Figure 3. Effects of Pb²⁺ treatment on mRNA psi modification and machinery in SH-SY5Y cells.

a. Timeline illustrating the stages and duration of the Pb²⁺ treatment applied to SH-SY5Y cells.

b. A representative photomicrograph of untreated and Pb²⁺ treated SH-SY5Y cells is shown.

c. We used Deseq2 to identify the transcripts with the highest fold change between the untreated and Pb²⁺ treated samples. 3 biological replicates of each condition were used. The color scale shows a Z score based on the relative fold change.

d. DRS determined the TPM of various PUS enzymes in untreated and Pb²⁺-treated SH-SY5Y cells. Individual colored bars represent each experimental condition, with error bars describing the standard error of the mean (SEM) across downsampled replicates. Individual replicates are shown as black dots.

e. SNR was calculated by using beta-binomial distribution and Jeffrey’s prior, plotting points against the difference in U-to-C error between the untreated and Pb²⁺ treated libraries. Orange dots represent uridine positions that are validated TRUB1 substrates, and blue dots represent uridine positions that are validated PUS7 substrates.

f. Putative psi-positions determined by Mod-p ID are plotted according to the difference in U-to-C basecalling error in the untreated and Pb²⁺ treated samples against the reads for each position. A dotted line at the +5% and −5% marks indicate the cutoff for a position to be changed in response to perturbation. The inlet shows the sequencing logo for positions within the TRUB1 motif, grey points above the threshold line, and total points above the threshold line.

g. Annotation of genes containing a psi modification that changed in response to perturbation and validated by PUS7 or TRUB1 KD (Figure 2) and orthogonal methods.

h. TPM of the transcripts bearing a validated psi modification that had the most significant differences between conditions determined by DRS. Individual colored bars represent each experimental condition, with error bars describing the standard error of the mean (SEM) across downsampled replicates. Individual replicates are shown as black dots.

i. Correlation of the differential U-C mismatch number of reads vs. the enzyme: substrate product for untreated and Pb²⁺ treated SH-SY5Y cells. Lines are Pearson correlation fits showing weak positive correlations for both PUS enzymes (R²=0.62 for TRUB1 and R²=0.23 for PUS7).