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A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions.

Maitra R, Sadofsky MJ - Nucleic Acids Res. (2009)

Bottom Line: We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66.Phylogenetic analysis shows the WW-like module to be highly conserved.The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.

ABSTRACT
More than one-third of the RAG1 protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination in vitro or in a mouse. What, then, is the function of the N-terminal domain? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other proteins in the cell, remains an open question. We revisited the issue of additional protein-protein interactions between RAG1 and other proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a protein complex containing RAG proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

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One-hybrid assay adapted to show GMEB binding to RAG proteins at the RSS. (A) The original one-hybrid assay (ref. 22). The RAG protein complex, fused to VP16, activates luciferase when bound to the 12RSS. (B) Modified one-hybrid assay is portrayed in which VP-16 is only present fused to GMEB1. Activation of luciferase requires protein–protein interaction between GMEB1 and the RAG complex on the 12RSS. (C) Luciferase activity (machine units) obtained from the listed combinations of proteins transfected into COS7 cells.
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Figure 5: One-hybrid assay adapted to show GMEB binding to RAG proteins at the RSS. (A) The original one-hybrid assay (ref. 22). The RAG protein complex, fused to VP16, activates luciferase when bound to the 12RSS. (B) Modified one-hybrid assay is portrayed in which VP-16 is only present fused to GMEB1. Activation of luciferase requires protein–protein interaction between GMEB1 and the RAG complex on the 12RSS. (C) Luciferase activity (machine units) obtained from the listed combinations of proteins transfected into COS7 cells.

Mentions: The luciferase assay was also used in a second context to measure GMEB1 interaction with the RAG protein complex. We adapted the one-hybrid assay (22) that had been previously used to detect RAG1 and RAG2 binding to the RSS. The authors of the original study used VP16 transactivator fusions to RAG1 and RAG2 to address the occupancy of the RAG protein complex on DNA. Here we extend that assay to demonstrate that GMEB1 fused to VP16 can be recruited to the RSS through interaction with the RAG1-NTD. Figure 5A shows the original scheme. Three reporter plasmids encode luciferase driven by a minimal promoter region and differ by the number of 12RSS elements in the intervening spacer (zero, three or eight RSS repeats). The previous study (22) showed that binding of RAG proteins to the RSS depended on both RAG1 and RAG2 coexpression. Panel 5B presents a variation on this assay, where only GMEB1 carries the VP16 transactivator. GMEB1 binding to RAG1 localized to the RSS activates transcription of the luciferase reporter. The results are tabulated in Figure 5C. We see, using full-length or core RAG1-VP16, that the RAG complex binds to the reporter plasmids, with luciferase activity increasing with the number of RSS copies (lines 1 and 2). GMEB1-VP16, in the absence of the RAG protein complex, does not transactivate the luciferase (line 3). GMEB-VP16 strongly transactivates the reporter when full-length RAG1 protein (absent VP-16), coupled with RAG2, binds the RSS elements (line 4). In contrast, core RAG1 plus RAG2 do not recruit GMEB1-VP16 (line 5). These data extend the results of the mammalian two-hybrid by showing that GMEB1 is capable of binding to RAG1 in the context of the RAG1/RAG2 complex situated on an RSS. One is struck by the magnitude by which the GMEB-VP16 fusion drives transcription compared to the relatively weak signal elicited by RAG1-VP16. Alternatives include the possibility that the RAG1-VP16 fusion is intrinsically a poor transcriptional activator for structural reasons, while the GMEB1-VP16 bound to RAG1 is better able to activate the promoter. It is also possible that the complex including GMEB1 binds DNA better than the RAG complex alone. These questions are under further study.Figure 5.


A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions.

Maitra R, Sadofsky MJ - Nucleic Acids Res. (2009)

One-hybrid assay adapted to show GMEB binding to RAG proteins at the RSS. (A) The original one-hybrid assay (ref. 22). The RAG protein complex, fused to VP16, activates luciferase when bound to the 12RSS. (B) Modified one-hybrid assay is portrayed in which VP-16 is only present fused to GMEB1. Activation of luciferase requires protein–protein interaction between GMEB1 and the RAG complex on the 12RSS. (C) Luciferase activity (machine units) obtained from the listed combinations of proteins transfected into COS7 cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691831&req=5

Figure 5: One-hybrid assay adapted to show GMEB binding to RAG proteins at the RSS. (A) The original one-hybrid assay (ref. 22). The RAG protein complex, fused to VP16, activates luciferase when bound to the 12RSS. (B) Modified one-hybrid assay is portrayed in which VP-16 is only present fused to GMEB1. Activation of luciferase requires protein–protein interaction between GMEB1 and the RAG complex on the 12RSS. (C) Luciferase activity (machine units) obtained from the listed combinations of proteins transfected into COS7 cells.
Mentions: The luciferase assay was also used in a second context to measure GMEB1 interaction with the RAG protein complex. We adapted the one-hybrid assay (22) that had been previously used to detect RAG1 and RAG2 binding to the RSS. The authors of the original study used VP16 transactivator fusions to RAG1 and RAG2 to address the occupancy of the RAG protein complex on DNA. Here we extend that assay to demonstrate that GMEB1 fused to VP16 can be recruited to the RSS through interaction with the RAG1-NTD. Figure 5A shows the original scheme. Three reporter plasmids encode luciferase driven by a minimal promoter region and differ by the number of 12RSS elements in the intervening spacer (zero, three or eight RSS repeats). The previous study (22) showed that binding of RAG proteins to the RSS depended on both RAG1 and RAG2 coexpression. Panel 5B presents a variation on this assay, where only GMEB1 carries the VP16 transactivator. GMEB1 binding to RAG1 localized to the RSS activates transcription of the luciferase reporter. The results are tabulated in Figure 5C. We see, using full-length or core RAG1-VP16, that the RAG complex binds to the reporter plasmids, with luciferase activity increasing with the number of RSS copies (lines 1 and 2). GMEB1-VP16, in the absence of the RAG protein complex, does not transactivate the luciferase (line 3). GMEB-VP16 strongly transactivates the reporter when full-length RAG1 protein (absent VP-16), coupled with RAG2, binds the RSS elements (line 4). In contrast, core RAG1 plus RAG2 do not recruit GMEB1-VP16 (line 5). These data extend the results of the mammalian two-hybrid by showing that GMEB1 is capable of binding to RAG1 in the context of the RAG1/RAG2 complex situated on an RSS. One is struck by the magnitude by which the GMEB-VP16 fusion drives transcription compared to the relatively weak signal elicited by RAG1-VP16. Alternatives include the possibility that the RAG1-VP16 fusion is intrinsically a poor transcriptional activator for structural reasons, while the GMEB1-VP16 bound to RAG1 is better able to activate the promoter. It is also possible that the complex including GMEB1 binds DNA better than the RAG complex alone. These questions are under further study.Figure 5.

Bottom Line: We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66.Phylogenetic analysis shows the WW-like module to be highly conserved.The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.

ABSTRACT
More than one-third of the RAG1 protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination in vitro or in a mouse. What, then, is the function of the N-terminal domain? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other proteins in the cell, remains an open question. We revisited the issue of additional protein-protein interactions between RAG1 and other proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a protein complex containing RAG proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

Show MeSH