Prp4 Kinase Research Articles

The prp4 kinase gene and related spliceosome factor genes in Trichophyton rubrum respond to nutrients and antifungals.

Trichophyton rubrum is a dermatophyte that causes most human superficial mycoses worldwide. The spliceosome, a large ribonucleoprotein complex responsible for pre-mRNA processing, may confer adaptive advantages to deal with different stresses. Here, we assessed the structural aspects of the Prp4 kinase protein and other pre-mRNA-splicing factors (Prps) in T. rubrum grown in different protein sources and exposed to antifungal drugs. Quantitative Reverse Transcription PCR (RT-PCR) assessed the modulation of prp1, prp31, prp8 and prp4 kinase genes after exposure of T. rubrum to sub-lethal doses of amphotericin B, caspofungin and acriflavine, or after T. rubrum growth on keratin sources for 48 and 72 h. We also performed the in silico analysis of the domain organization of Prps orthologues from filamentous fungi and yeasts. The prp4 gene was modulated in a time-dependent manner. Transcription levels were mostly up-regulated when T. rubrum was grown on keratin for 72 h, while exposure to amphotericin B promoted prp4 gene down-regulation at the same time point. We also observed co-expression of prp1 and prp31, and their down-regulation after amphotericin B exposure. In silico analysis revealed a conserved domain organization for most Prps orthologues with slight differences, which were mostly related to structural elements such as repetition domains in Prp1 and complexity in motif assembly for the Prp4 kinase. These differences were mainly observed in dermatophyte species and may alter protein interactions and substrate affinity. Our results improve the understanding of spliceosome proteins in fungi as well as their roles in adaptation to different environmental situations.

PRP4KA, a Putative Spliceosomal Protein Kinase, Is Important for Alternative Splicing and Development in Arabidopsis thaliana.

Splicing of precursor messenger RNAs (pre-mRNAs) is an essential step in the expression of most eukaryotic genes. Both constitutive splicing and alternative splicing, which produces multiple messenger RNA (mRNA) isoforms from a single primary transcript, are modulated by reversible protein phosphorylation. Although the plant splicing machinery is known to be a target for phosphorylation, the protein kinases involved remain to be fully defined. We report here the identification of pre-mRNA processing 4 (PRP4) KINASE A (PRP4KA) in a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase is the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals but it has not yet been studied in plants. In the same screen we identified mutants defective in SAC3A, a putative mRNA export factor that is highly coexpressed with PRP4KA in Arabidopsis Whereas the sac3a mutants appear normal, the prp4ka mutants display a pleiotropic phenotype featuring atypical rosettes, late flowering, tall final stature, reduced branching, and lowered seed set. Analysis of RNA-sequencing data from prp4ka and sac3a mutants identified widespread and partially overlapping perturbations in alternative splicing in the two mutants. Quantitative phosphoproteomic profiling of a prp4ka mutant detected phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, and other splicing-related factors. Tests of PRP4KB, the paralog of PRP4KA, indicated that the two genes are not functionally redundant. The results demonstrate the importance of PRP4KA for alternative splicing and plant phenotype, and suggest that PRP4KA may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.

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.

FgPrp4 Kinase Is Important for Spliceosome B-Complex Activation and Splicing Efficiency in Fusarium graminearum.

PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289.

Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation.

The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5’ splice site of both genes revealed that proper transient interaction with the 5’ end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5’ splice sites and weak branch sequences.

Evaluation of Cancer Dependence and Druggability of PRP4 Kinase Using Cellular, Biochemical, and Structural Approaches

PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.

Abstract 4376: Evaluation of PRP4 kinase as a potential drug target in cancer.

Abstract PRP4 kinase plays a crucial role in regulating pre-mRNA splicing, cell cycle progression, proliferation, and survival. The aforementioned functions have been well documented in Schizosaccharomyces pombe, Caenorhabditis elegans, and human cancer cell lines. For example, it was demonstrated that PRP4 kinase is essential for growth in fission yeast, and disruption of C. elegans PRP4 by RNAi resulted in a highly penetrant early embryonic lethality. In experiments utilizing siRNAs to screen for kinases essential for pancreatic cancer cell survival, PRP4 knockdown was demonstrated to increase apoptosis and decrease viability. In a genome-wide pooled shRNA screen, shRNAs against PRP4 was shown to reduce the viability of DLD-1, HCT-116, and HCC1954 cancer cell lines. Similarly, PRP4 kinase was identified as a potential therapeutic target in a pooled shRNA screen designed to identify genes required for proliferation and survival of diffuse large B-cell lymphoma cell lines. Moreover, in an effort to reveal potential kinase targets to treat multidrug resistance ovarian cancer, inhibition of PRP4 activity by shRNAs was shown to re-sensitize chemo-resistant human ovarian cancer to paclitaxel treatment. Interestingly, loss of PRP4 kinase was also demonstrated to enhance paclitaxel activity in breast cancer cells. To further investigate PRP4 kinase substrate spectrum and explore the druggability of PRP4 kinase, we utilize quantitative proteomics and structural biology to help achieve these objectives. In this report, we provided evidence that the kinase domain of PRP4 is essential for regulating cell growth and survival. In addition, through a global proteomics approach, we expanded the interactome and phosphoproteome of PRP4 kinase in cancer cells and identifed novel substrates of PRP4, including oncogenic PAK4 kinase. Subsequently, these substrates were substantiated in orthogonal biochemical and cellular assays. These new biological findings not only identify suitable biomarkers to monitor PRP4 kinase activity, but also provide interesting avenues for future in-depth interrogation of PRP4 functions in cancer biology and clinical development. Finally, we solve the X-ray structure of the PRP4 kinase domain and identify several features suitable for the rational design of PRP4 kinase inhibitors. We further provided the co-crystal structure of PRP4 kinase domain in complex with a small molecule and elucidated the exploitable mechanisms to synthesize potent and specific PRP4 inhibitors. Future efforts will be focused on understanding patient stratification strategy and assessing the utility of PRP4 kinase inhibitors in relevant pre-clinical models of cancer. Citation Format: Qiang Gao, Ingrid Mechin, Nayantara Kothari, Zhuyan Guo, Gejing Deng, Anlai Wang, Dmitri Wiederschain, Jennifer Rocnik, Werngard Czechtizky, Feng Liu, Tahir Majid, Vinod Patel, Christoph Lengauer, Carlos Garcia-Echeverria, Bailin Zhang, Hong Cheng, Marion Dorsch, Shih-Min A. Huang. Evaluation of PRP4 kinase as a potential drug target in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4376. doi:10.1158/1538-7445.AM2013-4376

Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation

Reversible protein phosphorylation has an essential role during pre-mRNA splicing. Here we identify two previously unidentified phosphoproteins in the human spliceosomal B complex, namely the pre-mRNA processing factors PRP6 and PRP31, both components of the U4/U6-U5 tri-small nuclear ribonucleoprotein (snRNP). We provide evidence that PRP6 and PRP31 are directly phosphorylated by human PRP4 kinase (PRP4K) concomitant with their incorporation into B complexes. Immunodepletion and complementation studies with HeLa splicing extracts revealed that active human PRP4K is required for the phosphorylation of PRP6 and PRP31 and for the assembly of stable, functional B complexes. Thus, the phosphorylation of PRP6 and PRP31 is likely to have a key role during spliceosome assembly. Our data provide new insights into the molecular mechanism by which PRP4K contributes to splicing. They further indicate that numerous phosphorylation events contribute to spliceosome assembly and, thus, that splicing can potentially be modulated at multiple regulatory checkpoints.

The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes.

Multiple genetic and biochemical interactions of Brr2, Prp8, Prp31, Prp1 and Prp4 kinase suggest a function in the control of the activation of spliceosomes in Schizosaccharomyces pombe

The spliceosomal component Prp1 (U5-102 kD) is found in Schizosaccharomyces pombe, a physiological substrate of Prp4 kinase. Here, we identify, spp41-1, a previously isolated extragenic suppressor of Prp4 kinase. The gene encodes an ATP-dependent RNA helicase homologous to the splicing factor Brr2 of Saccharomyces cerevisiae and U5-200 kD of mammalia. The suppressor allele, spp41-1, interacts genetically with alleles of prp1. We show that Prp1 and Brr2 are complexed in vivo with spliceosomal particles containing the five snRNAs U1, U2, U5, and base-paired U4/U6. Prp1 was found exclusively in small ribonucleoprotein particle (snRNP) complexes sedimenting in the range of 30S-60S, whereas Brr2 was also found sedimenting lower than 30S and free of snRNAs. Moreover, we find that the splicing factor Prp31 is complexed with Prp1 in the same spliceosomal particles containing the five snRNAs. These data indicate that in fission yeast spliceosomal particles larger than 30S exist, which can be considered as pre-catalytic spliceosomes. In addition, we show that S. pombe cells lacking Prp1 still contain these large pre-catalytic spliceosomal particles associated with Prp31. These data are consistent with the notion that in fission yeast phosphorylation of Prp1 by Prp4 kinase is involved in the activation of pre-catalytic spliceosomes.

Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes.

A growing body of evidence supports the coordination of pre-mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles. RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans. In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP-associated kinase. In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K. PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase. Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity. We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.

Spp42, identified as a classical suppressor of prp4-73, which encodes a kinase involved in pre-mRNA splicing in fission yeast, is a homologue of the splicing factor Prp8p.

We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4(+) gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73(ts) at the restrictive temperature (36 degrees ). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73(ts), indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73(ts) mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes.

Prp4 kinase is required for proper segregation of chromosomes during meiosis in Schizosaccharomyces pombe.

Chromosome segregation during meiosis is a complex process, which leads to production of four haploid gametes from two precursor cells. Reversible phosphorylation of proteins plays a crucial role in this process. The Schizosaccharomyces pombe Prp4 is an essential serine/threonine protein kinase, which belongs to the Clk/Sty family. To study the role of Prp4 in meiosis, we analysed chromosome segregation in a strain carrying conditional analog-sensitive allele of Prp4 protein kinase (prp4-as2). Our data show, that Prp4 protein kinase plays important role in chromosome segregation during meiosis, as revealed by enhanced missegregation of chromosomes in prp4-as2 mutant cells.