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Oral Presentation - Session1 Oral presentation - Session1 - Polypyrimidine tract binding proteins link alternative splicing 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 exons 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 polyadenylation 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 intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons (Marquez et al. 2012). As these events involve introns 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 exon 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,
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