Poster abstracts
Poster number 107 submitted by Sonia Ling
A pipeline for Bisulfite Pseudouridine Detection (B-PsiD)
Sonia Ling (University of Michigan College of Literature, Science, and the Arts), Kira Holton (University of Michigan Department of Biological Chemistry), Chase Weidmann (University of Michigan Department of Biological Chemistry)
Abstract:
Pseudouridine (Ψ), an isomer of uridine, is a prevalent RNA modification driven by pseudouridine synthase (PUS) enzymes. Dysregulation of one PUS enzyme, PUS7, is implicated in various disorders such as autism and microcephaly. PUS7 modifies the central U within UNUAR motifs of RNA, but only a small fraction is modified within cells, and little is known about how PUS7 chooses which UNUAR substrates to modify. In turn, our laboratory is interested in defining PUS7 substrate specificity for mRNAs, which requires quantification of the number of Ψ sites in cells. Therefore, the Bisulfite Pseudouridine Detection (B-PsiD) pipeline is being developed for integration into our laboratory’s workflows. The core of this pipeline extends upon the bisulfite-induced deletion sequencing (BID-Seq) method from Zhang et al., which relies on bisulfite-based conversion of pseudouridines into ribose ring-openings that are then read as deletions during reverse transcription [Nature Protocols, 19, 517–538 (2024)]. B-PsiD adapts BID-Seq using alternative library preparation methods, enabling orthogonal validation and quantification of the ratio of Ψ:U at every UNUAR site. Logistically, B-PsiD mirrors the five main parts of BID-Seq: (i) quality control and merging of the Illumina sequencing reads; (ii) contaminant RNA removal to isolate the mRNA; (iii) alignment to the human genome; (iv) realignment for more accurate deletion-only mapping; and finally, (v) the calculation of real Ψ modification stoichiometry at every UNUAR site. Once all high confidence modified Ψ sites are identified, our laboratory will compare the RNA structure around modified and unmodified sites via chemical probing and mutational profiling.
References:
Dai, Q., Zhang, LS, Sun, HL, et al. (2022). Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution. Nature Biotechnology, 41, 344–354. https://doi.org/10.1038/s41587-022-01505-w
Kim, K. Q., Burgute, B. D., Tzeng, SC, et al. (2022). N1-methylpseudouridine found within COVID-19 mRNA vaccines produces faithful protein products. Cell Reports, 40(9), 1–14. https://doi.org/10.1016/j.celrep.2022.111300
Ye, C. (2022). pseudoU-BIDseq [Source code]. http://doi.org/10.5281/zenodo.8158036
Zhang, LS, Ye, C., Ju, CW, et al. (2024). BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution. Nature Protocols, 19, 517–538. https://doi.org/10.1038/s41596-023-00917-5
Keywords: Pseudouridine, Bioinformatics, PUS7