Organism:
Homo sapiens
Platform:
GPL20301
Samples:
6
Experiment Types:
Expression profiling by high throughput sequencing
Submitted:
Apr 22 2023
Last Updated:
Apr 11 2025
Status:
Public on Apr 11 2025
Contact:
Haissi,,Cui (University of Toronto)
Summary
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 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.
Overall Design
Ribosome profiling library construction Ribosome profiling libraries were generated as previously described (37, 38) with some minor modifications. Briefly, two 10 cm dishes of cells were used for each biological replicate, and three biological replicates were prepared for each cell line (MDA-MB-231-empty vector, MDA-MB-231-SARS). Cell homogenization was performed in 1 ml lysis buffer (20mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml CHX, 1% (v/v) TritonX-100, 50 units/ml Turbo DNaseI). RNase I-treated lysates were overlaid on top of a sucrose cushion in 5 ml Beckman Ultraclear tubes and centrifuged in an SW55Ti rotor for 4 hours at 4ºC at 46,700 rpm. Pellets were resuspended and RNA was extracted using the miRNeasy kit (Qiagen) according to manufacturerâs instructions. 26-34 nucleotide RNA fragments were purified by electrophoresis on a 15% denaturing gel. Linker addition, cDNA generation (first-strand synthesis was performed at 50°C for 1 h), circularization, rRNA depletion, and amplification of cDNAs with indexing primers were performed. Library quality and concentration were assessed using high sensitivity D1000 screen tape on the Agilent tape station, Qubit 2.0 Fluorometer, and qPCR. All libraries were pooled and run on HiSeq4000 (SR75). Ribosome profiling analysis Ribosomal footprints were analyzed as described by Ingolia et al. (37) with these modifications: Trimgalore was used to trim off adapters and clip the first nucleotide off the 5â end. Reads were then mapped to ribosomal RNA using bowtie2 (39) and unmapped reads were further mapped to the human transcriptome (v19) with STAR aligner (33). Expected read length distribution was tested with the R package RiboProfiling. To center ribosomes and obtain a list of genes with P-sites in their 3â UTR, we used functionalities within Ribowaltz (40) and a custom python script by Scott Adamson, UConn, and Jax Laboratories. Observed/expected ratios were calculated as described elsewhere (38, 41, 42) using a custom script by Scott Adamson. Obs/exp indicates the number of observed codons in the A-site of the ribosome vs the calculated hypothetical expected number of observations. If ribosome pausing occurs, obs/exp > 1.