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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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Alignments over the WW-L motif. (A) The mouse RAG1 WWL is aligned to Itch, an E3 ligase (genebank NM_008395, fourth WW domain). Residues that contribute to the definition of the WW domain are highlighted in larger type. (B) Alignment of a wide selection of representative mammals over the WWL region. (C) Alignment of selected RAG1 sequences from all vertebrates. Boxed residues in (B) and (C) correspond to the defining features in (A). Positions with a consensus that exceeds 90% are listed. Dots represent identities to the first sequence; dashes represent gaps. Genebank accession numbers for each sequence are listed on the right.
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Figure 6: Alignments over the WW-L motif. (A) The mouse RAG1 WWL is aligned to Itch, an E3 ligase (genebank NM_008395, fourth WW domain). Residues that contribute to the definition of the WW domain are highlighted in larger type. (B) Alignment of a wide selection of representative mammals over the WWL region. (C) Alignment of selected RAG1 sequences from all vertebrates. Boxed residues in (B) and (C) correspond to the defining features in (A). Positions with a consensus that exceeds 90% are listed. Dots represent identities to the first sequence; dashes represent gaps. Genebank accession numbers for each sequence are listed on the right.

Mentions: Figure 6A shows an alignment between the RAG1 NTD and one well-studied WW domain, the fourth WW domain in the mouse protein Itch (Genebank NM_008395). The larger type emphasizes the important conserved features. These include the two tryptophan residues (W) as well as an internal hydrophobic cluster often containing a tyrosine (Y) and commonly, a proline two residues C-terminal to the second tryptophan. The spacing of the aligned residues within the RAG1 sequence does not correspond precisely to the current definition so we suggest calling the motif in RAG1 WW-like or WWL for now. This sequence is highly conserved across all mammalian RAG1 sequences, as summarized in Figure 6B (an alignment to 343 mammalian sequences is provided in supplement 2 of the Supplementary Data). The consensus is set at 90% stringency and shows complete conservation of the defining elements (although the tyrosine residue is replaced by cystine in the dog). The conservation is less absolute across the full vertebrate phylogeny. Figure 6C shows representatives of the major vertebrate radiations. Additional alignments are also presented in Supplementary Data.Figure 6.


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)

Alignments over the WW-L motif. (A) The mouse RAG1 WWL is aligned to Itch, an E3 ligase (genebank NM_008395, fourth WW domain). Residues that contribute to the definition of the WW domain are highlighted in larger type. (B) Alignment of a wide selection of representative mammals over the WWL region. (C) Alignment of selected RAG1 sequences from all vertebrates. Boxed residues in (B) and (C) correspond to the defining features in (A). Positions with a consensus that exceeds 90% are listed. Dots represent identities to the first sequence; dashes represent gaps. Genebank accession numbers for each sequence are listed on the right.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Alignments over the WW-L motif. (A) The mouse RAG1 WWL is aligned to Itch, an E3 ligase (genebank NM_008395, fourth WW domain). Residues that contribute to the definition of the WW domain are highlighted in larger type. (B) Alignment of a wide selection of representative mammals over the WWL region. (C) Alignment of selected RAG1 sequences from all vertebrates. Boxed residues in (B) and (C) correspond to the defining features in (A). Positions with a consensus that exceeds 90% are listed. Dots represent identities to the first sequence; dashes represent gaps. Genebank accession numbers for each sequence are listed on the right.
Mentions: Figure 6A shows an alignment between the RAG1 NTD and one well-studied WW domain, the fourth WW domain in the mouse protein Itch (Genebank NM_008395). The larger type emphasizes the important conserved features. These include the two tryptophan residues (W) as well as an internal hydrophobic cluster often containing a tyrosine (Y) and commonly, a proline two residues C-terminal to the second tryptophan. The spacing of the aligned residues within the RAG1 sequence does not correspond precisely to the current definition so we suggest calling the motif in RAG1 WW-like or WWL for now. This sequence is highly conserved across all mammalian RAG1 sequences, as summarized in Figure 6B (an alignment to 343 mammalian sequences is provided in supplement 2 of the Supplementary Data). The consensus is set at 90% stringency and shows complete conservation of the defining elements (although the tyrosine residue is replaced by cystine in the dog). The conservation is less absolute across the full vertebrate phylogeny. Figure 6C shows representatives of the major vertebrate radiations. Additional alignments are also presented in Supplementary Data.Figure 6.

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
Related in: MedlinePlus