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. 2021 Dec 13;16(12):e0257503.
doi: 10.1371/journal.pone.0257503. eCollection 2021.

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Affiliations

The influence of 4-thiouridine labeling on pre-mRNA splicing outcomes

Jessie A C Altieri et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. In vitro splicing kinetics of 4sU labeled RNA.
(A) Schematic depictions of the AdML and β-Globin minigenes used. Exon and intron sizes, splice sites and strengths (MaxEnt score in red) are indicated. (B) Autoradiogram representing time course behavior of 0%, 2.5%, 30%, and 100% 4sU containing β-Globin pre-mRNAs. Precursor RNA and spliced products are defined on the right of the gel. The numbers under each lane represents the efficiency of splicing. (C) Graphical representation of β-Globin time-course splicing assay splicing efficiencies over time (n = 3) and the corresponding splicing rate. ‘**’ indicates statistically significant difference (ordinary one-way ANOVA, P = 0.0031). (D) Graphical representation of AdML time-course splicing assay splicing efficiencies over time (n = 3) and the corresponding splicing rate. ‘ns’ indicates no statistically significant difference (ordinary one-way ANOVA, P = 0.14).
Fig 2
Fig 2. In vitro degradation assay of AdML minigene.
(A) Representative autoradiograph of degradation time-course for unmodified and fully modified 4sU AdML pre-mRNA. Time ‘0’ represents input RNA. Full-length input RNA is defined by the cartoon to the right of the gel. (B) Quantitation of the data shown in (A). First order decay regression of time course data was used for rate determination and statistical analysis.
Fig 3
Fig 3. In vitro transcription of AdML and β-Globin.
Autoradiograph depicting the T7 polymerase transcription profiles of (A) AdML and (B) β-Globin. Four transcripts of each minigene were created with varying amounts, 0%, 2.5%, 30%, and 100%, of 4sU incorporated. The full pre-mRNA strands and abortive transcripts in each lane are labeled.
Fig 4
Fig 4. Splicing behavior in cell culture in the presence of 4sU.
PCR analysis of (A) constitutive exons within the genes ADAP2, DOLPP1, and ZNF711 and (B) alternatively included exons in genes ADAP2, CLK2, TRA2B, ZNF711, and DOLPP1. Alternative exon inclusion in DOLPP1 (‘**’, P = 0.0024). ‘ns’ indicates no statistically significant difference P>0.05). The cartoon to the left of each image indicates exon inclusion or exclusion. cDNA from biological triplicates were analyzed after 2 and 24 hours of 4sU incubation.
Fig 5
Fig 5. Alternative splicing behavior in cell culture in the presence of 4sU.
PCR analysis of (A) intron retention in DDX5 (‘ns’, P = 0.08; ‘**’, P = 0.0045) and (B) alternative 5’ss selection in RIOK3 (‘ns’, P = 0.012; ‘**’, P = 0.042). The cartoon to the right of each image defines the alternative splicing products. cDNA samples from biological triplicates were analyzed after 2 and 24 hours of 4sU incubation.

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