Oral presentation - Session1 -

Polypyrimidine tract binding proteins link with development in Arabidopsis

Christina RUHL, Gabriele WAGNER, Dorothee LAMBERT, and Andreas WACHTER Center for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 32, 72076 Tübingen, Germany

Alternative splicing (AS) is a major determinant of transcriptome diversity in higher eukaryotes, and has been demonstrated to be critically involved in the regulation of development as well as stress responses. The AS outcome is defined by the splicing code, referring to the interplay of varying sets of splicing regulators and their target precursor mRNAs. To gain a better understanding of this process, we have analyzed the splicing regulatory functions of Polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana. We found several hundred AS events to be controlled by PTBs, and both developmental processes, such as seed germination and organ formation, and drought resistance were altered in ptb mutants. Combining in vitro interaction assays and in vivo splicing reporter studies, we started to define PTB binding motifs in target RNAs containing cassette that are either included or skipped in a PTB-dependent manner. Our work aims at deciphering central components of the plant splicing code, thereby providing a mechanistic basis for the control of AS and its links to intriguing biological functions in plants.

A survey of the sorghum transcriptome using the single- molecule long reads

Salah E. ABDEL-GHANY1*, Michael HAMILTON2*, Jen JACOBI3, Peter NAGM3, Nico DEVITT3, Faye SCHILKEY3, Asa BEN-HUR2, Anireddy S.N. REDDY1 1: Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO, 80523, 2: Department of Computer Sciences, Colorado State University, Fort Collins, CO, 80523, 3: National Center for Genome Resources, 2935 Rodeo Park Dr. East, Santa Fe, NM 87505, *These authors contributed equally.

Alternative splicing and of pre-mRNAs greatly contributes to transcriptome diversity and gene regulation in eukaryotes. Short-read sequencing of transcriptomes using next generation sequencing (RNA-seq) has been extensively used to analyze gene expression and the extent of alternative splicing in eukaryotes. However, a major limitation with short- read data is that it is difficult to accurately predict the full-length splice isoforms produced from a gene. Single-molecule long-read sequencing offers considerable advantage in identifying full-length splice isoforms and other forms of post-transcriptional regulatory events such as alternative polyadenylation. Here we sequenced the sorghum transcriptome using Pacific Biosciences single molecular real time long-read sequencing (Iso-Seq) and developed a pipeline (TAPIS - Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and alternative polyadenylation sites. These data provide transcriptome-wide full-length isoforms at an unprecedented scale and this is the first application of this technology in plants. In addition to providing a global view of full-length transcripts, the data allowed us to uncover alternative polyadenylation genome-wide, and has led to the identification of novel genes that were not previously annotated and to the extensive reannotation of the sorghum genome. The pipeline developed here would be useful to analyze Iso-seq data from any organism. The results of this study will be presented at the meeting.

Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

1 1 1 1 2 Yamile Marquez , Markus Höpfler , Zahra Ayatollahi , Andrea Barta , Maria KALYNA 1 Max F. Perutz Laboratories, Medical University of Vienna, Vienna, A-1030, Austria 2 Department of Applied Genetics and Cell Biology, BOKU – University of Natural Resources and Life Sciences, Vienna, A-1190, Austria

Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons (Marquez et al. 2012). As these events involve with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. Exitron splicing occurs in about 3.3% and 3.7% of Arabidopsis and human protein-coding genes, respectively. Intriguingly, intronless genes can be also alternatively spliced via exitron usage. Splicing of exitrons affects protein domains, disordered regions and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. At least some exitrons originate from ancestral coding exons and their evolution involves intron loss. We propose a “splicing memory” hypothesis whereby intron loss and imprints of former borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals complementing the repertoire of AS events.

