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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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(Top) Linear representation of the mouse RAG1 protein. The central core region (384–1008) is marked in gray, with essential acidic residues. The N-terminal region (residues 1–383) includes a previously identified RING motif, several clusters of basic residues, and (in this report) the WW-like domain. (Bottom) Representation of the two-hybrid assay. The GAL4 DNA-binding domain (GAL4 DNA-BD) is fused to the RAG1 N-terminal domain (NTD) or smaller peptides. A library of target proteins is obtained with each member fused to the GAL4 activation domain (GAL4 AD). In yeast, the GAL4 DNA-BD binds to the UAS element on a reporter construct. A protein–protein interaction between a target protein and the RAG1 NTD protein positions the GAL4 AD to activate transcription of the His3 marker gene.
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Figure 1: (Top) Linear representation of the mouse RAG1 protein. The central core region (384–1008) is marked in gray, with essential acidic residues. The N-terminal region (residues 1–383) includes a previously identified RING motif, several clusters of basic residues, and (in this report) the WW-like domain. (Bottom) Representation of the two-hybrid assay. The GAL4 DNA-binding domain (GAL4 DNA-BD) is fused to the RAG1 N-terminal domain (NTD) or smaller peptides. A library of target proteins is obtained with each member fused to the GAL4 activation domain (GAL4 AD). In yeast, the GAL4 DNA-BD binds to the UAS element on a reporter construct. A protein–protein interaction between a target protein and the RAG1 NTD protein positions the GAL4 AD to activate transcription of the His3 marker gene.

Mentions: RAG1 protein is large, with 1040 amino acid residues in the mouse (Figure 1).Figure 1.


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)

(Top) Linear representation of the mouse RAG1 protein. The central core region (384–1008) is marked in gray, with essential acidic residues. The N-terminal region (residues 1–383) includes a previously identified RING motif, several clusters of basic residues, and (in this report) the WW-like domain. (Bottom) Representation of the two-hybrid assay. The GAL4 DNA-binding domain (GAL4 DNA-BD) is fused to the RAG1 N-terminal domain (NTD) or smaller peptides. A library of target proteins is obtained with each member fused to the GAL4 activation domain (GAL4 AD). In yeast, the GAL4 DNA-BD binds to the UAS element on a reporter construct. A protein–protein interaction between a target protein and the RAG1 NTD protein positions the GAL4 AD to activate transcription of the His3 marker gene.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: (Top) Linear representation of the mouse RAG1 protein. The central core region (384–1008) is marked in gray, with essential acidic residues. The N-terminal region (residues 1–383) includes a previously identified RING motif, several clusters of basic residues, and (in this report) the WW-like domain. (Bottom) Representation of the two-hybrid assay. The GAL4 DNA-binding domain (GAL4 DNA-BD) is fused to the RAG1 N-terminal domain (NTD) or smaller peptides. A library of target proteins is obtained with each member fused to the GAL4 activation domain (GAL4 AD). In yeast, the GAL4 DNA-BD binds to the UAS element on a reporter construct. A protein–protein interaction between a target protein and the RAG1 NTD protein positions the GAL4 AD to activate transcription of the His3 marker gene.
Mentions: RAG1 protein is large, with 1040 amino acid residues in the mouse (Figure 1).Figure 1.

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