Alternative splicing in plants - novel regulatory mechanisms and its biological implications in the control of gene expression
Final Report Abstract
Alternative pre-mRNA splicing (AS) represents a major means to increase transcriptome diversity in higher eukaryotes. Recent studies revealed that more than 60 % of all multi-exon genes from the model plant Arabidopsis thaliana produce AS variants, with important functions in plant development and stress responses. Despite tremendous progress in the AS research, many questions about the regulation and functional implications of this process remain to be addressed in plants and other eukaryotes. In the course of this project, we could demonstrate that Polypyrimidine tract binding proteins (PTBs) are major regulators of AS in plants, affecting splicing patterns of several hundred genes in A. thaliana. PTBs preferentially regulate the skipping of cassette exons, removal of regulated introns, and usage of upstream 5’ splice sites. Utilizing both RNA-protein in vitro interaction studies and in vivo reporter analyses, critical PTB binding motifs of first model substrates were characterized. Furthermore, putative PTB-interacting proteins and splicing factors from the family of serine/argininerich proteins, displaying PTB-antagonizing activity, were identified. Extending this work in the future will allow defining the interplay between cis-regulatory elements and trans-acting splicing factors as basis of AS decisions. PTBs trigger AS of their own pre-mRNAs to NMD-targeted splicing variants as part of auto- and crossregulatory feedback loops. Further functions of PTB-mediated AS include regulation of genes involved in flowering time control and of Phytochrome interacting factor 6 (PIF6), which was previously shown to affect seed germination in a splicing-variant-specific manner. Accordingly, mutants with decreased and elevated PTB levels display accelerated and delayed abscisic acid-dependent seed germination, respectively, correlating with opposite shifts in PIF6 splicing. Additional physiological functions of PTBs were revealed by the analysis of mutants with decreased levels of the two highly similar homologs PTB1 and PTB2 from A. thaliana. These mutants were strongly impaired in development, displaying stunted growth, delayed flowering, increased leaf serration, and altered silique morphology. Intriguingly, PTB downregulation also strongly enhanced drought resistance, revealing a role of these splicing regulators in the plant stress response. Current work aims at identifying the molecular basis of these important PTB functions in plant development and stress physiology. Many AS variants contain features such as long 3’ untranslated regions, which are expected to trigger degradation of these transcripts via the RNA surveillance mechanism nonsense-mediated decay (NMD). In line with this hypothesis, our transcriptome-wide comparison of AS patterns in control and NMD-impaired A. thaliana seedlings revealed that almost one fifth of all genes generate at least one splicing variant that is targeted by NMD. Further it was demonstrated that the expression of three NMD factors is negatively regulated by NMD, which is expected to balance NMD activity. Our finding that NMD activity is strongly impaired upon salt stress and recent reports from other researchers on functions of NMD in development and stress responses in animals and plants suggests that tight regulation of this decay mechanism has important physiological implications. In conclusion, our investigations have provided novel insight into the regulation of AS and revealed widespread coupling of AS and NMD in plants. The PTB splicing factors were identified as a central component of the splicing code, with fundamental functions in plant development and stress responses. These intriguing links between PTB-mediated AS and agronomically relevant traits provide an excellent starting point for developing novel strategies in crop engineering.
Publications
- (2009) A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs. Nat Struct Mol Biol 16: 541-549
Hammond MC, Wachter A, Breaker, RR
- (2010). Polypyrimidine tract-binding protein homologues from Arabidopsis underlie regulatory circuits based on alternative splicing and downstream control. Plant J 64: 243-255
Stauffer E, Westermann A, Wagner G, Wachter A
- (2010). Riboswitch-mediated control of gene expression in eukaryotes. RNA Biol 7: 66- 76
Wachter A
- (2012). Polypyrimidine tract-binding protein homologues from Arabidopsis are key regulators of alternative splicing with implications in fundamental developmental processes. Plant Cell 24: 4360-4375
Rühl C, Stauffer E, Kahles A, Wagner G, Drechsel G, Rätsch G, and Wachter A
(See online at https://doi.org/10.1105/tpc.112.103622) - (2012). The role of Polypyrimidine tract-binding proteins and other hnRNP proteins in plant splicing regulation. Frontiers in Plant Science 3, 81
Wachter A, Rühl C, and Stauffer E
(See online at https://doi.org/10.3389/fpls.2012.00081) - (2013). Accurate detection of differential RNA processing. Nucleic Acids Res 41: 5189-5198
Drewe P, Stegle O, Hartmann L, Kahles A, Bohnert R, Wachter A, Borgwardt K, Rätsch G
(See online at https://doi.org/10.1093/nar/gkt211) - (2013). Nonsense-mediated decay of alternative pre-mRNA splicing variants is a major determinant of the Arabidopsis transcriptome. Plant Cell 25: 3726-3742
Drechsel G, Kahles A, Kesarwani A, Stauffer E, Behr J, Drewe P, Rätsch G, Wachter A
- (2014). Gene regulation by structured mRNA elements. Trends in Genetics 30: 172-181
Wachter A
(See online at https://doi.org/10.1016/j.tig.2014.03.001) - (2014). NMD: Nonsense-mediated defense. Cell Host & Microbe 16: 273-275
Wachter A, Hartmann L
(See online at https://doi.org/10.1016/j.chom.2014.08.015) - (2014). The Arabidopsis class II sirtuin is a lysine deacetylase and interacts with mitochondrial energy metabolism. Plant Physiol 164: 1401-1414
König A, Hartl M, Pham P, Laxa M, Boersema P, Orwat A, Kalitventseva I, Plöchinger M, Braun H, Leister D, Mann M, Wachter A, Fernie A, Finkemeier I
(See online at https://doi.org/10.1104/pp.113.232496)
