Pseudouridine reprogramming in the human T-cell epitranscriptome: from primary to immortalized states

Abstract

Immortalized cell lines are commonly used as proxies for primary cells in human biology research. For example, Jurkat leukemic T cells fundamentally contributed to uncovering T-cell signaling, activation, and immune responses. However, the immortalization process can alter key cellular properties, and researchers widely believe this process could significantly change RNA modification machinery and modification sites. In this study, we focus on pseudouridine (ψ), one of the most abundant mRNA modifications, and compare ψ profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS). Surprisingly, 87% of ψ-sites were shared between the two cell types, primarily in transcripts encoding proteins involved in essential cellular processes, including RNA-modification regulation. Furthermore, the analysis of the 13% of sites unique to each cell type reveals that Jurkat cells contained transcripts linked to immune activation and oncogenesis, while primary T cells contained transcripts associated with calcium signaling and intracellular trafficking. We provide a list of these genes, which should be considered when using immortalized cells to study RNA modifications in immunology contexts. Most differences were driven by whether the mRNA was present or absent in the immortalized or primary cell type. Interestingly, RNA-modification enzyme expression levels were highly conserved in both cell types. This suggests that site-specific differences in ψ levels arise from regulatory processes acting in trans rather than differences in modification enzyme levels.

Keywords: RNA modifications; immunology; nanopore direct RNA sequencing; primary T cells; pseudouridine.

FIGURE 1.

Pseudouridine profiles on coexpressed transcripts have mainly conserved positions with different occupancy levels and a limited set of unique sites in Jurkat and Naive libraries. (A, top left) Positional occupancy (mm%) for ψ sites in primary (orange) and immortalized (blue) cells, classified as unique (colored backgrounds) or shared (turquoise) based on U-to-C mismatch thresholds. The modification rate is a relative rate that can only be used to compare the same position within two cell types. (Top right) Venn diagram of unique and common ψ sites. (Bottom left) Δ occupancy (%) for shared ψ sites (primary minus immortalized) with a 20% threshold separating stable and variable sites; inset shows sequence logos by differential occupancy. (Bottom right) Pie chart of stable (light turquoise) versus variable (dark turquoise) shared sites. (B) Histogram showing a bimodal distribution of differential occupancy (U-to-C mismatch differences) for shared ψ sites. (C) Distribution of common ψ sites across the 5′ UTR, CDS, and 3′ UTR. (D) Common ψ-site distribution within the CDS by codon position (first, second, third nucleotide or stop codon). (E) Volcano plot of enriched GO Biological Process 2023 terms: odds ratio versus −log₁₀(P-value); larger, darker points indicate higher significance (gray points: P > 0.05; labeled: P < 0.01). (F) Heat map of the top 10 unique ψ sites in immortalized and primary libraries (ranked by differential occupancy) with insets showing sequence logos for unique primary (orange) and immortalized (blue) sites; CLAP gel confirms ψ incorporation in Jurkat cells (see Supplemental Fig. 4A). (G) Distribution of ψ sites across the 5′ UTR, CDS, and 3′ UTR. (H) ψ-site distribution within the CDS by codon position (first, second, or third nucleotide). (I) Enriched GO Reactome Pathways 2024 terms for immortalized (blue, top) and primary (orange, bottom) T cells.

FIGURE 2.

Pseudouridine profiles on transcripts uniquely expressed in immortalized or primary T cells. (A) Transcriptome-wide expression (TPMs) of unique transcripts in primary (orange) and immortalized T cells (blue). (B) Positional occupancy (U-to-C %mm) versus log-total reads for putative ψ sites (P < 0.001) in primary (top) and immortalized (bottom) cells. Red and green highlight known TRUB1 (GUUCN) and PUS7 (UNUAR) motifs, respectively; black points lack a motif. Dashed lines show minimum thresholds. (C) Volcano plot of GO Biological Process 2023 terms for ψ-sites on unique transcripts in primary (top) and immortalized (bottom) cells. Larger points indicate higher significance; gray points are nonsignificant (P > 0.05), with select terms labeled for P < 0.01.

FIGURE 3.

Comparison of immortalized and primary T cells to HeLa immortalized cell line. (A) Schematic representation of the comparison between immortalized state (immortalized Jurkat T cells vs. HeLa) and lineage (immortalized Jurkat T cells vs. Primary T cells). (B) Intersection of highly expressed transcripts (number of reads >20) across immortalized T cells, primary T cells, and HeLa cells. The histogram represents the number of transcripts shared among the indicated cell types. Black dots below each bar indicate which groups contribute to the intersection. (C) Intersection of ψ-sites detected in immortalized T cells, primary T cells, and HeLa cells (% U-to-C mismatch >10). The histogram represents the number of ψ modifications found in the indicated combinations of cell types. Black dots below each bar indicate which groups contribute to the intersection. (D) Heat map of hypermodified type I sites (% U-to-C mismatch >40) found in the three cell lines validated with orthogonal methods. For clarity, we only show the top five hypermodified type I sites found in two out of three conditions. Sites surrounded by a TRUB1 motif are highlighted in red, and sites surrounded by a PUS7 motif are highlighted in blue. (E) Sequencing logo of the hypermodified type I sites shared by immortalized T cells, primary T cells and HeLa.

FIGURE 4.

Hypermodified type II pseudouridine sites in immortalized and primary T cells. (A) Hypermodified type II transcripts with two to four putative ψ sites in primary (orange) and immortalized (blue) T cells; points represent in silico replicate detections. (B) Ternary plot showing the distribution of ψ sites across the 5′ UTR, CDS, and 3′ UTR in primary T cells; point size reflects the number of ψ sites on the same transcript, with transcripts harboring a TRUB1 motif in red and a PUS7 motif in green. Data points are annotated with the number of transcripts sharing the same UTR/CDS distribution. Transcript names are labeled for n = 1. (C) Ternary plot showing the distribution of ψ sites across the 5′ UTR, CDS, and 3′ UTR in immortalized T cells; point size reflects the number of ψ sites on the same transcript, with transcripts harboring a TRUB1 motif in red and a PUS7 motif in green. The number of hypermodified type II transcripts with the same UTR/CDS distribution is annotated next to each data point. Transcript names are labeled for n = 1. (D) Plot representing the distribution of Ψ sites and how they transition across transcript regions in primary T cells. Hypermodified type II transcripts have multiple Ψ sites, each localized in the 5′-UTR, CDS, or 3′-UTR regions of the transcript. (E) Plot representing the distribution of Ψ sites and how they transition across transcript regions in immortalized T cells. Hypermodified type II transcripts have multiple Ψ sites, each localized in the 5′-UTR, CDS, or 3′-UTR regions of the transcript.

Oleksandra Fanari