Modulation of transcription and alternative splicing by spliceosome-activating nineteen complex (NTC) in Arabidopsis thaliana

Components of the spliceosome activating NTC (Nineteen Complex) are conserved in eukaryotes and their mutations in mammals result in early embryonic lethality. In Arabidopsis, the core PRL (PLEIOTROPIC REGULATORY LOCUS) NTC subunit is encoded by two closely related genes PRL1 and PRL2. This provides the unique opportunity to study the regulatory roles of NTC using mutations of the PRL homologs. Whereas inactivation of PRL1 results in dwarfism, early flowering, altered leaf development and hypersensitivity to low temperature, sucrose, ABA and cytokinin, the prl2 mutations cause embryo lethality at the heart stage. Compared to PRL1, the PRL2 mRNA level is about 10-fold lower in both wild type and prl1 mutant seedlings. The viability of prl1 mutant is thus likely maintained by partially overlapping function of PRL2, which secures reduced but still sufficient level of spliceosome activation. Main aim of this work was to characterize the roles of PRL1 and PRL2 in the regulation of transcription and alternative splicing. Deep RNA-Seq analysis identified 5,814 differential expression (DE) and alternative splicing (DAS) events indicating 5-fold higher representation of unspliced introns in upregulated versus downregulated mRNA isoforms in the prl1 mutant compared to wt. About 25% of DAS events identified altered splice sites, and 30% changed mRNA’s ORF removing either N-terminal organellar or secretion signal peptides, or increasing C-terminal distance between polyA and translational stop codons to enhance mRNA degradation by nonsense-mediated decay (NMD). 25% of DE mRNAs carried alternative 5’ or 3’ ends indicating that the prl1 mutation also influences the positions of transcription start and polyadenylation/cleavage. Functional reannotation and GO analysis of dataset indicated upregulation of genes involved in cell division, UV response, DNA repair, iron uptake, anthocyanin biosynthesis, and ABA/oxidative stress responses, in contrast to downregulation of genes in cell elongation, vesicular transport, auxin signaling and redox-regulated pathogen defense and cell death pathways correlating with known phenotypic traits of the prl1 mutant. In a subset of DE genes, we characterized the effect of prl1 mutation on alternative splicing of ribosomal protein genes. To obtain prl2 mutant cell populations, we used a conditional genetic complementation system for generation of genetic mosaics, in which the prl2 mutant sectors were labelled by expression of the green fluorescent protein (GFP). Following enrichment of mosaic sectors in callus and cell suspension cultures, the GFP+ prl2 and GFP- wild type cell populations were separated by cell sorting and subjected to a similar deep RNA-Seq analysis. This study identified 6314 DE/DAS events, which were compared to those observed in prl1. This lead to the identification of numerous candidate genes implicated in meiotic recombination, fertilization, embryogenesis, DNA replication and post-embryonic development which are differentially downregulated in the prl2 mutant. These data suggest that NTC complexes carrying PRL1 and PRL2 control transcription and splicing of mRNAs coding for both overlapping and distinct cellular functions.