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Published on: September 28, 2018
Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3
Jiayun Wang1, Lingyan Yan1, Xu Wang1
1State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China.
Objective:
Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear.
Methods:
The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells.
Results:
HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well.
Conclusions:
Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.
Insights
Researchers identified heterogeneous nuclear ribonucleoprotein K (HNRNPK) as a key factor in regulating programmed cell death 1 (PD-1) expression. HNRNPK promotes PD-1 exon 3 skipping, decreasing surface PD-1 and increasing soluble PD-1, impacting immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Immunotherapy targeting PD-1 shows promise, necessitating understanding PD-1 expression regulation.
- PD-1 exon 3 skipping generates soluble PD-1 (sPD-1), enhancing immune response by competing with surface PD-1 (flPD-1).
- The precise mechanism of PD-1 exon 3 skipping remains unclear.
Purpose of the Study:
- To elucidate the regulatory mechanism of PD-1 exon 3 skipping.
- To identify splicing factors involved in PD-1 alternative splicing.
- To investigate the functional consequences of PD-1 exon 3 skipping on PD-1 expression.
Main Methods:
- Utilized SpliceAid program and minigene system to identify splicing factors.
- Employed site-directed mutagenesis and pulldown assays to verify HNRNPK binding.
- Synthesized and screened antisense oligonucleotides (ASOs) to inhibit exon 3 skipping.
- Analyzed alternative splicing via RT-PCR, and protein expression via Western blot and flow cytometry.
Main Results:
- Heterogeneous nuclear ribonucleoprotein K (HNRNPK) was identified as a key splicing factor promoting PD-1 exon 3 skipping.
- HNRNPK binds to an exonic splicing silencer (ESS) on PD-1 exon 3, facilitating skipping.
- ASO-mediated inhibition of HNRNPK-ESS interaction significantly reduced exon 3 skipping.
- HNRNPK-induced exon 3 skipping decreased flPD-1 and increased sPD-1 expression.
- This regulatory mechanism is conserved in mice.
Conclusions:
- A novel, evolutionarily conserved mechanism regulating PD-1 expression was discovered.
- HNRNPK promotes PD-1 exon 3 skipping by binding to an ESS.
- This splicing event leads to reduced flPD-1 and increased sPD-1 in T cells, impacting immune function.
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