Structural Biology of Gene Regulation - Richard Štefl

Research areas

• Protein-RNA and protein-protein interactions and their roles in the regulation of gene expression
• 3’-end processing and transcription termination
• NMR spectroscopy of proteins and nucleic acids and their complexes
• Development and application of new methods to aid the interpretation of NMR data

Main objectives

• Investigation of the role of RNA in gene expression, development and human diseases.
• Establishing an isotope laboratory for NMR studies and the development of new strategies for the preparation of isotopically labelled proteins in eukaryotic cells.

Content of research

RNA is essential for cell survival. Not only is it a messenger between the genomes and proteomes but it also carries out, or participates in, many functions such as RNA processing and protein translation, acting as structural scaffolds, transporters, gene regulators and biocatalysts. We will help to clarify molecular mechanisms underlying RNA quality control in eukaryotic cells through the investigation of the detailed biochemical principles of RNA recognition, processing and degradation. We will use a combination of biochemical, genetic, and structural methods to unravel the molecular mechanism of eukaryotic RNA surveillance.

Example of NMR spectroscopy investigation of RNA sequence- and structure-dependent recognition by proteins.

list / cards

SELECTED PUBLICATIONS

2017

• CERNA, H; CERNY, M; HABANOVA, H; SAFAROVA, D; ABUSHAMSIYA, K; NAVRATIL, M; BRZOBOHATY, B, 2017:Proteomics offers insight to the mechanism behind Pisum sativum L. response to pea seed-borne mosaic virus (PSbMV). JOURNAL OF PROTEOMICS 153 , p. 78 - 88.
• HUTER, P; ARENZ, S; BOCK, LV; GRAF, M; FRISTER, JO; HEUER, A; PEIL, L; STAROSTA, AL; WOHLGEMUTH, I; PESKE, F; NOVACEK, J; BERNINGHAUSEN, O; GRUBMULLER, H; TENSON, T; BECKMANN, R; RODNINA, MV; VAIANA, AC; WILSON, DN, 2017:Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P. MOLECULAR CELL 68 (3), p. 515 - +.
• JANCARIKOVA, G; HOUSER, J; DOBES, P; DEMO, G; HYRSL, P; WIMMEROVA, M, 2017:Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity. PLOS PATHOGENS 13 (8)
• JASNOVIDOVA, O; KLUMPLER, T; KUBICEK, K; KALYNYCH, S; PLEVKA, P; STEFL, R, 2017:Structure and dynamics of the RNAPII CTDsome with Rtt103. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (42), p. 11133 - 11138.
• JASNOVIDOVA, O; KREJCIKOVA, M; KUBICEK, K; STEFL, R, 2017:Structural insight into recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by Rtt103p. EMBO REPORTS 18 (6), p. 906 - 913.
• NECASOVA, I; JANOUSKOVA, E; KLUMPLER, T; HOFR, C, 2017:Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas Rap1 restores it (gkx812) (vol 45, pg 12170, 2017). NUCLEIC ACIDS RESEARCH 45 (21), p. 12599 - 12599.
• NECASOVA, I; JANOUSKOVA, E; KLUMPLER, T; HOFR, C, 2017:Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas Rap1 restores it. NUCLEIC ACIDS RESEARCH 45 (21), p. 12170 - 12180.
• PEKARIK, V; PESKOVA, M; GURAN, R; NOVACEK, J; HEGER, Z; TRIPSIANES, K; KUMAR, J; ADAM, V, 2017:Visualization of stable ferritin complexes with palladium, rhodium and iridium nanoparticles detected by their catalytic activity in native polyacrylamide gels. DALTON TRANSACTIONS 46 (40), p. 13690 - 13694.
• SKUBNIK, K; NOVACEK, J; FUZIK, T; PRIDAL, A; PAXTON, RJ; PLEVKA, P, 2017:Structure of deformed wing virus, a major honey bee pathogen. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (12), p. 3210 - 3215.

2016

• GRAF M; ARENZ S; HUTER P; DÖNHÖFER A; NOVÁČEK J; WILSON DN, 2016:Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions. NUCLEIC ACIDS RESEARCH , p. [Epub ahead of print] - .
• JASNOVIDOVA, O; KREJCIKOVA, M; KUBICEK, K; STEFL, R, 2016:Recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by 3 '-end processing apparatus. FEBS JOURNAL 283 , p. 208 - 209.
• KREJCIKOVA, M; GKIONIS, K; HEMZAL, D; PLATTS, JA; PAPADIA, P; MARGIOTTA, N; SPONER, J; KUBICEK, K, 2016:Combining NMR (Nuclear Magnetic Resonance) and Raman spectroscopy reveals structural and functional features of a new cisplatin derivative. FEBS JOURNAL 283 , p. 164 - 164.
• KUBICEK, K; KREJCIKOVA, M; BRAZDA, P; SMIRAKOVA, E; NOVACEK, J; CRAMER, P; STEFL, R, 2016:What are the key aspects of interaction between RNA polymerase II C-terminal domain phosphorylated on tyrosine-1 and the elongation factors. FEBS JOURNAL 283 , p. 21 - 21.
• MULLAPUDI E; NOVÁČEK J; PÁLKOVÁ L; KULICH P; LINDBERG AM; VAN KUPPEVELD FJ; PLEVKA P, 2016:Structure and genome release mechanism of human cardiovirus Saffold virus-3. JOURNAL OF VIROLOGY , p. [Epub ahead of print] - .
• MULLAPUDI, E; NOVACEK, J; PALKOVA, L; KULICH, P; LINDBERG, AM; VAN KUPPEVELD, FJM; PLEVKA, P, 2016:Structure and Genome Release Mechanism of the Human Cardiovirus Saffold Virus 3. JOURNAL OF VIROLOGY 90 (17), p. 7628 - 7639.
• NOVÁČEK J; ŠIBOROVÁ M; BENEŠÍK M; PANTŮČEK R; DOŠKAŘ J; PLEVKA P, 2016:Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (33), p. 9351 - 6.

