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

Area of Science:

• Molecular Biology
• Immunology
• RNA Biology

Background:

• Immortalized cell lines, like Jurkat T cells, are vital research tools but may differ from primary cells.
• The impact of immortalization on RNA modification machinery, specifically pseudouridine (ψ), is not fully understood.

Purpose of the Study:

• To compare pseudouridine (ψ) profiles in mRNA from primary and immortalized T cells using direct RNA sequencing (DRS).
• To identify differences in ψ-modified transcripts and understand their functional implications in different T cell types.

Main Methods:

• Direct RNA sequencing (DRS) was employed to profile pseudouridine (ψ) modifications.
• Comparative analysis of ψ-sites in mRNA from primary T cells and immortalized Jurkat T cells.

Main Results:

• A high conservation (87%) of ψ-sites was observed between primary and immortalized T cells, predominantly in essential cellular process transcripts.
• Unique ψ-sites in Jurkat cells were associated with immune activation and oncogenesis.
• Unique ψ-sites in primary T cells were linked to calcium signaling and intracellular trafficking.
• Differences in ψ-sites were mainly due to transcript presence/absence, not RNA-modification enzyme levels, which were conserved.

Conclusions:

• While most pseudouridine (ψ) modification sites are conserved, distinct differences exist between primary and immortalized T cells.
• These differences highlight specific cellular functions and warrant consideration when using immortalized cells in immunological research.
• Site-specific ψ level variations likely result from trans-acting regulatory factors rather than altered enzyme expression.