<b>PfNIF2 phosphatase dephosphorylates SR1 to ensure merozoite competence through mRNA splicing and genome stability</b>

Reversible protein phosphorylation is a central mechanism regulating cellular pathways in the malaria parasite Plasmodium falciparum. Here, we characterize PfNIF2, a perinuclear-localized protein phosphatase of the NIF family that contains a conserved ‘DLDET’ catalytic motif and exhibits peak expression during schizogony. Genetic disruption of pfnif2 impairs merozoite invasion efficiency, while site-directed mutagenesis demonstrates that aspartate 65 within the ‘DLD65ET’ motif is critical for both phosphatase activity and invasion competency. Integrated interactome and phosphoproteome analyses identify the serine/arginine-rich splicing factor 1 (SR1) as a key substrate. PfNIF2 interacts with the C-terminal region of SR1 through its CPDc domain and dephosphorylates S205 and S208. Loss of PfNIF2 results in SR1 hyperphosphorylation, leading to aberrant alternative splicing of ribosomal protein genes and genomic instability with elevated DNA strand breaks. Expression of a phosphoablative SR1 (S205A/S208A) restores normal splicing, reduces DNA damage, and rescues erythrocyte invasion capacity. Our findings establish PfNIF2 as a multifunctional regulator that, through direct dephosphorylation of SR1, coordinates nuclear RNA processing, preserves genome integrity, and facilitates host cell invasion in Plasmodium falciparum.