Promoted read-through and mutation against pseudouridine-CMC by an evolved reverse transcriptase

Abstract

Pseudouridine (Ψ) is an abundant RNA chemical modification that plays critical biological functions. Current Ψ detection methods are limited in identifying Ψs at base-resolution in U-rich sequence contexts, where Ψ occurs frequently. Here we report "Mut-Ψ-seq" that utilizes the classic N-cyclohexyl N'-(2-morpholinoethyl)carbodiimide (CMC) agent and an evolved reverse transcriptase ("RT-1306") for Ψ mapping at base-resolution. CMC selectively labels Ψs in RNA forming the CMC-Ψ adduct and we show that RT-1306 presents promoted read-through and mutation against the CMC-Ψ. We report a high-confidence list of Ψ sites in polyA-enriched RNAs from HEK-293T cells identified by orthogonal chemical treatments (CMC and bisulfite). The mutation signatures resolve the position of Ψ in UU-containing sequences, revealing diverse occurrence of Ψs in such sequences. This work provides methods and datasets for biological research of Ψ, and expands the toolkit for epitranscriptomic studies by combining the reverse transcriptase engineering and selective chemical labeling strategies.

Fig. 1. Structure and CMC-based detection of Ψ in RNA.

a Chemical structures showing the formation of Ψ upon the isomerization of U. b Reaction scheme of CMC with Ψ yielding the N3 CMC-Ψ adduct (“CMC-Ψ”). c Illustrations of RT stop and read-through events against the CMC-Ψ.

Fig. 2. Promoted read-through efficiencies and mutations against CMC-Ψ by RT-1306.

a Shown on the left are the fluorescence images of RT stop assay gels for SSIII, wtRT, and RT-1306 reading against Ψ-oligo1 and Ψ-oligo2 RNAs with and without CMC treatment. RT products were run and separated on 15% 8 M Urea-PAGE gels and imaged by the fluorescence imaging. Positions of the FAM-labeled RT primer, truncated cDNA at the Ψ site, and the full-length cDNA products are labeled with “P”, “T”, and “FL”, respectively. The same gels were also stained by SYBR-Gold and imaged (Supplementary Fig. 2). Shown on the right are quantified RT read-through based on the RT stop assay. The RT read-through efficiency over CMC-Ψ were quantified by the ratio of the fluorescence intensity of the “T” band over the sum of intensities of the “T” and “FL” bands. Error bars represent the standard deviations of n = 3 replicates for Ψ-oligo1, n = 2 replicates for Ψ-oligo2; full gel images of 2 replicates are presented in Supplementary Fig. 2. b Colony sequencing data of the cDNA products from RT-1306 processing Ψ-oligo1 and Ψ-oligo2 RNAs with (“CMC+”) and without (“CMC−”) CMC treatments (Methods). Shown are the clones that carry mutations at the Ψ sites and data for all sequenced colonies are shown in Supplementary Fig. 3.

Fig. 3. Characterization of Ψ into CMC-Ψ conversion and RNA loss by varying the duration of CMC reaction.

a Shown is the increasing Ψ into CMC-Ψ conversion efficiency of Ψ-oligo1 with the elongated CMC reaction duration, measured by the RT stop assay. b Dependence of the Ψ into CMC-Ψ conversion of Ψ-oligo1 RNA and the RNA recovery after CMC treatment, upon the CMC reaction duration.

Fig. 4. Promoted mutation signatures against CMC-Ψ in rRNAs by RT-1306 in “piloting” Ψ-seq libraries.

a Representative IGV views showing RT mutations at two example Ψ sites in 18S and 28S rRNAs. b Ψ1177 in 18S rRNA with 20-min or 2-h CMC reaction. c Profiling of RT mutation, stop and deletion signatures at all reported Ψ sites in rRNAs by RT-1306, with statistical comparisons to wtRT and SSIII. d Profiling of RT signatures by RT-1306 at Ψ sites in rRNAs detected with 20-min and 2-h CMC reaction. P values were calculated by two-sided Student’s t-test, with the significance levels noted where ns. not significant, *P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001.

Fig. 5. Development and results of Mut-Ψ-seq.

a Workflow and list of prepared libraries for Mut-Ψ-seq. b Comparison of Ψ identification efficiency by individual or combined RT signatures generated by RT-1306 or SSIII, through the AUC values derived from ROC curve analyses against reported Ψs and Us in rRNAs (Supplementary Fig. 9 and Supplementary Fig. 10). c Shown on the left is the ROC analyses for assessing Ψ identification efficiency of RT-1306 by 20-min or 2-h CMC reaction. Shown on the right are the distributions of observed combined rates generated by RT-1306 against reported 1088 Us (upper panel) and 105 Ψs (low panel) in rRNAs. P values were calculated by two-sided Student’s t-test, with the significance levels noted where ****P  <  0.0001. d Representative IGV views of the RT mutation signature suggesting the occurrence Ψ in GUΨC sequence in four mRNAs.