Regulation and evolution of alternative splicing in plants DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Zhihao Ling, M.S. geboren am 10.08.1988 in China Max-Planck-Institut für chemische Ökologie Gutachter: Prof. Ian T. Baldwin, Max Planck Institut für Chemische Ökologie, Jena Prof. Günter Theissen, Friedrich Schiller Universität Jena Prof. Dorothee Staiger, Universität Bielefeld Beginn der Promotion: 26. September 2012 Dissertation eingereicht am: 25. November 2016 Tag der Verteidigung: 28. June 2017 Table of Contents 1. General Introduction ................................................................................................................ 1 1.1 Alternative splicing is widespread in plants .......................................................................... 1 1.2 AS contributes to biological regulation processes in plants .................................................. 2 1.3 Abiotic stresses induced AS changes in plants ..................................................................... 4 1.4 Biotic stresses induced AS changes in plants ....................................................................... 7 1.5 The splicing code and determinants of AS in plants is largely unknown ............................. 8 1.6 The rapid evolution of AS ................................................................................................... 10 1.7 Thesis outline ...................................................................................................................... 11 References ................................................................................................................................. 12 2. Overview of Manuscripts ....................................................................................................... 20 3. Manuscripts ............................................................................................................................. 23 Manuscript I .............................................................................................................................. 23 Manuscript II ............................................................................................................................. 60 Manuscript III .......................................................................................................................... 152 4. General Discussion ................................................................................................................ 220 4.1 The function of environmental stress-induced AS in plants ............................................. 221 4.2 Genome-wide AS alterations in response to environmental stresses in plants may be largely due to the regulation of SR proteins ............................................................................ 224 4.3 The splicing code in plants is largely conserved ............................................................... 225 4.4 The rapid evolution of AS in plants is mainly cis-regulated ............................................. 227 References ............................................................................................................................... 230 Summary .................................................................................................................................... 235 Zusammenfassung..................................................................................................................... 237 i Bibliography .............................................................................................................................. 239 Eigenständigkeitserklärung ..................................................................................................... 249 Curriculum vitae ....................................................................................................................... 250 Acknowledgement ..................................................................................................................... 254 ii General Introduction 1. General Introduction 1.1 Alternative splicing is widespread in plants Alternative splicing (AS) is a mechanism by which two or more transcripts are generated by removing different introns or using different splice sites from the same pre-mRNA. In general, AS can be broadly classified into four fundamental types (Figure 1): alternative donor sites (AltD), alternative acceptor sites (AltA), intron retention (IR) and exon skipping (ES) (Black 2003). A given splice variant may also contain combinations of different AS types at different introns, thus generating a more complex AS pattern. The identification of two different polypeptides from ribulosebisphosphate carboxylase/oxygenase (rubisco) activase in spinach and Arabidopsis thaliana provided the first demonstration of AS in plants (Werneke et al. 1989). Before next generation sequencing (NGS) technology was developed, AS was thought to be rare in plants, even though it was known to be common in humans (Brett et al. 2002). The estimations of AS frequency in A. thaliana using expressed sequence tags (ESTs) revealed that only 11% to 30% of multi-exon genes underwent AS and the discovery of AS depended highly on the sequencing depth of ESTs (Campbell et al. 2006, Iida et al. 2004). Recently, by sequencing cDNA derived from poly (A) enriched mRNA using NGS technology, several recent genome-wide AS studies in A. thaliana (Marquez et al. 2012), rice (Zhang et al. 2010), grape (Zenoni et al. 2010), soybean (Shen et al. 2014), moss (Wu et al. 2014) and wild tobacco (Ling et al. 2015) demonstrated that AS is much more prevalent in plants than previously thought, and that AS can be found in up to 66% of intron-containing genes. Figure 1. The four fundamental types of alternative splicing 1 General introduction The above mentioned genome-wide AS analyses all indicated that the predominant type of AS in land plants is IR, while ES has the lowest frequency. The pattern is opposite to that in metazoans, where ES is the most abundant AS type and IR is least prevalent (Kim et al. 2007, Pan et al. 2008). This indicates that the regulatory machinery of AS in plants might be different from metazoans. Previous studies demonstrated that introns from a human gene cannot be processed in plants but introns from a plant gene can be efficiently spliced in humans, providing further evidence to support this (Barta et al. 1986, Hartmuth and Barta 1986). 1.2 AS contributes to biological regulation processes in plants One direct effect of AS is that the sequences of distinct splicing variants are different; if all these transcripts can be translated into proteins, proteome diversity can be expanded without corresponding expansion of the genome. In addition, the generated protein isoforms may also have different alterations, because AS can result in new transcripts that preserve the original open reading frame (if the difference caused by AS is in multiples of three nucleotides (nt)) or else cause a frameshift. The most commonly known non-frame shift AS is NAGNAG (N is any nucleotide) acceptor, which is a subset of AltA in which two splice sites (SS) are separated by three nt, thus resulting in two protein isoforms with only one amino acid difference (Iida et al. 2008, Schindler et al. 2008). In both plants and animals, NAGNAG events are enriched in genes encoding DNA-binding proteins and mainly influence polar amino acids that can change DNA- binding properties (Iida, et al. 2008, Schindler, et al. 2008, Vogan et al. 1996). When AS introduces reading frame shifts, it may generate either a protein isoform with novel functional domains or truncated protein isoforms, or else introduce premature termination codons (PTC) that result in transcript degradation. Protein isoforms with different domain arrangement may have different functions or differ in their subcellular localization and stability (Syed et al. 2012). The truncated proteins may lack certain functional domains or may have lost sites of post- translational modifications (Dinesh-Kumar and Baker 2000). For instance, several recent studies demonstrated that truncated proteins encoded by splicing variants can act as dominant-negative regulators by forming nonfunctional dimers that compete with functional dimers, which leads to physiological responses (Liu et al. 2013, Seo et al. 2012, Staudt and Wenkel 2011). However, functional studies on the proteins generated by AS are rare in plants, and some authors also raise 2 General Introduction the question of whether AS really played an important role in expanding proteome diversity in plants (Severing et al. 2009). As mentioned, another important effect of AS is that it can regulate transcript abundance by introducing PTC to transcripts, which may become subject to degradation by nonsense- mediated decay (NMD), a cytoplasmic RNA degradation machinery (Chang et al. 2007, Kalyna et al. 2012, Kervestin and Jacobson 2012, Schoenberg and Maquat 2012). NMD initiates rapid transcript decay when the termination of translation is perturbed, although, the detailed NMD pathway in plants is not yet well characterized (Belostotsky and Sieburth 2009, Chang, et al. 2007, Schoenberg and Maquat 2012). The orthologues of the core proteins of the NMD pathway, UPF1-3 and SMG-7 can be found in most of plants (but not SMG-1, SMG-5 and SMG-6), suggesting the NMD machinery