Differentiation-coupled intron retention reveals a candidate NKG2D-TR-like isoform at the murine Klrk1 locus

Abstract

NKG2D (encoded by KLRK1 in humans and Klrk1 in mice) is an activating receptor expressed by cytotoxic lymphocytes. In humans, NKG2D signaling is regulated post-transcriptionally: activated T cells retain intron 4 of KLRK1 to generate NKG2D-TR, a truncated dominant-negative isoform that limits receptor signaling. Whether mice, the principal preclinical model for NKG2D-directed therapies, possess an analogous regulatory mechanism remains unknown. Across four murine RNA-seq datasets comprising 50 samples and spanning T-cell differentiation, graft-versus-host disease, and acute and chronic LCMV infection, we examined the retained-intron isoform Klrk1-203. The transcript retains the canonical start codon, while its predicted stop codon lies within the terminal exon downstream of the final exon-exon junction, suggesting that it may escape nonsense-mediated decay. If translated, Klrk1-203 is predicted to encode a truncated product that retains the cytoplasmic and transmembrane domains but lacks most of the ligand-binding ectodomain. This predicted architecture resembles human NKG2D-TR, although the murine product contains a short C-terminal sequence encoded by the retained intron. Klrk1-203 was below the detection limit in unchallenged naive and early-effector T cells but was induced in differentiated effector and memory populations, reaching approximately one-fifth of total Klrk1 transcripts in one effector-memory sample. Read-level analyses independently demonstrated increased intron 4 retention with differentiation; however, short-read sequencing cannot fully distinguish Klrk1-203 from the co-retained Klrk1-204 transcript, making isoform-specific abundance dependent on model-based quantification. An independent coding-potential algorithm classified Klrk1-203 as non-coding, providing an important counterpoint to the structural predictions. Together, these findings identify Klrk1-203 as a candidate NMD-resistant, differentiation-associated regulator of murine NKG2D and a potential counterpart of human NKG2D-TR that warrants experimental validation.