INVESTIGATING THE ROLE OF PEPTIDYL ARGININE DEIMINASE 2 (PADI2) ON ALTERNATIVE SPLICING

ABSTRACT PADI2 (peptidyl arginine deiminase 2) is an enzyme involved in protein citrullination and has been shown to citrullinate RNA polymerase II (RNAPII), with a potential role in the regulation of transcription and co-transcriptional RNA processing. Given the close relationship between transcription and pre-mRNA splicing, this study aimed to investigate the role of PADI2 in alternative splicing and to elucidate the molecular mechanisms underlying this regulation. PADI2 expression was depleted by siRNA-mediated knockdown, and the effect of PADI2 loss on alternative splicing was investigated in selected target genes by RT-PCR and polyacrylamide gel electrophoresis. Efficient PADI2 depletion was confirmed at both the mRNA and protein levels. PADI2 knockdown resulted in significant alterations in alternative splicing, with both increased and decreased exon inclusion observed depending on the target transcript, supporting a transcript-specific regulatory role for PADI2. To investigate whether this effect was mediated by PADI2-dependent citrullination of RNAPII at residue R1810, wild-type and R1810 mutant RNAPII expressing cell were analyzed. Preventing R1810 citrullination did not reproduce the splicing alterations observed following PADI2 depletion, suggesting that the analyzed PADI2-dependent splicing regulation is not mediated by RNAPII citrullination. Since R1810 can also undergo methylation, the contribution of the methyltransferases PRMT5 and CARM1 were subsequently investigated. Their depletion did not reproduce the splicing phenotype, confirming the excluding R1810 methylation as the underlying mechanism. Together, these findings demonstrate that PADI2 regulates alternative splicing through a mechanism independent of RNAPII R1810 citrullination or methylation. Ongoing investigations aim to identify the molecular target(s) through which PADI2 exerts this effect. Elucidation of this mechanism may provide the basis for the identification of potential therapeutic targets and the future development of pharmacological strategies to modulate PADI2-dependent pathways.