Decoding the Function of Expansion Segments in Ribosomes

Abstract

Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. By employing quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.

Keywords: ES; ES27L; MetAP; expansion segment; methionine amino peptidase; rRNA; ribosome; translation fidelity.

Figure. 1.. A eukaryotie-specific rRNA expansion segment, ES27L, has a role in translation fidelity.

(A) Left: The secondary structure of S. cerevisiae 25S and 5.8S rRNAs are shown. ESs are highlighted in red. The arrows are showing the placement of ESs that cannot fit in the 2D rRNA representation. Right: Enlargement of ES27L. ES27L "b" and "c" stem-loops are in yellow. The E. coli ES27L equivalent stem-loop and ES27L "a" helix are in orange. (B) Left: Schematic workflow to make rDNA mutant strains is shown. Right: The spot assay indicates the sensitivity of ES27L mutant strains to Paromomycin, which induces translation error. The picture of the ES27L Δc strain was taken from the same plate shown in Figure S1G. (C) The percentage of amino acid misincorporation was measured by a tandem Rluc-Fluc reporters, in which each has various point mutations at Fluc active site (grey) under normal condition (w/o Paro) or Paromomycin treatment (Paro). (D) The percentage of stop codon read-through detected by reporters with an insertion of a stop codon (grey). (E) The percentage of frameshift induced by the PRF (Programed ribosomal frameshifting) sequence measured by a construct having the −1 PRF sequence or +1 PRF sequence (grey). Data are represented as mean + standard deviation (SD) (t-test, **P < 0.01; *P < 0.05; NS, not significant, n ≥ 3). See also Figure S1.

Figure 2.. ES27L recruits conserved N-terminal processing enzymes on the ribosome.

(A) Left: Schematic of the identification of ES27L associating factors. IP: Immunoprecipitation, MS: mass spectrometry Right: Comparison of ES27L Δb1-4 ribosome to the WT ribosome, proteins with log₂FC < −1 (FC: fold change) with FDR < 0.05 (FDR, false discovery rate) are defined as significantly depleted (black circle). Zuo1 protein is not significant (white circle). The average FC of two biological replicates is shown. (B) The ES27L-dependent ribosome association of hemagglutinin (HA)-tagged Map1 protein is shown by the shift in association in SDG fractions comparing the WT_rRNA strain to ES27L “b” stem loop mutants. (C) The ES27L dependent co-migration of HA-tagged Map2 protein with the ribosome is also shown. (D) ES27L independent ribosome association of Zuo1 protein is shown. Rps5/uS7, is shown as a control. See also Figure S2 and Table S1.

Figure. 3.. MetAP couples N-terminal processing and translation fidelity.

(A) Spot assay reveals that the MAP1 deletion strain phenocopies the ES27L Δb1-4 strain but the MAP2 deletion strain does not. Since Δmap1 has slower growth, a longer incubation is also shown. (B) The translation fidelity of wild type (WT: W303), Δmap1, and Δmap2 strains were evaluated with reporters containing an insertion of a stop codon between Rluc and Fluc gray under normal conditions (w/o Paro) or Paromomycin treatment (Paro). (C) Translation fidelity in WT: W303 and Δmap1 strains was also evaluated by amino acid misincorporation and both −1 and +1 frameshifting induced by the PRF sequence gray. Data are represented as mean + SD (t-test, **P < 0.01; *P < 0.05; NS, not significant, n ≥ 3). See also Figure S3.

Figure. 4.. Function of MetAP enzymatic activity for translation fidelity across evolution.

(A) The enzymatic activity of MetAP is required for translation fidelity. The cleavage of iMet (green) is catalyzed through a conserved His 301 (red). H₂O is in blue. SDG shows co-migration of the mutant Map1 protein at the catalytic Histidine (MAP1^H301N) with the ribosome. The RP (Rps5/uS7) is shown as a control. Right: percentage of the UGA stop codon gray readthrough in WT: W303 (white bar) and Δmap1 (black bar) strains with the overexpression of wild type Map1 protein (+MAP1-HA) or Map1 catalytic Histidine mutant (+MAP1^H301N-HA) is shown. (B) MetAP activity in vivo is assessed by iMet retention measured with a Rluc-HA reporter conjugated with the N-terminal peptide of human 14-3-3γ(dark gray box)r(dark gray box), a model substrate, wherein retained iMet that has not been processed can be detected with an iMet-14-3-3γ antibody. The HA antibody detects the entire reporter protein. (C) The SDG fractionation of the bacterial ribosome in C. crescentus, which does not possess ES27L followed by the western blotting for HA-tagged Map protein and ribosomal protein (Rps13/uS13) are shown. (D) The MetAPs activity and the percentage of the UGA stop codon gray read-through upon treatment with the MetAPs inhibitor Bengamide B (Beng) in mouse embryonic stem (ES) cells. Data are represented as mean + SD (t-test, **P < 0.01; *P < 0.05; NS, not significant, n ≥ 3). See also Figure S4.