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. 2025 Jan 15;39(1):e70287.
doi: 10.1096/fj.202401811RR.

Arginine demethylation of Serine/Arginine-rich splicing factor 1 enhances miRNA enrichment in small extracellular vesicles derived from pancreatic ductal adenocarcinoma cells

Kritisha Bhandari  1 , Jeng Shi Kong  1 , Wang-Ting Tina Ho  1 , Philip C Bourne  2 , Blaine H M Mooers  3 , Wei-Qun Ding  1
Affiliations

Arginine demethylation of Serine/Arginine-rich splicing factor 1 enhances miRNA enrichment in small extracellular vesicles derived from pancreatic ductal adenocarcinoma cells

Kritisha Bhandari et al. FASEB J. .

Abstract

Small extracellular vesicles (sEVs) are enriched in certain miRNAs, impacting the progression of pancreatic ductal adenocarcinoma (PDAC). The mechanisms involved in the selective sEV miRNA enrichment remain to be elucidated. We recently reported that Serine/Arginine-rich splicing factor 1 (SRSF1) regulates selective sEV miRNA enrichment in PDAC cells. SRSF1 is an onco-protein that is overexpressed in PDAC, and its function is dictated by posttranslational modifications such as phosphorylation and arginine methylation. The objective of this study was to examine the role of phosphorylation and arginine methylation in SRSF1-mediated sEV miRNA enrichment in PDAC cells. Treatment of PDAC cells with the protein arginine methyltransferase inhibitors AMI-5 and EPZ015666, but not with the phosphorylation inhibitor SRPIN340, selectively enhanced the level of sEV miR-1246, a miRNA known to be highly enriched in PDAC sEVs. Consistently, overexpression of the mutant SRSF1 with the three arginine residues R93, R97, and R109 being replaced with lysinaugmented sEV miR-1246 levels in both wild-type and SRSF1-knockdown PANC-1 cells. Interestingly, the binding of SRSF1 to miR-1246 was significantly reduced in PDAC cells overexpressing the mutant SRSF1, which was further confirmed using purified wild-type and the mutant SRSF1 proteins. We demonstrate that arginine demethylation of SRSF1 reduces SRSF1-miRNA binding in PDAC cells and enhances selective sEV miRNA enrichment, providing novel insight into SRSF1-mediated sEV miRNA enrichment in PDAC cells and opening up new avenues of investigation on the biology and function of extracellular vesicles in PDAC.

Keywords: SRSF1; miRNA; miR‐1246; pancreatic ductal adenocarcinoma (PDAC); protein arginine methylation; small extracellular vesicles (sEVs).

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Conflict of interest statement

