Quantitative proteomics reveals a role for SERINE/ARGININE-Rich 45 in regulating RNA metabolism and modulating transcriptional suppression via the ASAP complex in Arabidopsis thaliana

Authors: CHEN, SAMUEL - St Bonaventure University; ROONEY, TIMOTHY - St Bonaventure University; HU, ANNA - St Bonaventure University; Beard, Hunter; Garrett, Wesley; MANGALATH, LEANN - St Bonaventure University; POWERS, JORDAN - St Bonaventure University; Cooper, Bret; ZHANG, XIAO-NING - St Bonaventure University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/2019
Publication Date: 9/19/2019
Citation: Chen, S.L., Rooney, T.J., Hu, A.R., Beard, H.S., Garrett, W.M., Mangalath, L.M., Powers, J.J., Cooper, B., Zhang, X. 2019. Quantitative proteomics reveals a role for SERINE/ARGININE-Rich 45 in regulating RNA metabolism and modulating transcriptional suppression via the ASAP complex in Arabidopsis thaliana. Frontiers in Plant Science. 10:1116. https://doi.org/10.3389/fpls.2019.01116.
DOI: https://doi.org/10.3389/fpls.2019.01116

Interpretive Summary: In plant cells, RNA is made from DNA, the heritable genetic material. In turn, proteins, which have structural and enzymatic functions, are made from messenger RNA. In a cellular process called splicing, small parts of messenger RNA are cutaway from larger RNA molecules such that the smaller RNA encodes a protein that is different (smaller) than the protein from the larger RNA. It has been demonstrated in plant and animals that splicing helps make different proteins that then have different functions. A protein called SR45 is important for splicing in the model plant Arabidopsis. In this research, scientists studied an Arabidopsis plant missing SR45. This plant has a delayed flowering time and increased disease resistance. Flower proteins were measured by mass spectrometry, an analytical technique. The flowers were found to not only be missing SR45, but also SAP18 and ACINUS. In animals, proteins highly similar to SR45, SAP18, and ACINUS work together in the ASAP complex to regulate gene expression and RNA splicing. To study a correlation, the scientists showed that in the absence of SR45, SAP18 did not accumulate in the cell nucleus where the ASAP complex functions. The scientists also found that SAP18 altered the expression level of a gene that controls flowering and other genes involved in disease resistance. Hence, it appears that the loss of SR45 results in ASAP complex defects in the nucleus, leading to altered regulation of RNA for flowering and disease resistance genes. These results are most likely to influence scientists at universities, government agencies, and companies who study flowering time or and who try to find ways to improve disease resistance in crops such as tomatoes or soybeans.

Technical Abstract: 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.