Poster abstracts

Poster number 160 submitted by Yubin Kim

Multi-omic analysis of PUS7 D166E reveals alterations in RNA modification and gene expression

Yubin Kim (Department of Chemistry, University of Michigan), Grace Yoon (Division of Molecular Genetics, University of Toronto, The Hospital of Sick Children), Kristin S. Koutmou (Department of Chemistry, University of Michigan College of LSA)

Abstract:
When a single amino-acid substitution alters an RNA-modifying enzyme, its effects may extend beyond a single modified nucleotide. Pseudouridine synthase 7 (PUS7) catalyzes pseudouridine (Ψ) formation on diverse RNA substrates, and pathogenic PUS7 variants have been associated with neurodevelopmental phenotypes. We investigated a homozygous PUS7 c.498C>G (p.Asp166Glu; D166E) variant identified in a patient with neurodevelopmental features. This variant remains a variant of uncertain significance (VUS) and lies within the R3H-like binding domain rather than the catalytic TruD domain, making its functional consequences unclear.

To characterize the molecular effects of PUS7 D166E, we profiled tRNA modification landscapes using direct RNA nanopore sequencing and quantified global RNA modification levels by nucleoside LC-MS/MS, comparing patient cells with a heterozygous control. The tRNA sequencing analysis did not reveal a broad loss of pseudouridylation across the major tRNA sites examined. However, several sites showed altered modification patterns, with leucine tRNAs, particularly Leu-AAG and Leu-UAG, among the most prominent candidates. Consistent with the sequencing results, nucleoside LC-MS/MS did not detect a significant global reduction in Ψ. These findings suggest that PUS7 D166E may not cause a generalized loss of PUS7-dependent pseudouridylation but it could instead produce substrate- or site-specific alterations.

To further investigate downstream consequences of the variant, we are integrating mRNA sequencing and quantitative proteomics to assess changes in gene expression and protein abundance. Together, these analyses will provide a multi-omic framework for determining whether the PUS7 D166E variant alters RNA modification in a selective manner and whether such changes are associated with broader molecular consequences relevant to the patient's phenotype.

Keywords: PUS7, RNA modification, tRNA