Seryl-tRNA synthetase promotes translational readthrough by mRNA binding and involvement of the selenocysteine incorporation machinery

Abstract

Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.

Graphical Abstract

Figure 1.

SerRS binds to the 3′UTR of VEGFA to facilitate translational readthrough. (A) Scheme of VEGFA downstream of the first stop codon. VEGFA mRNA can be read through its first stop codon, resulting in VEGF-Ax with 22 appended amino acids. (B) Luciferase assay to quantify VEGFA translational readthrough. SerRS overexpression increased VEGFA TR while expression of two other aminoacyl-tRNA synthetases, GlyRS and TyrRS, did not impact VEGFA TR. (C) Upper panel: Secondary structure analysis of VEGFA predicts a stem-loop in the 3′UTR. Comparison between the VEGFA stem-loop motif and variable loops of tRNA^Ser and tRNA^Sec. Both variable loops and the VEGFA stem-loop contain G–C base pairs, which are recognized by SerRS (PDB-ID: 4RQF, lower panel). (D) Alignment of human tRNA^Sec and tRNA^Ser isoacceptor variable loops. The conserved G–C base pairs in each variable loop are highlighted. (E) Disruption of the VEGFA stem-loop motif abrogated SerRS-mediated TR, quantified by luciferase reporter assay. Exchanging G–C base pairs to A–U reduced TR, as did disrupting the stem-loop structure entirely. (F) Addition of 12 or 24 nucleotide spacers between the VEGFA UGA stop codon and stem-loop reduced TR as quantified by luciferase reporter assay. (G) Quantification of competitive EMSA results showing how different VEGFA constructs compete WT VEGFA mRNA off SerRS. (A–G) (n.s., not significant; * P< 0.05, ** P< 0.01, *** P< 0.001; **** P< 0.0001).

Figure 2.

SerRS-mediated translational readthrough is dependent on SerRS catalytic activity and selenocysteine incorporation elements. (A) Scheme of SerRS domain structure. (B) Domain mapping for SerRS-mediated translational readthrough. SerRS contains a tRNA-binding domain (TBD), a catalytic domain (CD) and a domain unique to SerRS (UNE-S) involved in nucleic acid binding. Expression of V5-tagged SerRS domains was confirmed by western blot. (C) EMSA showing binding of SerRS^WT but not SerRS^ΔUNE-S to the VEGFA mRNA. (D) EMSA showing binding of the catalytic mutant SerRST429A to the VEGFA mRNA. (E) A point mutation in the SerRS catalytic site (T429A), which renders SerRS catalytically inactive, abolished SerRS translational readthrough activity. (F) Mutation of the UGA stop codon to UAA or UAG abrogated increased translational readthrough upon SerRS overexpression in a VEGFA-based luciferase reporter assay. (G) SerRS-mediated TR measured by a VEGFA reporter assay with SerRS overexpression. TR is dependent on tRNA^Sec and eEFSec, as their knockdown abrogates the increase in translational readthrough by SerRS overexpression. SBP2 knockdown does not affect TR. Knockdown was verified by western blot. (A–G) (n.s., not significant; ** P< 0.01; *** P< 0.001; **** P< 0.0001)

Figure 3.

SerRS-mediated translational readthrough rescues protein levels reduced by a cancer-causing nonsense mutation found in a Taiwanese family. (A) Pedigree of the Taiwanese family with a hereditary MSH2 nonsense mutation (p.S612X, annotated as S611X in a previous study (16)). Female carriers are indicated by filled black circles, male carriers by filled black squares, and affected individuals (diagnosed with gastrointestinal cancers) by arrows. Open circles and squares indicate family members carrying the WT MSH2 gene. (B) Domain structure of MSH2. The monoallelic p.S612X nonsense mutation is located in the domain that interacts with MSH3 and MSH6. (C) qRT-PCR of MSH2 mRNA levels in patient-derived B cells (n = 3) showed no difference between mutant carriers and wild type individuals. (D) Western blot result of MSH2 protein levels in patient-derived B cells showed reduced full-length MSH2 protein in mutant carriers. (E) RNA structure prediction of the stem-loop motif following the p.S612X mutation on MSH2 that is recognized by SerRS. An EMSA showed SerRS binding to the stem-loop structure of MSH2. (F) Luciferase assay to quantify MSH2-S612X translational readthrough. Signal amplification was obtained from a reporter containing the N-terminal MSH2-S612X sequence including the 30 nucleotides downstream of the premature stop codon, followed by the coding sequence for luciferase. SerRS overexpression increased MSH2-S612X translational readthrough while expression of two other aminoacyl-tRNA synthetases, GlyRS and TyrRS, did not impact MSH2-S612X translational readthrough (n = 3). (G) Western blot results showing SerRS-mediated rescue of full-length MSH2-S612X protein levels after overexpression of SerRS, GlyRS and TrpRS with MSH2-S612X in HEK293 cells. (A–G) (n.s., not significant; ** P< 0.01).

Figure 4.

eCLIP-seq identifies a set of SerRS-bound RNAs that includes other translational readthrough genes. (A) A schematic showing the eCLIP-seq analysis workflow and filtering steps to reach the final list of 408 SerRS-bound windows within 50 nucleotides of a UGA stop codon (q < 0.05). Created with BioRender.com. (B) Venn diagram showing the overlap between the final 365 unique genes found in our eCLIP set (from 408 windows) with the confirmed translational readthrough genes identified in human foreskin fibroblasts by Dunn et al. (6). (C) Luciferase reporter assays were performed for the hits CFL1 and TIMP1, using VEGFA as a positive control. All three RNAs possess a G–C base pair-containing stem-loop after the canonical UGA stop codon. (D) Gene Ontology (GO) analysis was performed on RNAs from the final list of 408 windows. The number of genes enriched in each pathway are shown along with the overlapping translational readthrough genes from Figure 4B and our experimentally confirmed translational readthrough genes in Figure 4C. (A–D) (n.s., not significant; *** P< 0.001).