2015

• HROSSOVA, D; SIKORSKY, T; POTESIL, D; BARTOSOVIC, M; PASULKA, J; ZDRAHAL, Z; STEFL, R; VANACOVA, S, 2015:RBM7 subunit of the NEXT complex binds U-rich sequences and targets 3'-end extended forms of snRNAs. NUCLEIC ACIDS RESEARCH 43 (8), p. 4236 - 4248.

2014

• BACIKOVA, V; PASULKA, J; KUBICEK, K; STEFL, R, 2014:Structure and semi-sequence-specific RNA binding of Nrd1. NUCLEIC ACIDS RESEARCH 42 (12), p. 8024 - 8038.
• TUDEK, A; PORRUA, O; KABZINSKI, T; LIDSCHREIBER, M; KUBICEK, K; FORTOVA, A; LACROUTE, F; VANACOVA, S; CRAMER, P; STEFL, R; LIBRI, D, 2014:Molecular Basis for Coordinating Transcription Termination with Noncoding RNA Degradation. MOLECULAR CELL 55 (3), p. 467 - 481.

2013

• LABUTA, J; ISHIHARA, S; SIKORSKY, T; FUTERA, Z; SHUNDO, A; HANYKOVA, L; BURDA, JV; ARIGA, K; HILL, JP, 2013:NMR spectroscopic detection of chirality and enantiopurity in referenced systems without formation of diastereomers. NATURE COMMUNICATIONS 4

2012

• HOLUB, P; LALAKOVA, J; CERNA, H; PASULKA, J; SARAZOVA, M; HRAZDILOVA, K; ARCE, MS; HOBOR, F; STEFL, R; VANACOVA, S, 2012:Air2p is critical for the assembly and RNA-binding of the TRAMP complex and the KOW domain of Mtr4p is crucial for exosome activation. NUCLEIC ACIDS RESEARCH 40 (12), p. 5679 - 5693.
• JUNG, YW; BYUN, JS; KIM, YD; HEMZAL, D; HUMLICEK, J, 2012:Study of the Interaction Between HSA and Oligo-DNA Using Total Internal Reflection Ellipsometry. JOURNAL OF THE KOREAN PHYSICAL SOCIETY 60 (8), p. 1288 - 1291.
• KUBICEK, K; CERNA, H; HOLUB, P; PASULKA, J; HROSSOVA, D; LOEHR, F; HOFR, C; VANACOVA, S; STEFL, R, 2012:Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1. GENES & DEVELOPMENT 26 (17), p. 1891 - 1896.
• PORRUA, O; HOBOR, F; BOULAY, J; KUBICEK, K; D'AUBENTON-CARAFA, Y; GUDIPATI, RK; STEFL, R; LIBRI, D, 2012:In vivo SELEX reveals novel sequence and structural determinants of Nrd1-Nab3-Sen1-dependent transcription termination. EMBO JOURNAL 31 (19), p. 3935 - 3948.
• SIKORSKY, T; HOBOR, F; KRIZANOVA, E; PASULKA, J; KUBICEK, K; STEFL, R, 2012:Recognition of asymmetrically dimethylated arginine by TDRD3. NUCLEIC ACIDS RESEARCH 40 (22), p. 11748 - 11755.
• SUCHANKOVA, T; KUBICEK, K; KASPARKOVA, J; BRABEC, V; KOZELKA, J, 2012:Platinum-DNA interstrand crosslinks: Molecular determinants of bending and unwinding of the double helix. JOURNAL OF INORGANIC BIOCHEMISTRY 108 , p. 69 - 79.

2011

• HOBOR, F; PERGOLI, R; KUBICEK, K; HROSSOVA, D; BACIKOVA, V; ZIMMERMANN, M; PASULKA, J; HOFR, C; VANACOVA, S; STEFL, R, 2011:Recognition of Transcription Termination Signal by the Nuclear Polyadenylated RNA-binding (NAB) 3 Protein. JOURNAL OF BIOLOGICAL CHEMISTRY 286 (5), p. 3645 - 3657.
• PEKAROVA, B; KLUMPLER, T; TRISKOVA, O; HORAK, J; JANSEN, S; DOPITOVA, R; BORKOVCOVA, P; PAPOUSKOVA, V; NEJEDLA, E; SKLENAR, V; MAREK, J; ZIDEK, L; HEJATKO, J; JANDA, L, 2011:Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana. PLANT JOURNAL 67 (5), p. 827 - 839.

