Abstract
Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.
Highlights
4-thiouridine (4sU), a uridine analog, is a commonly used metabolic label that can be employed to investigate a wide variety of topics that range from pre-mRNA generation to mRNA degradation
Using complementary in vitro and cell culture experiments, we show that pre-mRNA splicing can be impacted by the incorporation of 4sU into pre-mRNAs
It is important to investigate whether the tools the scientific community use unintentionally create biases and side effects on the experiments being performed. 4sU labeling is a widely used tool in the RNA biology field to capture the processing of nascent RNAs and to evaluate RNA half-lives that is thought to have no consequence on the experimental outcome, aside from the previously reported inhibition of tRNA synthesis and nucleolar stress [7] at elevated concentrations
Summary
4-thiouridine (4sU), a uridine analog, is a commonly used metabolic label that can be employed to investigate a wide variety of topics that range from pre-mRNA generation to mRNA degradation. Indications of toxicity were observed at concentrations as low as 50μM [7], presumably because ribosomal RNA synthesis is inhibited, triggering cellular stress response [7] These observations suggested that 4sU incorporation can negatively impact translation and cellular homeostasis. Pre-mRNA splicing is an essential biological process that allows for proteomic complexity, despite the relatively small number of protein-coding genes within the human genome [8]. This process is carried out by the spliceosome, a multiprotein/snRNA complex that recognizes and binds to hallmark pre-mRNA sequences. The negative effects are only detectable at elevated levels of 4sU incorporation levels and in the context of weak splicing events
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