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A cytoplasm-specific activity encoded by the Trithorax-like ATX1 gene.

Ndamukong I, Lapko H, Cerny RL, Avramova Z - Nucleic Acids Res. (2011)

Bottom Line: It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET).It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A).Loss of SET, but not of the histone modifying ATX1-SET activity, affects cytoskeletal actin bundling illustrating that the two isoforms have distinct functions in Arabidopsis cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA.

ABSTRACT
Eukaryotes produce multiple products from a single gene locus by alternative splicing, translation or promoter usage as mechanisms expanding the complexity of their proteome. Trithorax proteins, including the Arabidopsis Trithorax-like protein ATX1, are histone modifiers regulating gene activity. Here, we report that a novel member of the Trithorax family has a role unrelated to chromatin. It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET). It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A). Loss of SET, but not of the histone modifying ATX1-SET activity, affects cytoskeletal actin bundling illustrating that the two isoforms have distinct functions in Arabidopsis cells.

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Interaction of the SET domain with eEF1A. (a) GST-SET pull-down of total protein extract from WT Arabidopsis leaves followed by immunoreaction with the maize antiEF1A antibody. The 50 kDa protein corresponds to the Arabidopsis EF1A (arrow). (b) Transiently co-expressed GFP-EF1A (green) and RFP-soloSET (red) fusion proteins in tobacco leaf cells. Overlapping signals appear in yellow. Arrows point to nuclei. (c) Two-dimensonal-distribution of proteins from WT and from RNAi-SET (RNAi) expressing cellular extracts (Coomassie blue staining). Arrows point to the EF1A spot, isoelectric point 9.20. (d) Immunostaining of the 2D-gel blots with the antibody against H3K4me3. (e) Western blots of protein extracts from SET-RNAi, WT and atx1 cells with the antiH3K4me3 antibody. Arrow indicates position of the 50 kDa protein band. Coomassie-stained proteins are shown as loading controls in the lower panel.
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Figure 5: Interaction of the SET domain with eEF1A. (a) GST-SET pull-down of total protein extract from WT Arabidopsis leaves followed by immunoreaction with the maize antiEF1A antibody. The 50 kDa protein corresponds to the Arabidopsis EF1A (arrow). (b) Transiently co-expressed GFP-EF1A (green) and RFP-soloSET (red) fusion proteins in tobacco leaf cells. Overlapping signals appear in yellow. Arrows point to nuclei. (c) Two-dimensonal-distribution of proteins from WT and from RNAi-SET (RNAi) expressing cellular extracts (Coomassie blue staining). Arrows point to the EF1A spot, isoelectric point 9.20. (d) Immunostaining of the 2D-gel blots with the antibody against H3K4me3. (e) Western blots of protein extracts from SET-RNAi, WT and atx1 cells with the antiH3K4me3 antibody. Arrow indicates position of the 50 kDa protein band. Coomassie-stained proteins are shown as loading controls in the lower panel.

Mentions: In an earlier screening of a yeast-two-hybrid expression library with the SET-domain sequence as bait, we have identified the cDNA from the At5g60390 gene as a SET-domain binding candidate. At5g60390 encodes the elongation factor 1A (EF1A) and, here, we tested the interaction of the two proteins by the pull-down assay. A GST-SET fusion protein was produced, immobilized on a column and reacted with protein extracts from wild-type leaf cells. A band of ∼50 kDa, corresponding to the molecular size of the Arabidopsis EF1A protein (Figure 5a), was detected by immunoblotting with antibodies against the maize protein. The band was positively identified as EF1A by MS (Supplementary Figure S5). We note that the SET-peptide selectively picked EF1A amongst the proteins present in whole cellular extracts confirming, thus, the interaction of EF1A and SET-ATX1 and their strong preference for each other as binding partners.Figure 5.


A cytoplasm-specific activity encoded by the Trithorax-like ATX1 gene.

