The Arabidopsis Genome Encodes Structurally and Functionally Diverse HMGB-type Proteins
Genome Research, Bielefeld University, Bielefeld, North Rhine-Westphalia, Germany Journal of Molecular Biology
(Impact Factor: 4.33).
06/2006; 358(3):654-64. DOI: 10.1016/j.jmb.2006.02.068
The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription and recombination. In addition to the previously identified HMGB1-HMGB6 proteins, the Arabidopsis genome encodes at least two other candidate family members (encoded by the loci At2g34450 and At5g23405) having the typical overall structure of a central domain displaying sequence similarity to HMG-box DNA binding domains, which is flanked by basic N-terminal and acidic C-terminal regions. Subcellular localisation experiments demonstrate that the At2g34450 protein is a nuclear protein, whereas the At5g23405 protein is found mainly in the cytoplasm. In line with this finding, At5g23405 displays specific interaction with the nuclear export receptor AtXPO1a. According to CD measurements, the HMG-box domains of both proteins have an alpha-helical structure. The HMG-box domain of At2g34450 interacts with linear DNA and binds structure-specifically to DNA minicircles, whereas the HMG-box domain of At5g23405 does not interact with DNA at all. In ligation experiments with short DNA fragments, the At2g34450 HMG-box domain can facilitate the formation of linear oligomers, but it does not promote the formation of DNA minicircles. Therefore, the At2g34450 protein shares several features with HMGB proteins, whereas the At5g23405 protein has different characteristics. Despite the presence of a region with similarity to the nucleosome-binding domain typical of HMGN proteins, At2g34450 does not bind nucleosome particles. In summary, our data demonstrate (i) that plant HMGB-type proteins are functionally variable and (ii) that it is difficult to predict HMG-box function solely based on sequence similarity.
Available from: Thomas Schubert
- "We expressed an UAP56-GFP fusion protein in BY2 cell protoplasts, to examine the subcellular distribution of UAP56 by confocal laser scanning microscopy (CLSM). The chromosomal HMGB protein encoded by the At2g34450 locus that localises to the cell nucleus  served as a reference (Figure 2A). The UAP56-GFP fusion protein accumulated in the nucleus (Figure 2B), indicating that as in other organisms , , 
Arabidopsis UAP56 is a nuclear protein. "
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ABSTRACT: The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.
Available from: Simon Arnold Mortensen
- "Thus, the Arabidopsis (Arabidopsis thaliana) genome encodes eight proteins that, according to their amino acid sequences, can be classified as HMGB proteins. However, experimental analyses demonstrate that the protein encoded by the Arabidopsis Genome Initiative locus At5g23405, despite marked sequence similarity to well-characterized HMGB proteins, does not share the features of bona fide HMGB proteins, including predominant nuclear localization and DNA-binding activity (Grasser et al., 2006). Therefore, it is required to test experimentally the functionality of predicted HMG-box domains, which may be of particular importance for domains that display a lower degree of sequence conservation such as the putative algal HMG-box proteins. "
Available from: Gerrit T S Beemster
- "The DNA bending activity of recombinant proteins was tested in DNA circularization experiments using a 32 Plabelled 78-bp DNA fragment, as described previously (Grasser et al., 2006). "
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ABSTRACT: The high mobility group (HMG)-box represents a DNA-binding domain that is found in various eukaryotic DNA-interacting proteins. Proteins that contain three copies of the HMG-box domain, termed 3 x HMG-box proteins, appear to be specific to plants. The Arabidopsis genome encodes two 3 x HMG-box proteins that were studied here.
DNA interactions were examined using electrophoretic mobility shift assays, whereas expression, subcellular localization and chromosome association were mainly analysed by different types of fluorescence microscopy.
The 3 x HMG-box proteins bind structure specifically to DNA, display DNA bending activity and, in addition to the three HMG-box domains, the basic N-terminal domain contributes to DNA binding. The expression of the two Arabidopsis genes encoding 3 x HMG-box proteins is linked to cell proliferation. In synchronized cells, expression is cell cycle dependent and peaks in cells undergoing mitosis. 3 x HMG-box proteins are excluded from the nuclei of interphase cells and localize to the cytosol, but, during mitosis, they associate with condensed chromosomes. The 3 x HMG-box2 protein generally associates with mitotic chromosomes, while 3 x HMG-box1 is detected specifically at 45S rDNA loci.
In addition to mitotic chromosomes the 3 x HMG-box proteins associate with meiotic chromosomes, suggesting that they are involved in a general process of chromosome function related to cell division, such as chromosome condensation and/or segregation.
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