Pre-mRNA splicing function of heat-inducible STABILIZED1 for stress responsive gene expression in the establishment of Arabidopsis thermotolerance

Young-Hee Cho, Geun-Don Kim, Sang-Dong YOO Department of Life Sciences, College of Life Sciences and Biotechnology, KOREA University, Seoul, Korea, 136-713

ABSTRACT Extracellular temperature fluctuation often leads to differential RNA splicing, resulting in the accumulation of different types and/or amounts of mature mRNAs in eukaryotic cells. However, our understanding of post-transcriptional regulatory mechanisms involved in cellular processes of pre-mRNA splicing under environmental stress conditions remains elusive in plants. We described a U5-snRNP-interacting protein homolog STABILIZED1 (STA1) was involved in pre-mRNA splicing of a key heat stress transcription factor HEAT STRESS TRANSCRIPTION FACTORA3 (HSFA3) for the establishment of heat stress tolerance in Arabidopsis. Our cell-based assay with intron-retained slicing reporters indicated that temperature fluctuation-inducible STA1 acted on specific pre-mRNA splicing for heat inducible HSFA3 in addition to cold inducible COR15A and IDD14. Cellular reconstitution of heat-inducible transcription cascades suggested that STA1-dependent pre-mRNA splicing had a pivotal role in DREB2A-dependent HSFA3 expression for heat responsive gene regulation. Genetic analysis with a loss-of-function mutant sta1-1 and its transgenic complementation lines as well as two mutant lines defective in non-sense mediated mRNA decay verified that STA1 was a necessary factor as a stress responsive splicing factor to acquire stress tolerance in response to a two-step heat treatment. Our findings have revealed that heat-inducible STA1 activity for heat-inducible pre-mRNA splicing serves as an additional regulatory tier for a fine tuning of heat stress adaptation in plants.

The Arabidopsis SR45 splicing factor, a negative regulator of sugar and ABA signaling, modulates stability of the energy- sensing SnRK1 protein kinase

Raquel F. Carvalho1, Dóra Szakonyi1, Craig G. Simpson2, John W.S. Brown2,3, Elena Baena- González1 and Paula DUQUE 1 1Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal2The James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK3University of Dundee at the James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK

ABSTRACT The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We have found that the plant-specific SR45 splicing factor negatively regulates glucose signaling during early development in Arabidopsis. SR45 appears to function in sugar responses independently of the hexokinase 1 (HXK1) conserved glucose sensor, relying instead on the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1). Although not affecting alternative splicing of the SnRK1 mRNA, the SR-like protein SR45 modulates SnRK1 protein degradation under glucose conditions. A high-resolution RT-PCR panel revealed that SR45 broadly targets alternative splicing in vivo, including that of its own pre-mRNA. Finally, we show that SR45 controls alternative splicing of an Arabidopsis inositol phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of the levels of the SnRK1 energy sensor in response to sugars.

NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis thaliana

Jakub Dolata1, Yanwu Guo2, Agnieszka Kolowerzo3,4, Dariusz Smolinski3,4, Grzegorz Brzyzek2, Szymon Swiezewski2, Artur JARMOLOWSKI1 1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland2Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland3Department of Cell Biology, Faculty of Biology and Environment Protection, Torun, Poland4Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Torun, Poland

ABSTRACT The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis thaliana homologue of disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5’ and 3’ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1- dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in the atntr1 genetic background resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis thaliana.

Uncovering the Role of SERRATE in General RNA Metabolism

Corinna Speth a,b, Eva-Maria Willingc, Xue Dongc, Korbinian Schneebergerc and Sascha LAUBINGER ZMBP, University of Tuebingen, Tuebingen, Germany b) Chemical Genomics Centre (CGC) of the Max Planck Society, Dortmund & MPI for Developmental Biology, Tuebingen, Gemany, c) MPI for Plant Breeding Research, Cologne, Germany

ABSTRACT Plant microRNAs (miRNA) are released from longer primary-miRNA (pri-miRNA) transcripts by a miRNA processing complex comprising several proteins including DICER- LIKE1 (DCL1), HYPONASTIC LEAVES1 (HYL1) and SERRATE (SE). In addition, the nuclear cap-binding complex (CBC) consisting of CAP-BINDING PROTEIN 20 (CBP20) and CBP80 binds to the 5_ap of pri-miRNA transcripts and interacts with SE to facilitate miRNA processing. Interestingly, SE and the CBC also ensure proper splicing of introns and therefore fulfill a more general function in RNA metabolism that is distinct from specialized miRNA-processing factors.To gain insights into the mechanistic functions of SE in general RNA metabolism, we conducted (1) extensive yeast two-hybrid screens, (2) mass-spec analysis of SE complexes purified form Arabidopsis, and (3) an in-depth transcriptome analysis of wild-type and semutants plants. We will present results showing that SE interacts with additional miRNAs factors, that it has additional functions in RNA metabolism other than miRNA processing and splicing, and that SE function is linked to the chromatin. Taken together, our results suggest that SE acts as a hub to coordinate RNA processing events and thereby shapes the Arabidopsis transcriptome.

