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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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Expression analysis of Rad51d alternative transcripts. (A) RT-PCR for Mus musculus Rad51d and selected splice variants was conducted in eight tissues. Expression of RAD51DΔ3 employing primers Rad51d F1 and R1 (upper panel); RAD51DΔ5 with Rad51d F2 and R2 (lower panel). (B) RT-PCR products of human RAD51D in normal breast. Arrows indicate transcripts corresponding to known human splice isoforms [16]. RAD51DΔ3 and RAD51DΔ5 transcript positions are underlined. Size markers in base pairs are shown on the left. Numbers in parenthesis represent transcript abundance as determined by ImageJ analysis of the band intensities (bk; background). (C) The RAD51D peptide is aligned with the predicted amino acid sequence of RAD51DΔ9,10 and RAD51DΔ10. RAD51DΔ9,10 contains the first 8 exons of RAD51D followed by four out of frame amino acids (underlined and italicized) encoded by intron 8. RAD51DΔ10 includes amino acids 1–302 of RAD51D followed by five novel amino acids encoded by intronic sequence 5980 bp downstream from the predicted Rad51d polyA site. Premature termination codons are indicated by an asterisk, and exon boundaries are represented by a gap in the alignment of the predicted amino acid sequences. (D) RT-PCR for RAD51DΔ9,10 (upper panel) and RAD51DΔ10 (lower panel) from Mus musculus tissues with primers Rad51d F3 and Rad51dΔ9,10 R1 or Rad51dΔ10 R1 respectively. Abbreviations: 51D; RAD51D-FL, Δ3; RAD51DΔ3, Δ5; RAD51DΔ5, Δ9,10; RAD51DΔ9,10, Δ10; RAD51DΔ10.
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Figure 5: Expression analysis of Rad51d alternative transcripts. (A) RT-PCR for Mus musculus Rad51d and selected splice variants was conducted in eight tissues. Expression of RAD51DΔ3 employing primers Rad51d F1 and R1 (upper panel); RAD51DΔ5 with Rad51d F2 and R2 (lower panel). (B) RT-PCR products of human RAD51D in normal breast. Arrows indicate transcripts corresponding to known human splice isoforms [16]. RAD51DΔ3 and RAD51DΔ5 transcript positions are underlined. Size markers in base pairs are shown on the left. Numbers in parenthesis represent transcript abundance as determined by ImageJ analysis of the band intensities (bk; background). (C) The RAD51D peptide is aligned with the predicted amino acid sequence of RAD51DΔ9,10 and RAD51DΔ10. RAD51DΔ9,10 contains the first 8 exons of RAD51D followed by four out of frame amino acids (underlined and italicized) encoded by intron 8. RAD51DΔ10 includes amino acids 1–302 of RAD51D followed by five novel amino acids encoded by intronic sequence 5980 bp downstream from the predicted Rad51d polyA site. Premature termination codons are indicated by an asterisk, and exon boundaries are represented by a gap in the alignment of the predicted amino acid sequences. (D) RT-PCR for RAD51DΔ9,10 (upper panel) and RAD51DΔ10 (lower panel) from Mus musculus tissues with primers Rad51d F3 and Rad51dΔ9,10 R1 or Rad51dΔ10 R1 respectively. Abbreviations: 51D; RAD51D-FL, Δ3; RAD51DΔ3, Δ5; RAD51DΔ5, Δ9,10; RAD51DΔ9,10, Δ10; RAD51DΔ10.

Mentions: Mouse Rad51d alternative transcripts were originally identified in brain tissue [16]. However, the isolation of corresponding expressed sequence tags in a variety of tissues and cell types suggest that RAD51D isoforms are widely expressed (Table 1). To further investigate, RNA isolated from C57BL/6J mouse tissues were used for RT-PCR expression analysis. A series of overlapping PCRs were designed to detect all Rad51d alternative transcripts (Figure 5A). RAD51DΔ3 and RAD51DΔ5 were consistently detected in most tissues examined and their identities verified by restriction enzyme and sequence analysis (not shown). However, RAD51DΔ3 was not detected in ovarian tissue. Additionally, RAD51DΔ5 was not detected in mammary gland and testis, suggesting that these splice isoforms are differentially expressed. Because these same isoforms were identified in human adult and fetal brain cDNA libraries [16], initially named HsTRAD-d1 and HsTRAD-d4 respectively, expression analysis was performed in normal human breast tissue using primers that span from exon 2 to exon 6. Consistent with the results in mouse tissue, 21% of the detectable products corresponded to RAD51DΔ3, whereas RAD51DΔ5 was not detectable above background levels (Figure 5B).


