, codons and corresponding amino acids are shown. During −1 frameshifting, the slippery site codons UUA (Leu) and AAC (Asn) are the last codons decoded in the 0 frame. Upon −1 frameshifting of the AAC codon to AAA, translation resumes at the CGG (Arg) triplet, where elongation proceeds uninterrupted to produce full-length Nsp12. (B) In vitro translation reaction depicting pausing at the frameshift site, as shown with Western blotting. Efficient frameshifting is observed for the WT template, consistent with our dual luciferase assays (see methods). Samples for cryo-EM originally intended to be trapped by dominant negative eRF1 (AAQ) show a tRNA-bound pause in proximity of the frameshift site. The tRNA-associated band is lost upon RNase treatment. Reactions without added eRF1 (AAQ) produce a similarly paused product. (C) Overview of the density low-pass filtered to 6 Å with the pseudoknot found close to the entry of the mRNA channel on the small subunit (SSU). The SSU proteins are colored in yellow, the large subunit (LSU) proteins in blue, and the rRNA in gray. The pseudoknot is colored according to its secondary structure as in (F), and the P-site tRNA is colored in green. (D) Close-up view of the pseudoknot from the solvent-exposed side of the SSU. Helix h16 of the 18S rRNA interacts with the base of Stem 1. Unpaired loop-forming nucleotides are colored in cyan. (E) P-site codon-anticodon interactions reveal a Phe (UUU) codon interacting with tRNA(Phe). yW37, wybutosine at position 37. (F) Schematic of the revised secondary structure elements in the pseudoknot necessary for −1 programmed ribosomal frameshifting, with different functional regions labeled and colored accordingly.
Because of its importance in the life cycle of many important viruses and coronaviruses in particular, programmed frameshifting has been extensively studied using a range of structural and functional approaches (4). The structure of a 3′ stimulatory pseudoknot in isolation or in context of the viral genome has been proposed recently by various groups using techniques that include molecular dynamics, nuclease mapping, in vivo selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), nuclear magnetic resonance (NMR), and cryo–electron microscopy (cryo-EM) (7, 14–17). Furthermore, a ribosomal complex with a frameshift stimulatory pseudoknot from the avian infectious bronchitis virus was reported at low resolution (18). Here, to provide a structural and mechanistic description of the events during ribosomal frameshifting, we investigated mammalian ribosomes captured in distinct functional states during translation of a region of SARS-CoV-2 genomic RNA where −1 programmed frameshifting occurs.
Structure determination of a frameshifting-primed ribosomal complex
We captured a 0 frame, preframeshift ribosomal complex by introducing a stop codon in place of the second codon of the slippery site (U_UUA_AAC to U_UUA_UAA) (Fig. 1A) and adding mutant eukaryotic release factor 1 [eRF1 (AAQ)] that is unable to release the nascent polypeptide. Translating complexes were prepared in an in vitro translation reaction using an in-house–generated rabbit reticulocyte lysate (RRL) system that supported efficient frameshifting in the previously reported range of around 50% (19) according to dual luciferase experiments (see methods). The ribosomes were programmed with mRNA encoding an affinity tag and harboring a region of the SARS-CoV-2 genome that encodes proteins Nsp10 (C terminus), Nsp11, and most of Nsp12. Western blotting showed that when using the wild-type (WT) RNA template, frameshifting was efficient, whereas the stop codon mutation prevented frameshifting and led to ribosome pausing. This effect was further enhanced when eRF1 (AAQ) was present in excess over endogenous WT eRF1 (Fig. 1B).
The cryo-EM three-dimensional (3D) reconstruction of ribosome–nascent chain complexes affinity-purified from the reactions supplemented with eRF1 (AAQ) revealed two distinct ribosomal complexes captured in the process of translating the slippery sequence (figs. S1 and S2). One represented a termination complex that contained the ATP-binding cassette transporter 1 (ABCE1), which is known to be involved in termination and recycling together with mutant eRF1 interacting with the stop codon (fig. S3). The second reconstruction resolved translating 80S ribosomes containing bound P- and E-site tRNAs (fig. S2). This reconstruction at 2.2-Å resolution allowed us to build the most accurate structure of a mammalian 80S ribosome so far and directly visualize many protein and virtually all rRNA modifications identified for the human ribosome based on quantitative mass spectrometry and as interpreted in a recent human ribosome structure (20, 21), consistent with the complete conservation of all modified residues between rabbit and human ribosomal RNAs (rRNAs) (figs. S4 and S5; and tables S1 to S3). Importantly, this reconstruction also featured additional density at the entrance to the mRNA channel suggestive of a structured RNA, which, after focused classification, revealed a prominent density for a complete 3′ frameshifting stimulatory pseudoknot at the entry of the mRNA channel on the 40S subunit (Fig. 1, C and D). The resolution of this reconstruction ranged from 2.4 Å at the core of the ribosome to ~7 Å at the periphery, where the most flexible regions of the pseudoknot are located (figs. S2 and S6). Based on the high-resolution maps that allowed visualization of the codon-anticodon interactions and modifications in the tRNA (Fig. 1E and fig. S6, A and B), we could unequivocally determine that a Phe-tRNA(Phe) was bound at the P-site (22). The mRNA does not adopt any unusual structure in the A-site of the ribosome as was observed for the HIV-1 frameshifting sequence visualized on the bacterial ribosome (23). This implied that the ribosome is paused by the downstream pseudoknot located at the entrance to the mRNA channel such that the P-site tRNA interacts with the UUU codon just prior to the first codon, UUA, of the slippery site (Fig. 2A).
