DAGZ > IMPB > Kalyna



Cells tightly control gene expression during development and in response to external cues. We utilize computational and experimental approaches to study one of the most interesting and complex processes in gene expression regulation, alternative splicing. We aim at a better understanding of molecular mechanisms of alternative splicing, its interaction with different post-transcriptional processes, and its role in shaping transcriptomes and proteomes under normal conditions, during stress response and in disease.

Principal Investigator: Dr. Maria Kalyna


Alternative Splicing Landscape in Plants

Alternative splicing is a key mechanism to increase the diversity of expressed information in eukaryotic genomes. In many eukaryotic genes, protein information embedded in regions called exons is interrupted by introns. Introns have to be removed and exons spliced together to yield a mature protein-coding transcript (mRNA). During alternative splicing, differential inclusion of exons and introns or their parts in mRNAs results in multiple transcript and protein variants with different fates and functions from a single gene. In the model plant Arabidopsis, an important role of alternative splicing is evidenced by ~60% of multi-exon genes undergoing alternative splicing (Marquez et al., 2012; Zhang et al., 2017). The question remains to what extent alternative splicing contributes to regulation of gene expression and proteome diversity and what are the biological functions of alternative splicing.

Cover illustration (Marquez et al., 2015) by Dr. Maria Kalyna gives a kaleidoscopic view of the proteome, reflecting the diversity generated by alternative splicing.

Regulation of Alternative Splicing

Production of transcript variants is tightly regulated. It changes in different tissues and cell types, during development, and in response to environmental cues. For example, light regulates alternative splicing by retrograde signals from chloroplasts (Petrillo et al., 2014). Serine/arginine-rich (SR) proteins are major regulators of alternative splicing and are themselves regulated via alternative splicing. Chloroplasts signal the nucleus to modulate alternative splicing of SR genes. Regulation of some plant SR genes by alternative splicing is evolutionarily conserved over up to a billion years, from single-celled green alga Chlamydomonas reinhardtii and moss Physcomitrella patens to Arabidopsis and rice (Kalyna et al., 2006), suggesting ancient role of this regulation. SR proteins play important roles in various aspects of plant growth and response to environment, including the DNA damage response. Elucidating regulation and molecular functions of SR proteins is essential for understanding how they modulate gene expression and biological processes during plant development and adaptation.

Interplay between Alternative Splicing and Different Post-Transcriptional Processes

Alternative splicing controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). In Arabidopsis, about 30% of alternatively spliced genes are regulated by NMD, and 13-18% of intron-containing genes are regulated by alternative splicing coupled to NMD (Kalyna et al., 2012). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. Our recent research indicates that alternative splicing isoforms are differentially affected by RNAi (Fuchs et al., 2021). Some transcript isoforms, though possessing the target sites for the small RNAs, escape degradation due to their nuclear localization, others are degraded predominantly by NMD. These interactions between RNAi and splicing isoforms are conserved in plant and mammalian cells. Moreover, artificial microRNAs (also termed shRNAmiR) can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Alternative splicing and different post-transcriptional processes act together in defining transcript fates and regulating gene expression. 

Art by Dr. Luciana Giono (CONICET-Universidad de Buenos Aires, Argentina), a co-author of the Fuchs et al., 2021 study, depicts different alternative splicing isoforms and some cellular RNA degradation machineries as if they were playing a mission in a paintball game.

Exitrons: Hidden Alternative Splicing within Protein-Coding Exons

Closer examination of alternative splicing in Arabidopsis led to the discovery of unusual alternative splicing events present also in humans that involve introns with features of both introns and protein-coding exons. These introns, named exitrons, allow alternative splicing of the internal parts of annotated protein-coding exons. Exitron splicing occurs in at least 6.6% Arabidopsis and 3.7% human protein-coding genes (Marquez et al., 2015; Zhang et al., 2017). Majority of exitrons have sizes of multiples of three nucleotides. Splicing of these exitrons results in internally deleted protein isoforms and affects protein domains, disordered regions, and various types of post-translational modifications, thus broadly influencing protein function. Exitron splicing is regulated across tissues, in response to stress, and in carcinogenesis. Exitrons originate from ancestral coding exons. We propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing (Marquez et al., 2012; Marquez et al., 2015). Intriguingly, exitrons exhibit distinctive nucleosome positioning pattern compared to other alternatively spliced regions, and nucleosome patterns differ between exitrons and retained introns, pointing to their distinct regulation (Jabre et al., 2020).


Frontiers Research Topic e-book 

Alternative Splicing Regulation in Plants

Fuchs A, Riegler S, Ayatollahi Z, Cavallari N, Giono LE, Nimeth BA, Mutanwad KV, Schweighofer A, Lucyshyn D, Barta A, Petrillo E, Kalyna M. Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing. (2021) Nucleic Acids Res 49:1133-1151

Nimeth BA, Riegler S, Kalyna M. Alternative Splicing and DNA Damage Response in Plants. (2020) Front Plant Sci 11:91.

Zhang R, Calixto CP, Marquez Y, Venhuizen P, Tzioutziou NA, Guo W, Spensley M, Entizne JC, Lewandowska D, Ten Have S, Frei Dit Frey N, Hirt H, James AB, Nimmo HG, Barta A, Kalyna M, Brown JW. A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing. (2017) Nucleic Acids Res 45:5061-5073.

Marquez Y, Hopfler M, Ayatollahi Z, Barta A, Kalyna M. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity. (2015) Genome Res 25: 995-1007. 

Petrillo E, Godoy Herz MA, Fuchs A, Reifer D, Fuller J, Yanovsky MJ, Simpson C, Brown JW, Barta A, Kalyna M, Kornblihtt AR. A chloroplast retrograde signal regulates nuclear alternative splicing. (2014) Science 344: 427-430. 

Marquez Y, Brown JW, Simpson C, Barta A, Kalyna M. Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. (2012) Genome Res 22:1184-1195. 

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current projects

finished projects

COLLABORATIONS (last five years)

  • Andrea Barta, Max Perutz Laboratories, Medical University of Vienna, Austria
  • John WS Brown, University of Dundee, Dundee, Scotland, UK
  • Paula Duque, Instituto Gulbenkian de Ciência (IGC), Oeiras, Portugal
  • Heribert Hirt, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia
  • Jürgen Kleine-Vehn, University of Natural Resources and Life Sciences, Vienna, Austria
  • Doris Lucyshyn, University of Natural Resources and Life Sciences, Vienna, Austria
  • Marjori and Antonius JM Matzke, Institute of Plant and Microbial Biology, Taipei, Taiwan
  • Akila Mayeda, Fujita Health University, Toyoake, Japan
  • Ezequiel Petrillo, Universidad de Buenos Aires, Buenos Aires, Argentina
  • Andreas Sommer, Next Generation Sequencing (NGS) Unit, VBCF, Vienna, Austria 
  • Dorothee Staiger, Universität Bielefeld, Germany
  • Naeem H Syed, Canterbury Christ Church University, Canterbury, UK