We are studying how pre-mRNA splicing is organised within the cell nucleus. Focusing primarily on spliceosomal snRNPs, we have developed novel approaches to localising the assembly and function of these essential splicing factors.
On the one hand, we have used advanced light microscopy techniques to show that snRNPs traffic to sub-nuclear compartments called Cajal bodies (CBs) where they assemble into active complexes. On the other hand, we have adapted chromatin immunoprecipitation technology, in order to visualise the action of snRNPs in co-transcriptional splicing, which requires resolution beyond the light microscope.
Our pioneering studies in the yeast Saccharomyces cerevisiae established that snRNPs and non-snRNP splicing factors are recruited exclusively to intron-containing genes, following transcription of gene regions encoding splicing signals. Spliceosome assembly proceeds in a stepwise fashion, is completed within the time course of transcription, and is dependent on the cap-binding complex (CBC) that binds the 5’ end of RNA polymerase II transcripts. This shows that transcription, capping and splicing are coupled processes. We have recently transferred this approach to mammalian cells and are currently investigating the roles of the CBC and the SR protein family of alternative splicing factors in co-transcriptional spliceosome assembly and alternative splicing.
- Änkö, M-L., Morales, L., Henry, I., Beyer, A. and Neugebauer, K.M. (2010). Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells. Nature Structural and Molecular Biology 17, 962-70.
- Pabis, M., Neufeld, N., Shav-Tal, Y. and Neugebauer, K.M. (2010). Expression and dynamic localization of an alternative isoform of the nuclear cap-binding complex subunit CBP20. Nucleus 1, 412-421.
- Huranová, M., Ivani, I., Benda, A., Poser, I., Brody, Y., Hof, M., Shav-Tal, Y., Neugebauer, K.M. and Stanek, D. (2010). Differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. Journal of Cell Biology 191, 75-86.
- Strzelecka, M., Trowitzsch, S., Weber, G., Lührmann, R., Oates, A.C. and Neugebauer, K.M. (2010). Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis. Nature Structural and Molecular Biology 17, 403–409.
- Strzelecka, M., Oates, A.C. and Neugebauer, K.M. (2010). Dynamic control of Cajal body number in zebrafish embryogenesis. Nucleus 1, 96-108.
- Sapra, A.K., Ankö, M.L., Grishina, I., Lorenz, M., Pabis, M., Poser, I., Rollins, J., Weiland, E.M., Neugebauer, K.M. (2009). SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Molecular Cell 34(2), 179-90.
- Listerman, I., Bledau, A.S., Grishina, I. and Neugebauer, K. M. (2007) Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III. PLoS Genetics 3:e212.
- Klingauf, M., Stanek, D., and Neugebauer, K.M. (2006) Enhancement of U4/U6 snRNP association in Cajal bodies predicted by mathematical modeling. Molecular Biology of the Cell 17, 4972. Pubmed ID 16987958.
- Listerman, I., Sapra, A. K., and Neugebauer, K. M. (2006). Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nature Structural & Molecular Biology 13, 815-822. Pubmed ID 16921380.
- Neugebauer, K. M. (2006). Keeping tabs on the women: life scientists in Europe. PLoS Biol 4, e97. Pubmed ID 16602822.
- Neugebauer, K. M. (2006). Please hold--the next available exon will be right with you. Nature Structural & Molecular Biology 13, 385-386. Pubmed ID 16738604.
- Stanek, D., and Neugebauer, K. M. (2006). The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze. Chromosoma 115, 343-354. Pubmed ID 16575476.
- Görnemann, J., Kotovic, K., Hujer, K. and Neugebauer, K. (2005). Co-transcriptional spliceosome assembly occurs in a stepwise fashion and requires the cap-binding complex. Molecular Cell 19, 53-63.
- Kittler, R., Putz, G., Pelletier, L., Poser, I., Heninger, A., Drechsel, D., Fischer, S., Konstantinova, I., Habermann, B., Grabner, H., Yaspo, M., Himmelbauer, H., Korn, B., Neugebauer, K., Pisabarro, M., and Buchholz, F. (2004). A large-scale RNAi screen in human cells identifies genes essential for cell division. Nature 432, 1036-1040.
- Stanek, D. and Neugebauer, K.M. (2004). Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer. Journal of Cell Biology 166, 1015-1025.
- Kotovic, K.M., Lockshon, D., Boric, L. and Neugebauer, K.M. (2003). Co-transcriptional recruitment of U1 snRNP to intron-containing genes in yeast. Molecular and Cellular Biology 23, 5768-5779.
- Stanek, D., Rader, S., Klingauf, M. and Neugebauer, K.M. (2003). Targeting of U4/U6 snRNP assembly factor SART3/p110 to Cajal bodies. Journal of Cell Biology 160, 505-516.
Key lab techniques: chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), fluorescent tagging of splicing factors (GFP, CFP, YFP, photo-activatable GFP, others) for live imaging, FRAP, FRET, FCS etc. Tagging of splicing factors on bacterial artificial chromosomes (BACs) for transient and stable expression in mammalian cells. RNAi (especially esiRNA, retrovirus mediated RNAi).
Key lab reagents BAC clones, expression cDNAs, splicing factor antibodies, tag antibodies.
Lab contact: Karla Neugebauer: email@example.com
Lab website: http://www.mpi-cbg.de/research/research-groups/karla-neugebauer.html