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Figures

Figure 1.
Figure 1.. Treatment with AMI-5 enhances selective miRNA enrichment in sEVs derived from PANC-1 cells.
A. Western blot detection of the exosome markers CD63, ALIX, Flotillin, and the negative marker Calnexin. B. Western blot of PANC-1 cell-derived nuclear and cytoplasmic proteins upon AMI-5 treatment. C. sEV and cellular miRNA levels upon AMI-5 treatment of PANC-1 cells. (n=15 for sEV, n=12 for cellular). ****p<0.0001, ***p<0.001, **p<0.01, One-way ANOVA, multiple comparisons.
Figure 2.
Figure 2.. Treatment with AMI-5 enhances selective miRNA enrichment in sEVs derived from MIA PaCa-2 cells but not from HPNE cells.
A-B. sEV and cellular miRNA levels upon AMI-5 treatment of MIA PaCa-2 cells (n=5-6). C-D. sEV and cellular miRNA levels upon AMI-5 treatment of HPNE cells (n=9 for sEV, n=6 for cellular). *p<0.05, One-way ANOVA, multiple comparisons.
Figure 3.
Figure 3.. EPZ treatment enhances selective miRNA enrichment in sEVs derived from PANC-1 cells.
A. Western blot detection of PANC-1 nuclear and cytoplasmic proteins upon EPZ treatment. B-C. sEV and cellular miRNA levels 48 and 72 hours post treatment of PANC-1 cells (n=5-6). ***p<0.001, **p<0.01, One-way ANOVA, multiple comparisons. EPZ= EPZ015666.
Figure 4.
Figure 4.. SRPIN treatment does not alter selective miRNA enrichment in sEVs derived from PANC-1 cells.
A. Western blot detection of PANC-1 cell-derived nuclear and cytoplasmic proteins upon SRPIN treatment. B-C. sEV and cellular miRNA level upon SRPIN treatment of PANC-1 cells (n=9). **p<0.01, *p<0.05, One-way ANOVA, multiple comparisons. SRPIN= SRPIN340.
Figure 5.
Figure 5.. Overexpression of wild type and mutant SRSF1 proteins in PANC-1 cells alters sEV miRNA enrichment.
A-B. Diagrams depicting the site-directed mutagenesis design (R93, R97 and R109 replaced with lysine (K) or alanine (A)). Created with Biorender.com. C. Western blot detection of SRSF1 showing overexpression of Flag-tagged wild type and mutant SRSF1 in PANC-1 cells. D. Western blot detection of nuclear and cytoplasmic proteins isolated from PANC-1 cells expressing Flag-tagged wild type and mutant SRSF1. E. Expression level of miR-1246 (left) and miR-320d (right) in sEVs derived from PANC-1 cells overexpressing Flag-SRSF1, Mutant A Flag-SRSF1 or Mutant K Flag-SRSF1 (n=6). F. Expression level of miR-1246 in MIA PaCa-2 cells expressing Flag-tagged wild type and mutant SRSF1 (n=6). ****p<0.0001, **p<0.01, *p<0.05, One-way ANOVA, multiple comparisons.
Figure 6.
Figure 6.. Overexpression of wild type and mutant SRSF1 proteins in SRSF1-KD PANC-1 cells alters sEV miRNA enrichment.
A. Western blot detection of SRSF1 in whole cell lysates of SRSF1-KD cells. B. Western blot detection of SRSF1 upon overexpression of Flag-tagged wild type and mutant SRSF1 in SRSF1-KD PANC-1 cells. C. Expression level of miR-1246 (left) and miR-320d (right) in sEVs derived from SRSF1-KD PANC-1 cells overexpressing Flag-SRSF1, Mutant A Flag-SRSF1 and Mutant K Flag-SRSF1 (n=10). **p<0.01, *p<0.05, One-way ANOVA, multiple comparisons.
Figure 7.
Figure 7.. Replacement of the arginine residues (R93, R97 and R109) with alanine or lysine alters SRSF1-miRNA binding in wild type and SRSF1-KD PANC-1 cells.
A-B. Expression level of miR-1246 in PANC-1 cells detected by RNA immunoprecipitation assay (RIP) using anti-SRSF1 and anti-Flag antibody conjugated beads (n=6). ****p<0.0001, **p<0.01, One-way ANOVA, multiple comparisons. C-D. Expression level of miR-1246 in SRSF1-KD PANC-1 cells detected by RNA immunoprecipitation using anti-SRSF1 and anti-Flag antibody conjugated beads (n=6). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, One-way ANOVA, multiple comparisons.
Figure 8.
Figure 8.. Replacement of the arginine residues (R93, R97 and R109) with lysine reduces SRSF1-miR-1246 binding in test tubes.
A. Coomassie blue staining and Western blot detection of the purified wild type and Mut K SRSF1 proteins. Five μg and 3 μg of purified SRSF1 proteins were loaded for Coomassie blue staining and western blot analysis respectively. B. Ten and 100 ng purified wild type or Mut K SRSF1 proteins were incubated with small RNAs derived from PANC-1 cells. The reaction was immunoprecipitated using a SRSF1 antibody and miR-1246 expression was detected in the immunoprecipitants by qRT-PCR (n=6-12). ****p<0.0001, ***p<0.001, **p<0.01, One-way ANOVA, multiple comparisons.
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