(A) Overview of the frameshift-primed state. The stimulatory pseudoknot pauses the ribosome at the penultimate codon (UUU) of the slippery site (red), with P-sites (green) and E-sites (pink) occupied by tRNAs and an empty A-site awaiting decoding in the nonrotated state. The length of the spacer region (gray) is critical for exact positioning of the pseudoknot as the spacer exerts tension at the entry of the mRNA channel (fig. S6C). The inset shows a secondary structure depiction of the frameshift-stimulating pseudoknot colored accordingly. PTC, peptidyl transferase center. (B) The backbone of Loop 1 (UGC) (cyan) of the pseudoknot interacts with the N-terminal domain of uS3 (red) and the C-terminal tail of eS10 (orange). mRNA residue G13486 is flipped out and interacts with uS3 (fig. S6D). (C) Mutagenesis experiments using dual luciferase assays in HEK293T cells indicate that the G13486 interaction is specific. Mutation of G13486 to other residues leads to a marked reduction in frameshifting efficiency, and deletion of Loop 1 (ΔL1) completely abolishes frameshifting. Similarly, deletion of a single nucleotide (A13537) in Loop 2 reduces frameshifting, whereas deletion of the entire loop (ΔL2) abolishes frameshifting. Normalized (Firefly-Renilla) luciferase activities were calculated for each construct as a percentage of their individual normalized in-frame controls. Data are presented as mean values ± standard deviations of three biological replicates (sets of translation reactions) averaged after three measurements, with error bars representing standard deviations. ****P < 0.0001 by Student’s two-tailed t test. (D) Mutagenesis experiments using dual luciferase reporter assays in HEK293T cells show that the position of the 0 frame stop codon influences frameshifting. When leaving the pseudoknot unaltered, an incremental increase in the distance of the 0 frame stop codon from the frameshift site leads to a concomitant decrease in frameshifting levels. Loss of the stop codon in the 0 frame leads to a sharp decline in frameshifting levels. This reduction is rescued by ~45% upon decreasing ribosome loading levels by implementing weaker initiation codons. The graph is normalized relative to the WT frameshifting of 25%. Mutations and complementary mutations are shown in fig. S8. Error bars represent standard deviation. NS, not significant; *P < 0.1; and **P < 0.01 by Student’s two-tailed t test.
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(A) Overview of the frameshift-primed state. The stimulatory pseudoknot pauses the ribosome at the penultimate codon (UUU) of the slippery site (red), with P-sites (green) and E-sites (pink) occupied by tRNAs and an empty A-site awaiting decoding in the nonrotated state. The length of the spacer region (gray) is critical for exact positioning of the pseudoknot as the spacer exerts tension at the entry of the mRNA channel (fig. S6C). The inset shows a secondary structure depiction of the frameshift-stimulating pseudoknot colored accordingly. PTC, peptidyl transferase center. (B) The backbone of Loop 1 (UGC) (cyan) of the pseudoknot interacts with the N-terminal domain of uS3 (red) and the C-terminal tail of eS10 (orange). mRNA residue G13486 is flipped out and interacts with uS3 (fig. S6D). (C) Mutagenesis experiments using dual luciferase assays in HEK293T cells indicate that the G13486 interaction is specific. Mutation of G13486 to other residues leads to a marked reduction in frameshifting efficiency, and deletion of Loop 1 (ΔL1) completely abolishes frameshifting. Similarly, deletion of a single nucleotide (A13537) in Loop 2 reduces frameshifting, whereas deletion of the entire loop (ΔL2) abolishes frameshifting. Normalized (Firefly-Renilla) luciferase activities were calculated for each construct as a percentage of their individual normalized in-frame controls. Data are presented as mean values ± standard deviations of three biological replicates (sets of translation reactions) averaged after three measurements, with error bars representing standard deviations. ****P < 0.0001 by Student’s two-tailed t test. (D) Mutagenesis experiments using dual luciferase reporter assays in HEK293T cells show that the position of the 0 frame stop codon influences frameshifting. When leaving the pseudoknot unaltered, an incremental increase in the distance of the 0 frame stop codon from the frameshift site leads to a concomitant decrease in frameshifting levels. Loss of the stop codon in the 0 frame leads to a sharp decline in frameshifting levels. This reduction is rescued by ~45% upon decreasing ribosome loading levels by implementing weaker initiation codons. The graph is normalized relative to the WT frameshifting of 25%. Mutations and complementary mutations are shown in fig. S8. Error bars represent standard deviation. NS, not significant; *P < 0.1; and **P < 0.01 by Student’s two-tailed t test.