Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• Our laboratory has used the DOG1 gene as a model and described a new generic mechanism of the way in which alternative splicing is controlled by Pol II speed in Arabidopsis thaliana.
• In collaboration with others, we showed that Pol II speed is environmentally controlled by darkness in plants through the modulation of alternative splicing.
• Working on DOG1, we uncovered an lncRNA antisense-mediated regulation of this key seed dormancy regulator.
• By analyzing SWI/SNF complex activity at gene terminators, we showed that this antisense-based mechanism operates at 1800 genes in Arabidopsis thaliana.
• We showed that DOG1 antisense acts as an environmental sensor.

Opis badań

The capacity to ignore short spells of favorable conditions forms the basis of a fascinating seed phenomenon: seed dormancy. Studies in the model plant Arabidopsis thaliana have shown that a key regulator of this process in nature is the Delay of Germination 1 (DOG1) gene. Our laboratory focuses on this gene. Over the years, we have described several interesting mechanisms that operate in this gene.

We found that alternative polyadenylation results in the production of two DOG1 transcripts: a shorter two-exon short gene (shDOG1) and a longer three-exon long gene (lgDOG1). Our work showed that the shDOG1 transcript is evolutionary conserved at the level of amino acids, translated, and sufficient to complement the dog1 mutant.

The second intron of DOG1 is subject to alternative splicing, generating four different isoforms. By studying the regulation of this alternative splicing, we described tight coupling between transcription and alternative splice site selection. We showed that mutations of the transcription elongation factor TFIIS led to the selection of proximal splice sites not only on DOG1 but also at the majority of tested genome-wide targets, suggesting the existence of kinetic coupling between Pol II elongation and splicing in plants. We also showed that alternative splice sites are active players in Pol II elongation control. We hypothesized that alternative splicing may locally pause the elongation of Pol II using a chromatin-based mechanism that centers around the spliceosomal disassembly factor NTR1. We also described a mechanism by which alternative splicing that is controlled by light is mediated by changes in Pol II speed that depends on the elongation factor TFIIS.

Transcription generates an extensive array of non-coding RNA (ncRNA), the functional significance of which is mostly unknown. Studies in yeast, humans, Drosophila, bacteria, and plants have shown that the majority of nucleotides in these genomes are transcribed, generating a mixture of long and short RNAs, most of which are non-coding. This non-coding transcription changes in a complex manner during development and in response to environmental signals. One specific class of ncRNA is antisense transcripts. We recently characterized an antisense transcript that originates close to the DOG1 proximal (main) termination site that strongly inhibits dormancy strength and DOG1 expression. We further showed that this transcript can only act when transcribed from the same DNA copy because it is unable to silence DOG1 that is transcribed from a second allele in the same cell.

We also showed that DOG1 antisense in mature plants acts as a sensor for the plant hormone ABA, allowing the mature plant to upregulate DOG1 expression in response to drought. This allowed us to characterize DOG1 function outside the seed in conferring an increase in drought tolerance.

Metodologia

We use a wide range of molecular biology methods that we adapted for use in seeds, including chromatin immunoprecipitation, RNA sequencing, targeted NetSEQ, smFISH, clustered regularly interspaced short palindromic repeats (CRISPR), dCAS9, HiC, and 3C. We also have a dedicated NightShade luciferase imaging system (Berthold) and a Boxeed seed sowing/phenotyping robot (LabDeers).

Wybrane publikacje

• Light Regulates Plant Alternative Splicing through the Control of Transcriptional Elongation. Godoy Herz MA, Kubaczka MG, Brzyżek G, Servi L, Krzyszton M, Simpson C, Brown J, Swiezewski S, Petrillo E, Kornblihtt AR. Mol Cell. 2019 doi: 10.1016/j.molcel.2018.12.005.
• Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance. Yatusevich R, Fedak H, Ciesielski A, Krzyczmonik K, Kulik A, Dobrowolska G, Swiezewski S EMBO Rep. 2017doi: 10.15252/embr.201744862.
• Arabidopsis SWI/SNF chromatin remodeling complex binds both promoters and terminators to regulate gene expression. Archacki R, Yatusevich R, Buszewicz D, Krzyczmonik K, Patryn J, Iwanicka-Nowicka R, Biecek P, Wilczynski B, Koblowska M, Jerzmanowski A, Swiezewski S. Nucleic Acids Res. 2017. doi: 10.1093/nar/gkw1273.
• Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript. Fedak H, Palusinska M, Krzyczmonik K, Brzezniak L, Yatusevich R, Pietras Z, Kaczanowski S, Swiezewski S. Proc Natl Acad Sci U S A. 2016. doi: 10.1073/pnas.1608827113.
• NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis. Dolata J, Guo Y, Kołowerzo A, Smoliński D, Brzyżek G, Jarmołowski A, Świeżewski S. EMBO J. 2015 doi: 10.15252/embj.201489478.

Współpraca

• Sebastian Marquardt, Sebastian Marquardt's Lab, Copenhagen University, Denmark, ku.dk/meet-the-scientists-page/sebastian-marquardts-group/
• Alberto Kornblihtt, Kornblihtt lab, University of Buenos Aires, Argentina, ark.fbmc.fcen.uba.ar
• Artur Jarmołowski, Laboratory of Gene Expression, Adam Mickiewicz University, Poland, ibmib.amu.edu.pl/en/department-of-gene-expression/gene-expression-laboratory/
• Rafał Archacki, Plant Experimental Biology and Biotechnology, Warsaw University, Poland

Nagrody i wyróżnienia

• Szymon Świeżewski. Parnas prize. 2015. Polish Biochemical Society, Poland.
• Szymon Świeżewski. NCN Prize. 2017. National Science Centre, Poland.
• Szymon Świeżewski. PAS individual award. 2018. Polish Academy of Science (II department).
• Halina Fedak/Małgorzata Palusińska. prof. Wacław Szybalski Prize. Polish Academy of Science Biotechnology Committee.