Alternative splicing in the cold temperature response of Arabidopsis: an RNA-seq approach

Cristiane P. G. CALIXTO 1, Runxuan Zhang 2, Nikoleta A. Tzioutziou2 Allan B. James3, Craig G. Simpson1, Wenbin Guo1,2, Eduardo Eyras4,5, Hugh G. Nimmo3 and John W. S. Brown1,2 1University of Dundee, UK; 2James Hutton Institute, Dundee, UK; 3University of Glasgow, UK; 4,5 Universitat Pompeu Fabra and ICREA, Barcelona, Spain.

ABSTRACT Alternative splicing (AS) has been implicated in a wide range of developmental and physiological processes. We have shown that AS is important in regulating expression of key circadian clock genes and mediating the response of the clock to changes in temperature. The circadian clock is a cellular mechanism able to organize many physiological processes in anticipation/preparation to daily and seasonal changes. AS may therefore have roles in temperature perception, entrainment, compensation, regulation of downstream physiological responses and acclimation to exposure to low temperature. We have generated data to build transcription and splicing networks to identify genes that may regulate alternative splicing of core clock genes at low temperature. It also represents a high resolution time-course of the response of plants to cold. We are using Salmon to analyse the RNA-seq data and quantify transcripts. To use Salmon, we have developed a comprehensive, non-redundant reference transcript dataset covering more than 33,000 genes and 75,000 transcripts. This allows us to analyse expression at the level of individual transcripts. We have very good correlation between the individual transcript abundances and experimental data from our high resolution RT-PCR system (Spearman’s correlation coefficient = 0.902). The dynamic time-course data of individual transcripts has allowed the identification of genes which are regulated only by transcription, genes which are regulated only by AS and genes which are regulated by both. We have identified genes with major changes in AS in response to cold, including isoform switches that occur rapidly after transfer to cold.

The role of photoreceptors in regulating alternative splicing in Physcomitrella patens.

Chueh-Ju SHIH 1,2,3, and Shih-Long Tu1,2,4 1Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, 2 Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, 3Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan 4Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan.

ABSTRACT Light influences plant growth and development throughout all stages of life cycle. In response to light, plants have evolved photoreceptors to modulate photomorphological responses by sensing light fluctuations and regulating gene expression. Photoreceptor- mediated gene regulation at transcriptional, post-transcriptional and translational levels are well documented, however the research at the pre-mRNA splicing step remains poor. Our lab has previously reported the involvement of photoreceptors, especially red light photoreceptors, phytochromes, in light-responsive alternative splicing. To regulate alternative splicing, splicing regulators like serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) positively or negatively modulate splicing activity and alter splice sites. In order to further investigate how photoreceptors affect alternative splicing, we examined the relationship between photoreceptors and splicing regulators in the moss Physcomitrella patens. Among the tested photoreceptors and splicing factors, Physcomitrella phytochrome 4 displays a red light-dependent interaction with two hnRNPs in vitro and in vivo, suggesting that phytochrome 4 controls alternative splicing via hnRNPs. Over-express and knock out these two hnRNPs in the moss also differ the alternative splicing pattern of several genes, showing the involvement of them in splicing regulation. We conclude that phytochromes directly participate the control of alternative splicing. Elucidating how phytochrome 4 regulates splicing factors to alter the outcome of pre-mRNA splicing is undergoing.

REFERENCES Wu, H.-P., Su, Y.-s., Chen, H.-C., Chen, Y.-R., Wu, C.-C., Lin, W.-D., and Tu, S.-L. (2014). Genome-wide analysis of light-regulated alternative splicing mediated by photoreceptors in Physcomitrella patens. Genome Biol. 15, R10.