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GTPase ran in—and out
The DNA of eukaryotes, and especially of plants, is well known for containing a fifth base, 5-methyl-cytosine. The presence of m5C does not affect the double helix itself but it is involved in epigenetic regulation of gene expression and in chromatin organization. These effects are mediated via methyl CpG-binding proteins, which in turn interact with other regulatory proteins. Our knowledge of these proteins in plants has been extended significantly by the work of Yano et al. (Japan and Israel, pp. 1179–1187). They focused on a methyl CpG-binding protein in Arabidopsis thaliana, AtMBD5. Using the yeast two-hybrid assay system and affinity pull-down techniques, they demonstrated an interaction within the A. thaliana proteome between AtMBD5 and a member of the Ran-GTPase family of regulatory proteins, AtRAN3. The cDNAs encoding these two proteins were spliced, respectively, to the sequences encoding the N-terminal and the C-terminal halves of yellow fluorescent protein (YFP) and the constructs were used to transform tobacco BY2 cells. Interaction of the two proteins in vivo was then detected by bimolecular fluorescence complementation (BiFC): fluorescence is only detected if the two halves of YFP are brought together, via protein–protein interactions, to recreate functional YFP. Use of BiFC plus differential fluorescent labelling facilitated the visualization of the two proteins at different cell cycle stages. In ‘interphase’, AtMBD5 was located in the nucleus, especially with heterochromatin (which is rich in m5C). AtRAN3 (presumed to be the active GTP-bound form) was also located in the nucleus and associated with AtMBD5 during this phase. At anaphase, however, the complex was not directly associated with chromatin but instead was located on the spindle. During telophase, AtMBD5 (and a proportion of the AtRAN3) re-associated with heterochromatin while the bulk of AtRAn3 was located at the growing cell plate. These results thus imply a role or roles for AtMBD5, in association with AtRAN3, in chromatin dynamics during cell division.
Professor J. A. Bryant
University of Exeter, UK
j.a.bryant{at}exeter.ac.uk
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