Internal Coordination Maintains the Structural and Functional Integrity of ASAP Complex Components in Arabidopsis

Abstract

RNA binding proteins (RBPs) regulate every stage of the mRNA lifecycle – from transcription, processing, degradation to translation – to ensure the expression of target genes support the fitness of an organism. One such conserved RBP complex is the apoptosis-and splicing-associated protein (ASAP) complex. In Arabidopsis, the ASAP complex is composed of three components: Serine/Arginine-rich 45 (SR45), apoptotic chromatin condensation inducer in the nucleus (Acinus), and Sin3-associated protein 18 (SAP18). A current model suggests that within the ASAP complex, SAP18 recruits histone deacetylases to a flower suppressor gene locus, FLOWER LOCUS C (FLC), to silence FLC expression and promote plant reproduction. Besides transcriptional regulation, the ASAP complex is also involved in splicing and nonsense mediated decay by interacting with the Exon Junction Complex. Interestingly, a null mutant of SR45, sr45-1, exhibits an elevated level of FLC and delayed flowering, suggesting that SR45 regulates FLC either dependent or independent of SAP18. In addition, the sr45-1 mutant shows increased expression of many defense genes, heightened innate immunity and a significant decrease of SAP18 protein, especially in the nucleus. However, it is unclear whether a decrease in nuclear SAP18 can help explain the hyperimmune phenotype of the sr45-1 mutant. To answer this question, two experiments were set up: (1) To address whether SR45-suppressed genes are targets for SAP18-mediated gene silencing, the activity of SAP18 at a chromatin level was investigated by a chromatin immunoprecipitation (ChIP) assay followed by high throughput sequencing. SAP18-associated DNAs were identified in a subset of SR45-suppressed genes and genes that were differentially alternatively spliced in the sr45-1 mutant. As predicted, some were defense genes. One example is Pathogenesis-related protein 5 (PR5). Furthermore, the SAP18 footprint on the PR5 gene coincided with less acetylated histones. (2) To uncover a possible mechanism for the decrease in nuclear SAP18 in the sr45-1 mutant, sr45-1 mutant seedlings overexpressing SAP18-GFP were treated with 50 uM MG132, a proteasome inhibitor. With treatment, increased nuclear localization of SAP18-GFP and total accumulation of SAP18 protein was confirmed via confocal imaging and western blot, respectively. Thus, increased degradation may at least partially explain the decreased nuclear accumulation of SAP18 observed in the sr45-1 mutant. Altogether, these results suggest that SR45 likely maintains SAP18 protein stability by forming the ASAP complex and is dependent on SAP18 to suppress the expression of a subset of defense genes.