Poster abstracts
Poster number 5 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, University of Michigan Medicine, University of Michigan Rogel Cancer Center, University of Michigan Center for RNA Biomedicine)
Abstract:
RNA modifications are capable of influencing RNA function through changes in structure or stability, which can then influence gene expression. Pseudouridine (Ψ), an isomer of uridine, is one prevalent RNA modification driven by pseudouridine synthase (PUS) enzymes. Dysregulation of a specific PUS enzyme, PUS7, is implicated in various disorders such as autism, microcephaly, and several types of cancers. While PUS7 modifies the central U within UNUAR motifs of RNA, only a small fraction of these sites are actually modified within the cells, and not much 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 us to first quantify the number of Ψ sites in cells. Therefore, I am developing a Bisulfite Pseudouridine Detection (B-PsiD) pipeline 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 the existing BID-Seq pipeline using alternative library preparation methods, enabling orthogonal validation of the original pipeline and quantification of the ratio of Ψ:U at every UNUAR site. Logistically, the B-PsiD pipeline 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 intends to compare the RNA structure and protein-binding networks around modified and unmodified sites through chemical probing and mutational profiling. Meanwhile, further pipeline optimizations may be implemented in the future such as the usage of tools like Dask to scale workloads or Snakemake to manage workflows.
References:
Zhang, LS., Ye, C., Ju, CW. et al. BID-seq for transcriptome-wide quantitative sequencing of mRNA pseudouridine at base resolution. Nature Protocols 19, 517–538 (2024). https://doi.org/10.1038/s41596-023-00917-5
Keywords: Pseudouridine, Bioinformatics, PUS7