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Karla Neugebauer
Organization of the cell nucleus
Previous and current research
Biologists still seek to understand the basic organization of the nucleus, which contains the chromosomes and all of the factors required for DNA metabolism and gene expression. How are these biochemical functions organized in 3D space? Many factors involved in transcription and pre-mRNA processing are found concentrated in nuclear bodies, non-membrane bound objects ranging in diameter from 0.5 - 2 μm. These factors are also present in the nucleoplasm where genes are transcribed. What functions account for these distinct pools of factors within the nucleus? We are approaching the organization of the cell nucleus from the point of view of pre-mRNA splicing. One of our goals is to define mechanistically the role of nuclear organization in pre-mRNA splicing. A second goal is to use cell biology to complement biochemical and genetic experiments that have gone before us but which have left many unanswered questions about how pre-mRNA splicing is regulated in cells.
Within nucleoplasm, the pre-mRNA substrates and the factors essential for the splicing are highly concentrated at transcription sites, which are distributed apparently at random within the three-dimensional space of the nucleus. Additional pre-mRNA processing events, such as capping and polyadenylation also occur "co-transcriptionally", meaning during the process of transcription. Thus, transcription units can be seen as the sites within nuclei that nucleate the assembly of spliceosomes, likely through rapid binding of splicing factors to nascent RNA. We have developed in vivo assays in yeast and mammalian cells that allow us to detect co-transcriptional spliceosome assembly and splicing activity at genes of interest. We now use these tools to analyze the roles of specific splicing regulators and to address the elusive question of how alternative splicing is controlled during development.
In addition to their nucleoplasmic distribution and function, some essential splicing factors - the spliceosomal snRNPs - are concentrated in a nuclear body called the Cajal body (CB). Recently, our lab has provided evidence that CBs are the sites of snRNP assembly, by showing that intermediates in the snRNP assembly pathway are concentrated in CBs and by identifying one protein required for the trafficking of immature complexes to CBs. We recently showed by mathematical modeling that snRNP assembly is enhanced in cells containing CBs by up to 10-fold, due to the increased likelihood that immature snRNPs will be captured by CBs, where they meet a higher concentration of their partners than they would by random walk throughout the nucleoplasm. These data suggest that nuclear bodies function to make essential nuclear processes more efficient. We have recently established the zebrafish embryo as an excellent system for imaging the nuclear structures before, during and after the onset of zygotic genome transcription. The embryo is amenable to targeted knockdown of key factors in nuclear morphology and gene expression, and we are exploiting this system to understand the structure and function of CBs as well as to investigate the role of splicing in embryonic development.
Future prospects and goals
- Determine the mechanisms of splicing factor recruitment to transcription units in vivo
- Identify natural targets of alternative splicing factors in developing systems
- Determine the function of Cajal bodies and other nuclear compartments in vivo
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Selected publications
Strzelecka, M., A.C. Oates and K.M. Neugebauer. 2010: Dynamic control of Cajal body number during zebrafish embryogenesis. Nucleus, Vol. 1(1): 96-108.
Sapra, AK, M. Anko, I. Grishina, M. Lorenz, M. Pabis, I. Poser, J. Rollins, E. Weiland, and KM Neugebauer. 2009: SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo. Mol Cell, Vol. 34, no. 2, pp. 179-190.
Klingauf, M., D. Stanek, and K.M. Neugebauer. 2006: Enhancement of U4/U6 snRNP association in Cajal bodies predicted by mathematical modeling. Mol. Biol. Cell 17(12):4972-81.
Listerman, I., A.K. Sapra, and K.M. Neugebauer. 2006: Co-transcriptional coupling of splicing factor recruitment and pre-mRNA splicing in mammalian cells. Nature Structure and Molecular Biology 13:815-22.
Görnemann, J., K. Kotovic, K. Hujer, and K. Neugebauer (2005): Co-transcriptional spliceosome assembly occurs in a stepwise fashion and requires the cap-binding complex. Molecular Cell. 19:53-63.
Stanek, D. and Neugebauer, K. M. (2004): Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer. J. Cell Biol. 166:1015-1025.