Phosphorylation by Prp4 kinase releases the self-inhibition of FgPrp31 in Fusarium graminearum.

Prp31 is one of the key tri-snRNP components essential for pre-mRNA splicing although its exact molecular function is not well studied. In a previous study, suppressor mutations were identified in the PRP31 ortholog in two spontaneous suppressors of Fgprp4 mutant deleted of the only kinase of the spliceosome in Fusarium graminearum. To further characterize the function of FgPrp31 and its relationship with FgPrp4 kinase, in this study we identified additional suppressor mutations in FgPrp31 and determined the suppressive effects of selected mutations. In total, 28 of the 35 suppressors had missense or nonsense mutations in the C terminus 465-594 aa (CT130) region of FgPrp31. The other 7 had missense or deletion mutations in the 7-64 aa region. The nonsense mutation at R464 in FgPRP31 resulted in the truncation of CT130 that contains all the putative Prp4 kinase-phosphorylation sites reported in humans, and partially rescued intron splicing defects of Fgprp4. The CT130 of FgPrp31 displayed self-inhibitory interaction with the N-terminal 1-463 (N463) region, which was reduced or abolished by the L532P, D534G, or G529D mutation in yeast two-hybrid assays. The N463 region, but not full-length FgPrp31, interacted with the N-terminal region of FgBrr2, one main U5 snRNP protein. The L532P mutation in FgPrp31 increased its interaction with FgBrr2. In contrast, suppressor mutations in FgPrp31 reduced its interaction with FgPrp6, another key component of tri-snRNP. Furthermore, we showed that FgPrp31 was phosphorylated by FgPrp4 in vivo. Site-directed mutagenesis analysis showed that phosphorylation at multiple sites in FgPrp31 is necessary to suppress Fgprp4, and S520 and S521 are important FgPrp4-phosphorylation sites. Overall, these results indicated that phosphorylation by FgPrp4 at multiple sites may release the self-inhibitory binding of FgPrp31 and affect its interaction with other components of tri-snRNP during spliceosome activation.

Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation.

Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes.

Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis.

Although DNA methylation is known to play an important role in the silencing of transposable elements (TEs) and introduced transgenes, the mechanisms that generate DNA methylation-independent transcriptional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation (RdDM) is required for the silencing of the RD29A-LUC transgene in the Arabidopsis ros1 mutant background with defective DNA demethylase. Loss of function of ARGONAUTE 4 (AGO4) gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the ros1/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC transgene expression in the ros1/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcriptional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.