21 / 2026-02-27 15:30:04

Alternative polyadenylation landscapes and their switching mechanisms during T cell activation revealed by scPolyA-seq2

Single cell RNA sequencing; single cell polyadenylation sequencing; alternative polyadenylation; T cell activation; PAS motif

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Alternative polyadenylation (APA) is a critical post-transcriptional regulatory mechanism for generating transcript isoforms. However, our understanding of APA dynamics during T cell activation at single-cell resolution remains elusive.

Here, we developed scPolyA-seq2, a sensitive 3′ single-cell sequencing technology that enables precise identification and quantification of polyadenylation sites (PAS). We profiled the APA landscape of resting human CD4+ T cells and stimulated human CD4+ T cells at single-cell resolution. Integration of APA with gene expression data identified new cell subsets, indicating that APA provides a new layer of information. T cell activation resulted in longer transcripts, increased usage of canonical PAS motifs, and higher expression of polyadenylation factors. We defined APA events that contain a distal PAS and a proximal PAS within the same last exon as tandem 3′UTR APA, and the remaining events as exon-switch APA. The tandem 3′UTR APA prefers proximal PAS usage while the exon-switch APA prefers distal PAS usage during T cell activation, showing opposite temporal PAS usage patterns. Analysis of exon-switch APA revealed that switching to distal PAS is accompanied by the adoption of canonical PAS motifs, which are more efficient for polyadenylation.

We further found the T cell activation process is correlated with higher expression of polyadenylation factors, leading to more efficient binding and cleavage at PAS with canonical motifs. The increased usage of PAS with canonical motifs facilitates cellular efficiency, thereby enabling rapid responses to environmental stimuli. This study provides an APA atlas at single-cell resolution and reveals the PAS usage switch mechanism during T cell activation.