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Functional characterization and identification of mouse Rad51d splice variants.

Gruver AM, Yard BD, McInnes C, Rajesh C, Pittman DL - BMC Mol. Biol. (2009)

Bottom Line: In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2.These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10.These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina Campus, Columbia, SC 29208, USA. gruvera@ccf.org

ABSTRACT

Background: The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns.

Results: Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DDelta7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10.

Conclusion: These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

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Summary of alternatively spliced transcripts of Mus musculus Rad51d. (A) The ten exons of Rad51d are shown as numbered boxes drawn relative to base pair length with both the full-length transcript (upper panel) and each alternatively spliced transcript shown (lower panel). Shaded areas designate partial intron retention (+int3) or partial exon exclusion (Δ7b). (B) Predicted translation products of the RAD51D isoforms are illustrated. Black and yellow boxes indicate the location of Walker Motifs A and B respectively. A core helix-hairpin-helix-GP rich domain (blue) and linker region (red) are also indicated. Amino acids introduced by splicing induced frameshift in the transcripts of RAD51DΔ8, RAD51DΔ3, and RAD51DΔ3,7b are colored green with the corresponding novel sequence following. Asterisks represent sites of premature termination codons.
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Figure 1: Summary of alternatively spliced transcripts of Mus musculus Rad51d. (A) The ten exons of Rad51d are shown as numbered boxes drawn relative to base pair length with both the full-length transcript (upper panel) and each alternatively spliced transcript shown (lower panel). Shaded areas designate partial intron retention (+int3) or partial exon exclusion (Δ7b). (B) Predicted translation products of the RAD51D isoforms are illustrated. Black and yellow boxes indicate the location of Walker Motifs A and B respectively. A core helix-hairpin-helix-GP rich domain (blue) and linker region (red) are also indicated. Amino acids introduced by splicing induced frameshift in the transcripts of RAD51DΔ8, RAD51DΔ3, and RAD51DΔ3,7b are colored green with the corresponding novel sequence following. Asterisks represent sites of premature termination codons.

Mentions: Multiple Rad51d transcripts were first detected by Northern blot analysis [25], and seven splice variants were later identified by RT-PCR in both mouse and human brain tissues [15]. The Rad51d gene consists of 10 exons, and a summary of the current evidence for each alternative transcript for the human and mouse Rad51d gene from the ASD and EASED databases is presented in Table 1[17,26]. The Mus musculus Rad51d alternative transcripts are summarized in Figure 1A and for clarity are referred to as RAD51DΔ (exon excluded) or RAD51D+(intron included). The highly conserved ATP binding Walker Motifs A and B, present in all members of the RAD51 family, are contained within exons 4 and 7 of Rad51d respectively (Figure 1B). RAD51D full length (FL) includes both exons 7a and 7b in contrast to the RAD51DΔ7b alternative transcript in which the final 15 base pairs of exon 7 are excluded. Previously, this 3' portion of exon 7 as well as the retained intron in RAD51D+int3 were labeled as additional exons [15]. Internal deletions are also predicted in RAD51DΔ7,8 and RAD51DΔ5 (residues 193–246, and 116–159 respectively), while stretches of novel amino acid sequence and premature stop codons are predicted for the RAD51DΔ8, RAD51DΔ3 and RAD51D+int3 isoforms as a result of splicing induced frameshift mutations (residues 224–233, 49–53, and 88–109 respectively). The splice variants RAD51DΔ3 and RAD51DΔ3,7b are predicted to encode identical peptides.


Functional characterization and identification of mouse Rad51d splice variants.

Gruver AM, Yard BD, McInnes C, Rajesh C, Pittman DL - BMC Mol. Biol. (2009)

Summary of alternatively spliced transcripts of Mus musculus Rad51d. (A) The ten exons of Rad51d are shown as numbered boxes drawn relative to base pair length with both the full-length transcript (upper panel) and each alternatively spliced transcript shown (lower panel). Shaded areas designate partial intron retention (+int3) or partial exon exclusion (Δ7b). (B) Predicted translation products of the RAD51D isoforms are illustrated. Black and yellow boxes indicate the location of Walker Motifs A and B respectively. A core helix-hairpin-helix-GP rich domain (blue) and linker region (red) are also indicated. Amino acids introduced by splicing induced frameshift in the transcripts of RAD51DΔ8, RAD51DΔ3, and RAD51DΔ3,7b are colored green with the corresponding novel sequence following. Asterisks represent sites of premature termination codons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667185&req=5

Figure 1: Summary of alternatively spliced transcripts of Mus musculus Rad51d. (A) The ten exons of Rad51d are shown as numbered boxes drawn relative to base pair length with both the full-length transcript (upper panel) and each alternatively spliced transcript shown (lower panel). Shaded areas designate partial intron retention (+int3) or partial exon exclusion (Δ7b). (B) Predicted translation products of the RAD51D isoforms are illustrated. Black and yellow boxes indicate the location of Walker Motifs A and B respectively. A core helix-hairpin-helix-GP rich domain (blue) and linker region (red) are also indicated. Amino acids introduced by splicing induced frameshift in the transcripts of RAD51DΔ8, RAD51DΔ3, and RAD51DΔ3,7b are colored green with the corresponding novel sequence following. Asterisks represent sites of premature termination codons.
Mentions: Multiple Rad51d transcripts were first detected by Northern blot analysis [25], and seven splice variants were later identified by RT-PCR in both mouse and human brain tissues [15]. The Rad51d gene consists of 10 exons, and a summary of the current evidence for each alternative transcript for the human and mouse Rad51d gene from the ASD and EASED databases is presented in Table 1[17,26]. The Mus musculus Rad51d alternative transcripts are summarized in Figure 1A and for clarity are referred to as RAD51DΔ (exon excluded) or RAD51D+(intron included). The highly conserved ATP binding Walker Motifs A and B, present in all members of the RAD51 family, are contained within exons 4 and 7 of Rad51d respectively (Figure 1B). RAD51D full length (FL) includes both exons 7a and 7b in contrast to the RAD51DΔ7b alternative transcript in which the final 15 base pairs of exon 7 are excluded. Previously, this 3' portion of exon 7 as well as the retained intron in RAD51D+int3 were labeled as additional exons [15]. Internal deletions are also predicted in RAD51DΔ7,8 and RAD51DΔ5 (residues 193–246, and 116–159 respectively), while stretches of novel amino acid sequence and premature stop codons are predicted for the RAD51DΔ8, RAD51DΔ3 and RAD51D+int3 isoforms as a result of splicing induced frameshift mutations (residues 224–233, 49–53, and 88–109 respectively). The splice variants RAD51DΔ3 and RAD51DΔ3,7b are predicted to encode identical peptides.

Bottom Line: In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2.These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10.These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina Campus, Columbia, SC 29208, USA. gruvera@ccf.org

ABSTRACT

Background: The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns.

Results: Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DDelta7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10.

Conclusion: These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.

Show MeSH
Related in: MedlinePlus