Abstracts (poster) - Wissenschaft Online

Rudolf Engelke, Gerhard Mittler
Proteomic analysis of the nuclear matrix in pre-B cells.
The nuclear matrix (NM) is an operationally defined structure that was introduced by
Berezney and Coffey in 1974 to denote a residual nuclear scaffold devoid of bulk
chromatin obtained by sequential salt extractions, detergent and DNase I treatment (1).
Further analysis suggested that this subnuclear structure is a ribonucleoprotein network
comprising the interchromatin space and the nuclear lamina and might therefore play an
important role in compartmentalization of the nucleus as well as the three dimensional
organization of the genome. Consistent with this, nuclear matrix proteins have been
implicated in almost all aspects of nucleic acid metabolism including DNA replication and
repair, transcription as well as RNA processing (2, 3).
In this work we have improved the classical biochemical nuclear matrix preparation
methods utilizing 250 mM ammonium sulfate, 2 M NaCl or 25 mM lithium-3,5,diiodosalicylate
(LIS) as nuclear extraction reagent and analyzed the NM proteome by
nanoLC tandem mass spectrometry leading to the identification of more than 950 NM
protein candidates. Our NM preparation maintains the morphological integrity of the
nucleus as assessed by phase contrast and immunofluorescence microscopy. Cherry-NLS
and DNA DAPI staining served as purification control demonstrating the removal of
chromatin and nucleoplasm. Implications of a quantitative SILAC-based comparison of
the NM versus the nuclear proteome in order to define bona fide NM constituents are
discussed.
Literature
(1) Berezney, R. and D. S. Coffey (1974). "Identification of a nuclear protein matrix."
Biochem Biophys Res Commun 60(4): 1410-7.
(2) Pederson, T. (1998). "Thinking about a nuclear matrix." J Mol Biol 277(2): 147-59.
(3) Zaidi, S. K., et al. (2005). "The dynamic organization of gene-regulatory machinery
in nuclear microenvironments." EMBO Rep 6(2): 128-33.
contact:
Rudolf Engelke
MPI of Immunobiology
engelke@immunbio.mpg.de
Stübeweg 51
79108 Freiburg (Germany)