Transcriptional control of stress responses
Although many components of the RNA polymerase II (RNAPII) associated regulatory factors are conserved in eukaryotes, so far little is known about their specific roles in interpretation and integration of inputs from different signalling pathways at the level of transcription in higher plants. Our research focuses on understanding the regulatory roles of three functionally interlinked RNAPII transcription regulatory modules that carry subunit homologs of TFIIH general transcription factor, SAGA transcriptional co-activator and spliceosome-activating NTC complexes in Arabidopsis. We aim to identify those molecular mechanisms by which mutations affecting key components of these RNAPII regulatory modules cause pleiotropic defects through influencing the functions of their targets in stress, hormone, metabolic or developmental pathways. With other words, we try to dissect the complexity of pleiotropic changes caused by inactivation of key components of RNA polymerase II transcription modules by identifying their selective genome-wide targets and thereby reconstituting from the individual defects the observed pleiotropic consequences. On this way, interdependent changes in transcription, splicing, RNA stability, nuclear export, translation, phosphorylation etc. can unravel coordinate and hierarchical control of specific pathways providing new insights into cellular regulation at the level of systems biology. In particular, we are interested to uncover elements of regulatory circuits that govern plant responses to biotic and abiotic stress.
1. TFIIH-associated protein kinases and regulation of RNAPII CTD code in Arabidopsis
The general transcription factor TFIIH is recruited to RNAPII during transcription initiation. TFIIH-associated protein kinases play a pivotal role in phosphorylation of serine 5 and 7 residues of Y1S2P3T4S5P6S7 heptapeptide repeats in the carboxy-terminal domain of RNAPII largest subunit (RNAPII CTD). During transcription, RNAPII CTD serves as loading platform for timely ordered assembly of associated regulatory factors governing transcription initiation, pausing, elongation, termination; splicing, nuclear RNA export and degradation, chromatin modifications and gene silencing. TFIIH is also essential for global genome and transcription-coupled nucleotide excision repair. In Arabidopsis, three CDKD homologs of yeast Kin28/human CDK7 are implicated in CTD phosphorylation and their activities are regulated by the upstream activating kinase CDKF;1, which is functionally similar to yeast Cak1. RNAPII CTD is also phosphorylated by the CDKC class of Arabidopsis protein kinases, which show functional similarity to yeast Ctd1/Bur1 and human CDK9. We use T-DNA insertion mutants, inducible artificial microRNA and epitope-labelled kinase constructs expressed by precisely modified genes to determine the roles of CTD kinases in regulation of the CTD code and transcription, splicing and stability of protein coding and non-coding pre-mRNAs in correlation with developmental and signalling effects of their stable or conditional inactivation in Arabidopsis.
2. Regulation of stress, hormone and metabolic responses by SnRK1 kinases
Arabidopsis SnRK1 kinases belong to the AMP-activated protein kinase (AMPK) family. Trimeric core of SnRK1 is composed of catalytic α (AKIN10, 11 and 12), substrate targeting β (AKINβ1, 2 and 3) and activating γ (SNF4) subunits. In analogy to yeast Snf1 and mammalian AMPKs, plant SnRK1 kinases play a central role in controlling key metabolic enzymes and transcription in response to glucose availability and changes in cellular energy homeostasis. Yeast Snf1 is found in association with the GCN5 histone acetylase in the SAGA RNAPII co-activator, while RNAPII association of plant SnRK1 is still an open question. In our previous work, we identified SnRK1α subunits in association with SKP1, a common subunit of SCF (SKP1-CULLIN1-F-box protein) E3 ubiquitin ligases, and showed that SnRK1 mediates targeting the SCF complexes to the Arabidopsis α4/PAD1 subunit of 20S proteasome. We have also studied differential regulation and cellular localization of Arabidopsis SnRK1 subunits. Our current studies focus on the definition of SCF-related regulatory roles, genome-wide transcription regulatory targets, and composition and interactions of SnRK1 kinase complexes in Arabidopsis
3. Regulatory roles of spliceosome activating NineTeen Complex
NTC, named after its Prp19 E3 ubiquitin ligase subunit, is required for co-transcriptional assembly and activation of the splicesome. Components of the NTC are loaded onto RNAPII by their contacts to initiation, elongation and chromatin modifying factors. Several components of Arabidopsis NTC are encoded by duplicated genes, differential regulation of which permits the isolation of partial loss of function mutations and studying their pleiotropic effects on cellular pathways. As a consequence of such NTC mutations, specific introns are either retained or show alternative splicing in polyadenylated pre-mRNAs, which provides a tool for identification of pathway specific targets. Whether defective splicing allows the translation of truncated protein products, conferring dominant positive or negative regulatory functions or the aberrantly spliced mRNAs are targeted to destruction by nonsense-mediated decay is still an open question. Association of NTC with elongation and chromatin modifying factors suggests intriguing alternative regulatory functions. In yeast and other metazoans, components of the NTC are also implicated general genome and transcription coupled repair. In Arabidopsis, the prl1 mutation characterized by our group earlier suppresses the induction of cell death response triggered by either accumulation of singlet oxygen or constitutive activation of innate immunity pathways. The PRL1 NTC subunit is also identified as substrate receptor subunit of CUL4-DDB1 E3 ubiquitin ligase, which is implicated in interaction with TFIIH in the DNA repair pathways. In addition, PRL1 acts as in vitro inhibitor of SnRK1 and CUL4-DDB1-PRL1 is reported to control the stability of SnRK1α subunit AKIN10 indicating cross-talk between the three studied RNAPII regulatory modules. Major goals of our current studies are to compile genome-wide splicing defects caused by the prl1 mutation; to study regulatory interactions of NTC with RNAPII initiation and elongation factors and components of DNA repair pathways, including TFHII as potential candidate; and to identify the mechanisms by which NTC mutations alter the control of flowering time and circadian clock pathways.
Information on past studies and collaborations of our research group is provided by the enclosed list of Publications. The group offers a collaboration-based help in the identification of T-DNA insertion mutations, which are absent from public mutant depositories; in the application of recombineering technologies for precise modification of plant genes; in the exploitation of a novel affinity purification technology for isolation of nuclear protein complexes; and in the use of Agrobacterium T-DNA vectors, Ti plasmid helper strains GV3101 (pMP90) and GV3101 (pMP90RK), plant transformation technologies and Arabidopsis mutants constructed by the PI while working in the past with two great friends and mentors, Jeff Schell and George P. Rédei.