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Research topics

RNA molecules are not just messengers acting between DNA and proteins but rather required factors that play an active role in the expression of genes encoded in our genome. An RNA processing step called splicing can dramatically increase the diversity of proteins in human cells and tissues. RNA splicing is catalyzed by a large macromolecular complex, the spliceosome, which is formed from several RNA-protein complexes called snRNPs. In our group we are interested in spliceosome assembly, the organization of RNA splicing in the cell nucleus and regulation of alternative splicing. We also aim to determine how mutations in splicing factors can cause retinitis pigmentosa, a human genetic disease characterized by photoreceptor cell degeneration.

1. Formation of splicing complexes in living cells. Using advanced microscopy techniques (e.g. live cell imaging, FRET, FCS) we explore where and when the spliceosome assembles in the cell nucleus. Experimental data are then used for modelling of spliceosome assembly in the 3D space of the nuclear landscape. We identified the conserved nuclear compartment, the Cajal body, as the site of snRNP assembly and recycling, and proposed a model stating that the presence of Cajal bodies increases the efficiency of snRNP formation.

Spliceosome

Assembly of the spliceosome in vivo. Currently, it is unknown how splicing machinery assembles in the cell nucleus. To analyze spliceosome assembly in situ we created a battery of stable cell lines expressing splicing factors tagged with fluorescent proteins. These cells are used for measurements of interactions and dynamics of individual proteins directly in living cells.

2. Role of splicing mutation in an eye degenerative disease. The autosomal dominant disorder retinitis pigmentosa (RP) is characterized by progressive peripheral vision loss and night vision difficulties that can eventually lead to total loss of vision. With advances in molecular research, it is now known that RP can be caused by molecular defects in many different genes mostly involved in a light detection process. However, several mutations were found in snRNP specific proteins that are essential for every cell in our body. Why this mutation specifically affects retina cells remains mystery. In our research, we concentrate on a role of RP mutations on formation of snRNP complexes and assembly of the spliceosome.

3. Regulation of alternative splicing. Pre-mRNA splicing is an essential step in gene expression and its regulation vastly increases the coding potential of our genome. In our lab we are interested inconnection between splicing, chromatin and nuclear architecture and mainly how chromatin modifications and nuclear structure influence splicing outcome.

Current Grant Support

GACR grant no.: GA204/07/0133; 2007-2011: Self-organization principles of non-membrane-bound organels in eukaryotic cells (collaboration with Jan Malínský from IEM AS CR)
AS CR project KAN200520801 - Targeted expression and transport of bioactive molecules
GACR grant no.: P305/10/0424; 2010-2013: Regulation of alternative splicing via chromatin acetylation
GACR grant no.: P302/11/1910; 2011-2014: Formation of splicing machinery in the context of the cell nucleus
GA Charles University, grant no.: 153310; 2010-2011: Cajal body as a proofreading factor in spliceosomal snRNP formation (I. Novotný)
GA Charles University, grant no.: 274111; 2011-2012: The role of promotor in the regulation of RNA splicing (E. Dušková)

Most important recent papers

(2004-present; names of the lab members underlined)

Dundr M, Hebert MD, Karpova TS, Stanek D, Xu H, Shpargel KB, Meier UT, Neugebauer KM, Matera AG, Misteli T. In vivo kinetics of Cajal body components. J Cell Biol 164(6): 831-42, 2004. (Pubmed) (DOI)

Stanek D, Neugebauer KM. Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer. J Cell Biol 166(7): 1015-25, 2004. (Pubmed) (DOI)

Klingauf M, Stanek D, Neugebauer KM. Enhancement of U4/U6 small nuclear ribonucleoprotein particle association in Cajal bodies predicted by mathematical modeling. Mol Biol Cell 17(12): 4972-81, 2006. (Pubmed) (DOI)

Cvackova Z, Albring KF, Koberna K, Ligasova A, Huber O, Raska I, Stanek D. Pontin is localized in nucleolar fibrillar centers. Chromosoma 117(5): 487-97, 2008. (Pubmed) (DOI)

Stanek D, Pridalova-Hnilicova J, Novotny I, Huranova M, Blazikova M, Wen X, Sapra AK, Neugebauer KM. Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies. Mol Biol Cell 19(6): 2534-43, 2008. (Pubmed) (DOI)

Cvackova Z, Masata M, Stanek D, Fidlerova H, Raska I. Chromatin position in human HepG2 cells: although being non-random, significantly changed in daughter cells. J Struct Biol 165(2): 107-17, 2009. (Pubmed) (DOI)

Huranova M, Hnilicova J, Fleischer B, Cvackova Z, Stanek D. A mutation linked to retinitis pigmentosa in HPRP31 causes protein instability and impairs its interactions with spliceosomal snRNPs. Hum Mol Genet 18(11): 2014-23, 2009. (Pubmed) (DOI)

Huranova M, Jablonski JA, Benda A, Hof M, Stanek D, Caputi M. In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer. RNA 15(11): 2063-71, 2009. (Pubmed) (DOI)

Huranova M, Ivani I, Benda A, Poser I, Brody Y, Hof M, Shav-Tal Y, Neugebauer KM, Stanek D. The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J Cell Biol 191(1): 75-86, 2010. (Pubmed) (DOI)

Novotny I, Blazikova M, Stanek D, Herman P, Malinsky J. In vivo kinetics of U4/U6·U5 tri-snRNP formation in Cajal bodies. Mol Biol Cell 22(4): 513-23, 2011. (Pubmed) (DOI)

Hnilicova J, Hozeifi S, Duskova E, Icha J, Tomankova T, Stanek D. Histone deacetylase activity modulates alternative splicing. PLoS One 6(2): e16727, 2011. (Pubmed) (DOI)

Reviews

Neugebauer KM, Geiger JA, Kotovic KM, Stanek D. Pre-mRNA processing in the nuclear landscape. In: Visions of the Cell Nucleus - Eukaryotic DNA. P Hemmerich and S Diekmann, Eds: American Scientific Publishers. 36 pages.

Stanek D, Neugebauer KM. The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze. Chromosoma 115(5): 343-54, 2006. (Pubmed) (DOI)

Hnilicova J, Stanek D. Where splicing joins chromatin. Nucleus 2(3): 182-188, 2011. (Pubmed) (DOI)

Teaching, lectures, public presentations

MB150P91E - RNA structure and function - download presentations and recordings (password protected access)

FRAP fitting (MATLAB scripts)

Staněk D. (2009) Překvapivá souvislost - degenerace oční sítnice a pre-mRNA sestřih. Akademický bulletin 09/2009 (PDF).

Staněk D. (2010) Za vším hledej RNA. Vesmír 89: 322, 2010/5. (web).

Advanced Techniques in Fluorescence Microscopy - practical course, Institute of Molecular Genetics

Alumni

Tereza Tománková, M.Sc.
current position: PhD student, Department of Immunology, Medical Faculty, Palacky University in Olomouc

Ivan Ivani, M.Sc.
current position: PhD student, Department of Life - Molecular Modelling and Bioinformatics, Barcelona Supercomputing Center

Petr Tešina, M.Sc.
current position: PhD student, Department of Structural Biology, Institute of Molecular Genetics ASCR, Prague

Viola Hausnerová, M.Sc.
current position: PhD student, Department of Cellular Biology and Pathology, First Medical Faculty, Charles University in Prague