2010

• PERGOLI, R; KUBICEK, K; HOBOR, F; PASULKA, J; STEFL, R, 2010:H-1, C-13, and N-15 chemical shift assignments for the RNA recognition motif of Nab3. BIOMOLECULAR NMR ASSIGNMENTS 4 (1), p. 119 - 121.
• RICHARD STEFL, FLORIAN C. OBERSTRASS, JENNIFER L. HOOD, MURIEL JOURDAN, MICHAL ZIMMERMANN, LENKA SKRISOVSKA, CHRISTOPHE MARIS, LI PENG, CTIRAD HOFR, RONALD B. EMESON, FRÉDÉRIC H.-T. ALLAINE, 2010:The solution structure of the ADAR2 dsRBM-RNA complex reveals a sequence-specific read out of the minor groove. CELL 143 (2), p. 225 - 237.

GRANTY

• Dynamic assembly and exchange of RNA polymerase II CTD factors (649030), H2020 - Excellent science - ERC - Consolidator grant, 2015 - 2020
• The importance of having YS phosphorylated. Investigation of interaction between CTD and transcription elongation factor Spt6 (GA15-24117S), Czech Science Foundation - Standard Grants, 2015 - 2017
• Molecular basis for site-specific read-out of histone H3K4me2 by the Set3 PHD finger (GA15-17670S), Czech Science Foundation - Standard Grants, 2015 - 2017
• Structural studies of protein-RNA complexes involved in RNA quality control (GA204/08/1212), Howard Hughes Medical Institute, 2008 - 2010
• Structural studies of protein-RNA complexes involved in RNA quality control (1263), Howard Hughes Medical Institute, 2007 - 2009
• Structural basis of transcription termination of nonpolyadenylated transcripts (IAA401630903), Academy of Sciences of the Czech Republic - Grants of distinctly investigative character focused on the sphere of research pursued at present particularly in the Academy of Sciences of the Czech Rep., 2009 - 2011
• Structural studies of protein-RNA interactions involved in RNA quality control (CDA0049/2006), Human Frontier Science Program - Career Development Award, 2007 - 2009
• Structural basis for poly(A) independent transcription termination pathway (GAP305/10/1490), Czech Science Foundation - Standard Grants, 2010 - 2014
• Centre of excellence – Centre for RNA biology (GAP305/12/G034), Czech Science Foundation - Projects of Excellence, 2012 - 2018

1. A structural basis for the cross-talk between histones and RNA Polymerase II

Supervisor: doc. Mgr. Richard Štefl, Ph.D.
Consultants: Mgr. Karel Kubíček, PhD., doc. Mgr. Štěpánka Vaňáčová, Ph.D.

Annotation:

Chromatin is a highly flexible architecture in which spatially and temporally coordinated changes between structurally condensed states (transcriptionally repressive), and structurally accessible states (transcriptionally active) regulate gene expression. Posttranslational modifications of histones play the fundamental role in maintaining the dynamic equilibrium of these two chromatin states. Individual histone modifications are associated with a given stage of chromatin remodelling and transcriptional cycle. Interestingly, the C-terminal domain (CTD) of RNA polymerase II (RNAPII) is also posttranslationally modified which serves as a signal for the recruitment of appropriate processing factors in coordination with the transcription cycle. Recent findings revealed that these is an overlap between the occurrence of the histone and CTD marks, suggesting the existence of a cross-talk between the chromatin remodelers (orchestrated by histone modifications) and transcription/processing factors (orchestrated by RNAPII CTD modifications). We will identify protein adaptors that spatially and temporally mediate interactions between chromatin and RNAPII. These proteins will be structurally characterized and we will reveal the structural basis for the cross-talk between histones and RNAPII that is orchestrated through their posttranslational modifications.

Keywords: RNA, posttranslational modifications, CTD, RNAPII, histones, chromatin, C-terminal domain, gene expression

2. Cracking the CTD code

Supervisor: doc. Mgr. Richard Štefl, Ph.D.
Consultants: Mgr. Karel Kubíček, PhD., Mgr. Pavel Plevka, Ph.D.

Annotation:

The concept of the CTD code that specifies the position of RNAPII in the transcriptional cycle and thus recruits specific processing factors, was suggested almost a decade ago. However, how the C-terminal domain (CTD) of RNA polymerase II recruits, activates, and displaces appropriate processing factors in coordination with the transcription cycle, remains obscure. Association of specific factors with the CTD is dictated by different post-translational modification patterns and conformational changes in the CTD. To reveal the CTD code, a combination of structural, biochemical, and genetic methods will be used to study important complexes of the CTD with CTD code reader proteins. The project will help to decipher the basic rules that govern the readout of the CTD code.

Keywords: CTD, TNAPII, C-terminal domain, CTD code, post-translational modification