RESUMO
BACKGROUND: Regulation of pre-mRNA splicing diversifies protein products and affects many biological processes. Arabidopsis thaliana Serine/Arginine-rich 45 (SR45), regulates pre-mRNA splicing by interacting with other regulatory proteins and spliceosomal subunits. Although SR45 has orthologs in diverse eukaryotes, including human RNPS1, the sr45-1 null mutant is viable. Narrow flower petals and reduced seed formation suggest that SR45 regulates genes involved in diverse processes, including reproduction. To understand how SR45 is involved in the regulation of reproductive processes, we studied mRNA from the wild-type and sr45-1 inflorescences using RNA-seq, and identified SR45-bound RNAs by immunoprecipitation. RESULTS: Using a variety of bioinformatics tools, we identified a total of 358 SR45 differentially regulated (SDR) genes, 542 SR45-dependent alternative splicing (SAS) events, and 1812 SR45-associated RNAs (SARs). There is little overlap between SDR genes and SAS genes, and neither set of genes is enriched for flower or seed development. However, transcripts from reproductive process genes are significantly overrepresented in SARs. In exploring the fate of SARs, we found that a total of 81 SARs are subject to alternative splicing, while 14 of them are known Nonsense-Mediated Decay (NMD) targets. Motifs related to GGNGG are enriched both in SARs and near different types of SAS events, suggesting that SR45 recognizes this motif directly. Genes involved in plant defense are significantly over-represented among genes whose expression is suppressed by SR45, and sr45-1 plants do indeed show enhanced immunity. CONCLUSION: We find that SR45 is a suppressor of innate immunity. We find that a single motif (GGNGG) is highly enriched in both RNAs bound by SR45 and in sequences near SR45- dependent alternative splicing events in inflorescence tissue. We find that the alternative splicing events regulated by SR45 are enriched for this motif whether the effect of SR45 is activation or repression of the particular event. Thus, our data suggests that SR45 acts to control splice site choice in a way that defies simple categorization as an activator or repressor of splicing.
Assuntos
Arabidopsis/genética , Arabidopsis/imunologia , Perfilação da Expressão Gênica , Imunidade Inata/genética , Splicing de RNA , Arabidopsis/microbiologia , Flores/genéticaRESUMO
Pre-mRNA alternative splicing is a conserved mechanism for eukaryotic cells to leverage existing genetic resources to create a diverse pool of protein products. It is regulated in coordination with other events in RNA metabolism such as transcription, polyadenylation, RNA transport, and nonsense-mediated decay via protein networks. SERINE/ARGININE-RICH 45 (SR45) is thought to be a neutral splicing regulator. It is orthologous to a component of the apoptosis and splicing-associated protein (ASAP) complex functioning to regulate RNA metabolism at multiple levels. Within this context, we try to understand why the sr45-1 mutant Arabidopsis has malformed flowers, delayed flowering time, and increased disease resistance. Prior studies revealed increased expression for some disease resistance genes and the flowering suppressor Flowering Locus C (FLC) in sr45-1 mutants and a physical association between SR45 and reproductive process-related RNAs. Here, we used Tandem Mass Tag-based quantitative mass spectrometry to compare the protein abundance from inflorescence between Arabidopsis wild-type (Col-0) and sr45-1 mutant plants. A total of 7,206 proteins were quantified, of which 227 proteins exhibited significantly different accumulation. Only a small percentage of these proteins overlapped with the dataset of RNAs with altered expression. The proteomics results revealed that the sr45-1 mutant had increased amounts of enzymes for glucosinolate biosynthesis which are important for disease resistance. Furthermore, the mutant inflorescence had a drastically reduced amount of the Sin3-associated protein 18 (SAP18), a second ASAP complex component, despite no significant reduction in SAP18 RNA. The third ASAP component protein, ACINUS, also had lower abundance without significant RNA changes in the sr45-1 mutant. To test the effect of SR45 on SAP18, a SAP18-GFP fusion protein was overproduced in transgenic Arabidopsis Col-0 and sr45-1 plants. SAP18-GFP has less accumulation in the nucleus, the site of activity for the ASAP complex, without SR45. Furthermore, transgenic sr45-1 mutants overproducing SAP18-GFP expressed even more FLC and had a more severe flowering delay than non-transgenic sr45-1 mutants. These results suggest that SR45 is required to maintain the wild-type level of SAP18 protein accumulation in the nucleus and that FLC-regulated flowering time is regulated by the correct expression and localization of the ASAP complex.