A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution

Abstract Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.

Table S1.Name and sequence of the RNA used in the experiment.The nucleotides marked in red were the selected detection sites.

Figure S2 .
Figure S2.Preliminary experimental validation of pseU-TRACE.(a) Two single-stranded DNA and two probes complementary to the DNA were designed, and ligase ligation and qPCR amplification were performed.(b-d) Optimization of the ligation reaction time (b), ligation temperature (c), and ligase concentration (d).(e) 60 nt RNA containing Ψ or U was subjected to pseU-TRACE analysis.(f) Different up probes were designed to investigate whether BS treatment of Ψ does result in one base deletion rather than a mismatch.The relative amount was determined using 2 −ΔCT .∆CT was calculated by the differences in the threshold cycle of amplification (CT values) between "Control" and "BS treat".Error bars indicated mean±s.d. for 2 or 3 technical replicates.*p<0.05;**p<0.01;***p<0.001;ns, nonsignificant by t-test (one-tailed).

Figure S3 .
Figure S3.The performance and detection limits of pseU-TRACE.(a) A set of 59-or 60-nt DNA templates with different nucleotides beside the ligation site were conducted pseU-TRACE analysis to determine the influences of sequences to the ligation efficiency.The relative amount of ligation products of T-G DNA was used as the reference.(b-d) Different amounts of input RNA were performed pseU-TRACE to determine the baseline.The nucleotides surrounding the Ψ site of RNA were 5' C and 3' A (60-Ψ, b), 5' C and 3' G (60-Ψ2, c), 5' U and 3' U (60-Ψ3, d), respectively.And the cDNA sequence of these RNA oligos were corresponded to T-G, C-G and A-A cDNA in Figure S3 a. ∆CT was calculated by the differences in the threshold cycle of amplification (CT values) between "Control" and "BS treat".Error bars indicated mean±s.d. for 3 technical replicates.

Figure S4 .
Figure S4.pseU-TRACE enabled the selective detection of Ψ in rRNA.(a-d) Real-time fluorescence amplification threshold cycle (CT values) (top) and the relative amount (bottom) showed that pseU-TRACE could detect Ψ in 28S and 18S.(e-g) Real-time fluorescence amplification threshold cycle (CT values) (top) and the relative amount (bottom) showed pseU-TRACE results of other nucleotides including A, C and G in 28S.Specific Ψ sites were marked on the top left of the graphs.Each experiment was replicated twice (Rep1 and Rep2).Error bars indicated mean±s.d. for 3 technical replicates.*p<0.05;**p<0.01;***p<0.001;ns, non-significant by t-test (one-tailed).

Figure S5 .
Figure S5.pseU-TRACE enabled the detection of Ψ in mRNA from total RNA samples.(a-d) Real-time fluorescence amplification threshold cycle (CT values) (top) and the relative amount (bottom) demonstrated that pseU-TRACE detected Ψ in mRNA from HEK293T and HeLa total RNA.Specific Ψ sites were marked on the top left of the graphs.Each experiment was replicated twice (Rep1 and Rep2).Error bars indicated mean±s.d. for 3 technical replicates.*p<0.05;**p<0.01;***p<0.001;ns, non-significant by t-test (one-tailed).

Figure S6 .
Figure S6.pseU-TRACE enabled the selective detection of Ψ in mRNA.(a-c) Real-time fluorescence amplification threshold cycle (CT values) (top) and the relative amount (bottom) showed that pseU-TRACE could specifically identify Ψ in mRNA after Oligo(dT)25 magnetic beads purification.Specific Ψ sites were marked on the top left of the graphs.Each experiment was replicated twice (Rep1 and Rep2).Error bars indicated mean±s.d. for 3 technical replicates.*p<0.05;**p<0.01;***p<0.001;ns, non-significant by t-test (one-tailed).

Figure S7 .
Figure S7.Detection of mRNA level after siRNA transfection.(a-b) The relative mRNA level was measured by qPCR after transfection of si-TRUB1 437, 680, 962, 533 or si-NC for 24 h (a) or 48 h (b).Using si-NC sample as a reference, the relative mRNA level was calculated by comparing with si-NC.(c-d) The relative mRNA level was measured by qPCR after transfection of si-PUS7 998, 1477, 1676, 1780 or si-NC for 24 h (c) or 48 h (d).Using si-NC sample as a reference, the relative mRNA level was calculated by comparing with si-NC.Error bars indicated mean±s.d. for 3 technical replicates.

Figure S8 .
Figure S8.Detection of protein level after siRNA transfection.(a-b) Western blot showed that protein level was significantly reduced in samples transfected with si-TRUB1 437, 680, 962 and 533 compared to si-NC for 48 h (a) or 72 h (b).(c-d) Western blot showed that protein level was significantly reduced in samples transfected with si-PUS7 998, 1477, 1676 and 1789 compared to si-NC for 48 h (c) or 72 h (d).Two biological replicates were performed simultaneously.

Figure S9 .
Figure S9.pseU-TRACE used for functional studies of Ψ regulation and metabolism.pseU-TRACE combined with siRNA interference used for the identification of Ψ under PUS7 regulation.(a-b) Real-time fluorescence amplification threshold cycle (CT values) (top) and the amount (bottom) showed pseU-TRACE results for detecting Ψ in NRDC and FSCN1 mRNA, comparing si-NC transfection with si-PUS7 1676 transfection.Specific Ψ sites were marked on the top left of the graphs.Each experiment was replicated twice (Rep1 and Rep2).Error bars indicated mean±s.d. for 3 technical replicates.*p<0.05;**p<0.01;***p<0.001;ns, non-significant by t-test (one-tailed).