Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique


<jats:title>Abstract</jats:title><jats:p>Pseudouridine (Ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of Ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based Ψ mapping technique called photo-crosslinking assisted Ψ sequencing (PA-Ψ-seq) and use it to map Ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add Ψ residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of Ψ addition on cellular mRNAs to each of these three PUS enzymes, the Ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of Ψ residues detected on total human mRNA below the ∼0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of Ψ residues to human mRNAs remains to be defined.</jats:p>






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Publication Info

Campos, Cecilia Martinez, Kevin Tsai, David G Courtney, Hal P Bogerd, Christopher L Holley and Bryan R Cullen (n.d.). Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique. 10.1101/2021.05.01.442255 Retrieved from

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Christopher Lee Holley

Associate Professor of Medicine

The Holley Laboratory is focused on the role of non-coding RNA (ncRNA) in cardiovascular health and disease, with a special emphasis on snoRNA (small nucleolar RNA).  snoRNAs are canonically known to guide the chemical modification of other RNAs, with ribosomal RNA being the primary target.  Dr. Holley’s research has helped to uncover a novel biologic role for the Rpl13a snoRNAs in the regulation of reactive oxygen species (ROS) and oxidative stress.  These four snoRNAs (U32a, U33, U34, and U35a) have a critical role in the oxidative stress response to a variety of stimuli, including saturated fatty acids, lipopolysaccharide, doxorubicin, and hydrogen peroxide.

The Holley Lab has shown that at least one mechanism linking the Rpl13a snoRNAs to ROS and oxidative stress is snoRNA-guided methylation of mRNA. This methylation in an mRNA coding sequence inhibits subsequent protein translation. We have also shown that snoRNA-guided methylation alters RNA conformational ensembles, which can stabilize short-lived structures.

Currently, the lab is studying the role of Rpl13a snoRNAs in atherosclerosis, where loss or inhibition of these snoRNAs reduces athero by ~50%. We are actively pursuing translational research opportunities to design "RNA therapeutics" targeting these snoRNAs for potential clinical use.

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