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

Abstract

Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding 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 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, 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.

Keywords: HIV-1; epitranscriptomics; post-transcriptional gene regulation; pseudouridine.

FIGURE 1.

Validation of the novel PA-Ψ-seq mapping technique. (A) Validation of the Ψ antibody. A dot blot assay was performed using RNA in vitro transcribed with either UTP or pseudo-UTP (upper panel). RNA integrity was confirmed by gel electrophoresis (lower panel). (B) Comparison of PA-Ψ-seq-mapped rRNA Ψ sites with previously reported Ψ residues on human 28S rRNA in CEM T cells and 293T cells pulsed with 4SU or 6SG. The T > C conversions derived from 4SU crosslinking are shown as red/blue bars. On transcripts that are encoded in the reverse orientation to the reference genome, T > C conversions are shown as the reverse complement: A > G, as a green/orange bar. Similarly, 6SG crosslinking results in G > A conversions shown as orange/green bars, with C > T conversions shown as blue/red bars in the reverse orientation. Blue bars below the figure indicate the location of Ψ residues reported previously, as labeled. (C) Mapping of Ψ residues on human RPL15 mRNA in CEM T cells and 293T cells pulsed with 4SU or 6SG. The previously reported single Ψ residue is indicated by an arrow. (D) Similar to C, detection of a Ψ residue previously identified in SRSF1 mRNA. (E) Detection of previously reported Ψ residues in tRNA-Glu-CTC. (F) Similar to E, previously reported Ψ on tRNA-Gln-CTG. (G) Mapping the location of the two Ψ residues previously identified in U4 snRNA. The Ψ residue at position 4 is too close to the 5′ end of the U4 snRNA to be captured by the PA-Ψ-seq technique. (H) Similar to G, but mapping the location of the three Ψ residues previously identified in U5 snRNA.

FIGURE 2.

Using PA-Ψ-seq to map Ψ residues located on HIV-1 transcripts. (A) HIV-1-infected CEM T cells or 293T cells were pulsed with 4SU and virion RNA produced by CEM cells, or poly(A)+ RNA isolated from CEM or 293T cells, analyzed for the presence of Ψ using PA-Ψ-seq. (B) Similar to panel A, except that these data were obtained using total HIV-1 virion RNA isolated from virions released by 6SG-pulsed wild-type or PUS-KO 293T cells, as indicated. All these data used the PA-Ψ-seq technique except for the last lane, which used an m⁶A-specific antibody to perform the very similar PA-m⁶A-seq technique as a specificity control. Highly reproducible Ψ peaks are numbered and indicated by beige lines and are shown relative to a schematic of the ORFs present on the HIV-1 genome.

FIGURE 3.

Validation of cellular PUS enzyme knockout cell lines. (A) Western blot of WT and two ΔPUS1 clonal cell lines. (B) Western blot of WT and two ΔPUS7 clonal cell lines. (C) Western blot of WT and two ΔTRUB1 clonal cell lines. (D) Analysis of Ψ levels in total RNA samples derived from WT or PUS-deficient 293T clones using UPLC-MS/MS. N = 4 biological replicates with each PUS knockout cell line analyzed twice. Individual values, average and SD indicated. (E) Similar to D, except analyzing highly purified mRNA samples derived from the indicated WT or PUS-deficient 293T clones.

FIGURE 4.

Representative PUS1, PUS7, and TRUB1-dependent Ψ residues on cellular RNAs. PA-Ψ-seq mapping of Ψ residues on human tRNAs known to be deposited by TRUB1 (A,B) or PUS7 (C–E). PA-Ψ-seq was used to identify de novo Ψ residues on cellular mRNAs that are dependent on PUS1 (F,G), PUS7 (H,I), or TRUB1 (J,K). The location of Ψ residues in mRNA exons (indicated by blue boxes) is indicated by arrows. Whenever the read counts in the Ψ site of the ΔPus or ΔTRUB lane was not zero, the sequencing depth-normalized fold change (FC) in site read count is shown.

FIGURE 5.

Effect of loss of PUS enzymes on HIV-1 gene expression. (A) Western blot of p55 and p24 Gag produced after infection of WT or PUS mutant cell lines at 48 h post-infection (hpi) with VSV-G pseudotyped NL4-3ΔEnv virions. Cellular GAPDH was used as loading control. (B) Similar to A, except this shows the quantification of total (p55 plus p24) HIV-1 Gag production in HIV-1-infected WT or PUS mutant 293T cell lines at 48 hpi across three independent biological replicates. Data were normalized to cellular GAPDH and are given relative to the parental 293T cells, which were set at 1.0. (**) P < 0.01.