Ndamukong I, Lapko H, Cerny RL, Avramova Z - Nucleic Acids Res. (2011)

Interaction of the SET domain with eEF1A. (a) GST-SET pull-down of total protein extract from WT Arabidopsis leaves followed by immunoreaction with the maize antiEF1A antibody. The 50 kDa protein corresponds to the Arabidopsis EF1A (arrow). (b) Transiently co-expressed GFP-EF1A (green) and RFP-soloSET (red) fusion proteins in tobacco leaf cells. Overlapping signals appear in yellow. Arrows point to nuclei. (c) Two-dimensonal-distribution of proteins from WT and from RNAi-SET (RNAi) expressing cellular extracts (Coomassie blue staining). Arrows point to the EF1A spot, isoelectric point 9.20. (d) Immunostaining of the 2D-gel blots with the antibody against H3K4me3. (e) Western blots of protein extracts from SET-RNAi, WT and atx1 cells with the antiH3K4me3 antibody. Arrow indicates position of the 50 kDa protein band. Coomassie-stained proteins are shown as loading controls in the lower panel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Interaction of the SET domain with eEF1A. (a) GST-SET pull-down of total protein extract from WT Arabidopsis leaves followed by immunoreaction with the maize antiEF1A antibody. The 50 kDa protein corresponds to the Arabidopsis EF1A (arrow). (b) Transiently co-expressed GFP-EF1A (green) and RFP-soloSET (red) fusion proteins in tobacco leaf cells. Overlapping signals appear in yellow. Arrows point to nuclei. (c) Two-dimensonal-distribution of proteins from WT and from RNAi-SET (RNAi) expressing cellular extracts (Coomassie blue staining). Arrows point to the EF1A spot, isoelectric point 9.20. (d) Immunostaining of the 2D-gel blots with the antibody against H3K4me3. (e) Western blots of protein extracts from SET-RNAi, WT and atx1 cells with the antiH3K4me3 antibody. Arrow indicates position of the 50 kDa protein band. Coomassie-stained proteins are shown as loading controls in the lower panel.
Mentions: In an earlier screening of a yeast-two-hybrid expression library with the SET-domain sequence as bait, we have identified the cDNA from the At5g60390 gene as a SET-domain binding candidate. At5g60390 encodes the elongation factor 1A (EF1A) and, here, we tested the interaction of the two proteins by the pull-down assay. A GST-SET fusion protein was produced, immobilized on a column and reacted with protein extracts from wild-type leaf cells. A band of ∼50 kDa, corresponding to the molecular size of the Arabidopsis EF1A protein (Figure 5a), was detected by immunoblotting with antibodies against the maize protein. The band was positively identified as EF1A by MS (Supplementary Figure S5). We note that the SET-peptide selectively picked EF1A amongst the proteins present in whole cellular extracts confirming, thus, the interaction of EF1A and SET-ATX1 and their strong preference for each other as binding partners.Figure 5.

Bottom Line: It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET).It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A).Loss of SET, but not of the histone modifying ATX1-SET activity, affects cytoskeletal actin bundling illustrating that the two isoforms have distinct functions in Arabidopsis cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA.

ABSTRACT
Eukaryotes produce multiple products from a single gene locus by alternative splicing, translation or promoter usage as mechanisms expanding the complexity of their proteome. Trithorax proteins, including the Arabidopsis Trithorax-like protein ATX1, are histone modifiers regulating gene activity. Here, we report that a novel member of the Trithorax family has a role unrelated to chromatin. It is encoded from an internal promoter in the ATX1 locus as an isoform containing only the SET domain (soloSET). It is located exclusively in the cytoplasm and its substrate is the elongation factor 1A (EF1A). Loss of SET, but not of the histone modifying ATX1-SET activity, affects cytoskeletal actin bundling illustrating that the two isoforms have distinct functions in Arabidopsis cells.

Show MeSH
Related in: MedlinePlus