A GFP splicing reporter in a coilin mutant background reveals links between alternative splicing, siRNAs, and coilin function in Arabidopsis thaliana.

Coilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins (RNPs) including spliceosomal snRNPs. To study coilin function in plants, we conducted a genetic suppressor screen using a coilin (coi1) mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in older coi1 seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants of GFP pre-mRNA. In the coi1 suppressor screen, we identified suppressor mutations in WRAP53, a putative coilin-interacting protein; SMU2, a predicted splicing factor; and ZCH1, an incompletely characterized zinc finger protein. These suppressor mutations return the hyper-GFP fluorescence of young coi1 seedlings to the intermediate wild-type level. Additionally, coi1 zch1 mutants display more extensive GFP silencing and elevated levels of GFP siRNAs, suggesting the involvement of wild-type ZCH1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced the roles of coilin in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. The participation of coilin in these processes, at least some of which incorporate small RNAs, supports the hypothesis that coilin provides a chaperone for small RNA trafficking. Our study demonstrates the usefulness of the GFP splicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.

A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana.

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications. Here we present the final five mutants, which are defective, respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME CRITICAL REGION 14 (dgcr14), CYCLIN-DEPENDENT KINASE G2 (cdkg2), INTERACTS WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing data and analyses of differential gene expression and alternative splicing patterns for the cbp80 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, for which such information was not yet available. Sequencing of small RNAs from the cbp80 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss the cumulative findings and their implications for the regulation of pre-mRNA splicing efficiency and alternative splicing in plants. The mutant collection provides a unique resource for further studies on a coherent set of splicing factors and their roles in gene expression, alternative splicing and plant development.

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.