PfNIF2 phosphatase dephosphorylates SR1 to ensure merozoite competence through mRNA splicing and genome stability

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.

可逆蛋白质磷酸化是调控恶性疟原虫（Plasmodium falciparum）细胞通路的核心机制。本研究对PfNIF2进行了系统表征：该蛋白属于NIF家族，定位于核周区域，含有保守的"DLDET"催化基序，并在裂体增殖期达到表达峰值。对pfnif2基因的遗传敲除会损害裂殖子的入侵效率，而定点诱变实验证实，"DLD65ET"基序内的天冬氨酸65位对该磷酸酶的活性及入侵能力均至关重要。整合互作组与磷酸化蛋白质组分析鉴定出富含丝氨酸/精氨酸剪接因子1（SR1）为其关键底物。PfNIF2通过自身的CPDc结构域与SR1的C端区域相互作用，并对S205和S208位点进行去磷酸化修饰。PfNIF2的缺失会导致SR1发生过度磷酸化，进而引发核糖体蛋白基因的异常可变剪接，同时造成基因组不稳定性及DNA链断裂水平升高。表达磷酸化失活型SR1（S205A/S208A）可恢复正常剪接、减轻DNA损伤，并挽救红细胞入侵能力。本研究证实PfNIF2是一种多功能调控因子，通过直接对SR1进行去磷酸化修饰，协调疟原虫细胞核内的RNA加工过程、维持基因组完整性，并促进恶性疟原虫对宿主细胞的入侵。