Nuclear phosphatase PfNIF2 governs erythrocyte invasion and genome integrity through invasion-related proteins and SR1 dephosphorylation

PfNIF2 is a nuclear protein phosphatase belonging to the NIF family in Plasmodium falciparum. It is characterized by a conserved ‘DLDET’ motif, which is essential for its catalytic activity. PfNIF2 primarily localizes to the perinuclear or nuclear compartment and shows peak expression during the schizont stage of the parasite's lifecycle. Genetic disruption of the pfnif2 gene significantly impaired the efficiency of merozoite invasion, resulting in a 1.65-fold reduction (P < 0.05). It also increased susceptibility to artemisinin derivatives, with the IC50 of dihydroartemisinin decreasing from 12.5 nM to 7.9 nM (P < 0.001), although the asexual development of the parasites remained viable. Site-directed mutagenesis confirmed that aspartate 65 within the ‘D63LD65ET’ motif is critical for enzymatic function and invasion competence. Transcriptomic profiling revealed extensive dysregulation of invasion-related genes, including RON4 and MSP1, in schizont-stage PfNIF2Del parasites. Integrated co-immunoprecipitation, LC-MS/MS, and phosphoproteomic analyses demonstrated that PfNIF2 interacts with DNA repair machinery, such as SR1, SUN2, and MSH2-1. It plays a role in dephosphorylating RNA-binding proteins and invasion effectors. Notably, the expression of a phosphoablative variant of serine/arginine-rich splicing factor 1 (SR1-S205A/S208A) in PfNIF2Del parasites substantially rescued defects in DNA damage repair, alternative splicing fidelity, artemisinin sensitivity, and erythrocyte invasion. These findings establish PfNIF2 as a multifunctional regulator that bridges nuclear RNA processing, genome integrity maintenance, and host cell invasion in malaria parasites. type (WT) 3D7 and PfNIF2Del parasites using TRIzol reagent (Thermo Fisher). To ensure biological replicates, the experiments were repeated at three-week intervals. mRNA was enriched through the application of poly(dT) oligonucleotide-conjugated magnetic beads, followed by reverse transcription employing SuperScript II (Thermo Fisher) in conjugation with random hexamer primers. RNA templates were subsequently removed via RNase H treatment, and cDNA was purified using a DNA Clean and Concentrator Kit (Zymo Research, CA, USA). Indexed libraries were constructed using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina, CA, USA) and sequenced on an Illumina platform. Raw sequencing reads underwent quality filtering in fastq format and were subjected to adapter trimming using cutadapt v1.13. High-quality reads were aligned to the Plasmodium falciparum 3D7 reference genome (PlasmoDB-68) employing HISAT2 v2.0.4]. Deduplication of reads was conducted with UMI-tools v1.0.0 [52], and quantification was achieved through HTSeq v0.9.1. Differential expression analysis was carried out using DESeq (v1.18.0), with P-values adjusted for false discovery rate (FDR <0.05) following the Benjamini-Hochberg method. Gene Ontology (GO) enrichment analysis was performed using GOseq.