Functional characterization and identification of mouse Rad51d splice variants.

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

Expression analysis of Rad51d alternative transcripts. (A) RT-PCR for Mus musculus Rad51d and selected splice variants was conducted in eight tissues. Expression of RAD51DΔ3 employing primers Rad51d F1 and R1 (upper panel); RAD51DΔ5 with Rad51d F2 and R2 (lower panel). (B) RT-PCR products of human RAD51D in normal breast. Arrows indicate transcripts corresponding to known human splice isoforms [16]. RAD51DΔ3 and RAD51DΔ5 transcript positions are underlined. Size markers in base pairs are shown on the left. Numbers in parenthesis represent transcript abundance as determined by ImageJ analysis of the band intensities (bk; background). (C) The RAD51D peptide is aligned with the predicted amino acid sequence of RAD51DΔ9,10 and RAD51DΔ10. RAD51DΔ9,10 contains the first 8 exons of RAD51D followed by four out of frame amino acids (underlined and italicized) encoded by intron 8. RAD51DΔ10 includes amino acids 1–302 of RAD51D followed by five novel amino acids encoded by intronic sequence 5980 bp downstream from the predicted Rad51d polyA site. Premature termination codons are indicated by an asterisk, and exon boundaries are represented by a gap in the alignment of the predicted amino acid sequences. (D) RT-PCR for RAD51DΔ9,10 (upper panel) and RAD51DΔ10 (lower panel) from Mus musculus tissues with primers Rad51d F3 and Rad51dΔ9,10 R1 or Rad51dΔ10 R1 respectively. Abbreviations: 51D; RAD51D-FL, Δ3; RAD51DΔ3, Δ5; RAD51DΔ5, Δ9,10; RAD51DΔ9,10, Δ10; RAD51DΔ10.
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Figure 5: Expression analysis of Rad51d alternative transcripts. (A) RT-PCR for Mus musculus Rad51d and selected splice variants was conducted in eight tissues. Expression of RAD51DΔ3 employing primers Rad51d F1 and R1 (upper panel); RAD51DΔ5 with Rad51d F2 and R2 (lower panel). (B) RT-PCR products of human RAD51D in normal breast. Arrows indicate transcripts corresponding to known human splice isoforms [16]. RAD51DΔ3 and RAD51DΔ5 transcript positions are underlined. Size markers in base pairs are shown on the left. Numbers in parenthesis represent transcript abundance as determined by ImageJ analysis of the band intensities (bk; background). (C) The RAD51D peptide is aligned with the predicted amino acid sequence of RAD51DΔ9,10 and RAD51DΔ10. RAD51DΔ9,10 contains the first 8 exons of RAD51D followed by four out of frame amino acids (underlined and italicized) encoded by intron 8. RAD51DΔ10 includes amino acids 1–302 of RAD51D followed by five novel amino acids encoded by intronic sequence 5980 bp downstream from the predicted Rad51d polyA site. Premature termination codons are indicated by an asterisk, and exon boundaries are represented by a gap in the alignment of the predicted amino acid sequences. (D) RT-PCR for RAD51DΔ9,10 (upper panel) and RAD51DΔ10 (lower panel) from Mus musculus tissues with primers Rad51d F3 and Rad51dΔ9,10 R1 or Rad51dΔ10 R1 respectively. Abbreviations: 51D; RAD51D-FL, Δ3; RAD51DΔ3, Δ5; RAD51DΔ5, Δ9,10; RAD51DΔ9,10, Δ10; RAD51DΔ10.
Mentions: Mouse Rad51d alternative transcripts were originally identified in brain tissue [16]. However, the isolation of corresponding expressed sequence tags in a variety of tissues and cell types suggest that RAD51D isoforms are widely expressed (Table 1). To further investigate, RNA isolated from C57BL/6J mouse tissues were used for RT-PCR expression analysis. A series of overlapping PCRs were designed to detect all Rad51d alternative transcripts (Figure 5A). RAD51DΔ3 and RAD51DΔ5 were consistently detected in most tissues examined and their identities verified by restriction enzyme and sequence analysis (not shown). However, RAD51DΔ3 was not detected in ovarian tissue. Additionally, RAD51DΔ5 was not detected in mammary gland and testis, suggesting that these splice isoforms are differentially expressed. Because these same isoforms were identified in human adult and fetal brain cDNA libraries [16], initially named HsTRAD-d1 and HsTRAD-d4 respectively, expression analysis was performed in normal human breast tissue using primers that span from exon 2 to exon 6. Consistent with the results in mouse tissue, 21% of the detectable products corresponded to RAD51DΔ3, whereas RAD51DΔ5 was not detectable above background levels (Figure 5B).

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