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TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

Harley ME, Murina O, Leitch A, Higgs MR, Bicknell LS, Yigit G, Blackford AN, Zlatanou A, Mackenzie KJ, Reddy K, Halachev M, McGlasson S, Reijns MA, Fluteau A, Martin CA, Sabbioneda S, Elcioglu NH, Altmüller J, Thiele H, Greenhalgh L, Chessa L, Maghnie M, Salim M, Bober MB, Nürnberg P, Jackson SP, Hurles ME, Wollnik B, Stewart GS, Jackson AP - Nat. Genet. (2015)

Bottom Line: Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism.TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes.Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, UK.

ABSTRACT
DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

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TRAIP mutations result in reduced cellular levels of TRAIP protein(a) The Arg185* mutation severely reduces TRAIP transcript levels in P1 and P2 patient cell lines. RT-PCR using primers in 5′ and 3′ UTR to amplify TRAIP transcripts in primary fibroblasts and lymphoblastoid cell lines (LCLs) demonstrates marked decrease in full length TRAIP transcript, consistent with nonsense-mediated decay. Additional low intensity PCR products are evident, that represent alternatively spliced transcripts, confirmed by subcloning and Sanger sequencing (lower panels). These include transcripts, which through omission of exon 7, or exons 6, 7 and 8, retain an open reading frame and result in protein products with small internal deletions. Loading control, GAPDH. (b) TRAIP protein levels are reduced in patients with Arg185* and the Arg18Cys mutations. Immunoblotting with an affinity purified rabbit anti-TRAIP antibody raised against recombinant TRAIP protein demonstrates reduced levels of the 53 kDa TRAIP protein in P3, and marked depletion in P1 and P2 where protein is only detectable on prolonged exposure (Supplementary Fig. 2). Loading controls, actin and vinculin.
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Figure 2: TRAIP mutations result in reduced cellular levels of TRAIP protein(a) The Arg185* mutation severely reduces TRAIP transcript levels in P1 and P2 patient cell lines. RT-PCR using primers in 5′ and 3′ UTR to amplify TRAIP transcripts in primary fibroblasts and lymphoblastoid cell lines (LCLs) demonstrates marked decrease in full length TRAIP transcript, consistent with nonsense-mediated decay. Additional low intensity PCR products are evident, that represent alternatively spliced transcripts, confirmed by subcloning and Sanger sequencing (lower panels). These include transcripts, which through omission of exon 7, or exons 6, 7 and 8, retain an open reading frame and result in protein products with small internal deletions. Loading control, GAPDH. (b) TRAIP protein levels are reduced in patients with Arg185* and the Arg18Cys mutations. Immunoblotting with an affinity purified rabbit anti-TRAIP antibody raised against recombinant TRAIP protein demonstrates reduced levels of the 53 kDa TRAIP protein in P3, and marked depletion in P1 and P2 where protein is only detectable on prolonged exposure (Supplementary Fig. 2). Loading controls, actin and vinculin.

Mentions: We obtained cell lines from all three patients and characterized these to confirm the deleterious effect of mutations on the TRAIP protein. The Arg185* mutation introduces a premature stop codon in exon 7 and would be expected to cause nonsense-mediated decay of the TRAIP transcript. RT-PCR was therefore performed on RNA extracted from patient cells to assess transcript levels. This demonstrated marked reduction in TRAIP mRNA both in the immortalized lymphoblastoid cell line from P1 and a primary fibroblast cell line from P2 (Fig. 2a). Notably, low levels of residual full-length mRNA as well as alternatively spliced transcripts were evident. Cloning and sequencing of these RT-PCR products demonstrated that the latter included transcripts that are predicted to produce in-frame deletions of the TRAIP protein of 37 or 99 amino acids (Fig. 2a).


TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

Harley ME, Murina O, Leitch A, Higgs MR, Bicknell LS, Yigit G, Blackford AN, Zlatanou A, Mackenzie KJ, Reddy K, Halachev M, McGlasson S, Reijns MA, Fluteau A, Martin CA, Sabbioneda S, Elcioglu NH, Altmüller J, Thiele H, Greenhalgh L, Chessa L, Maghnie M, Salim M, Bober MB, Nürnberg P, Jackson SP, Hurles ME, Wollnik B, Stewart GS, Jackson AP - Nat. Genet. (2015)

TRAIP mutations result in reduced cellular levels of TRAIP protein(a) The Arg185* mutation severely reduces TRAIP transcript levels in P1 and P2 patient cell lines. RT-PCR using primers in 5′ and 3′ UTR to amplify TRAIP transcripts in primary fibroblasts and lymphoblastoid cell lines (LCLs) demonstrates marked decrease in full length TRAIP transcript, consistent with nonsense-mediated decay. Additional low intensity PCR products are evident, that represent alternatively spliced transcripts, confirmed by subcloning and Sanger sequencing (lower panels). These include transcripts, which through omission of exon 7, or exons 6, 7 and 8, retain an open reading frame and result in protein products with small internal deletions. Loading control, GAPDH. (b) TRAIP protein levels are reduced in patients with Arg185* and the Arg18Cys mutations. Immunoblotting with an affinity purified rabbit anti-TRAIP antibody raised against recombinant TRAIP protein demonstrates reduced levels of the 53 kDa TRAIP protein in P3, and marked depletion in P1 and P2 where protein is only detectable on prolonged exposure (Supplementary Fig. 2). Loading controls, actin and vinculin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697364&req=5

Figure 2: TRAIP mutations result in reduced cellular levels of TRAIP protein(a) The Arg185* mutation severely reduces TRAIP transcript levels in P1 and P2 patient cell lines. RT-PCR using primers in 5′ and 3′ UTR to amplify TRAIP transcripts in primary fibroblasts and lymphoblastoid cell lines (LCLs) demonstrates marked decrease in full length TRAIP transcript, consistent with nonsense-mediated decay. Additional low intensity PCR products are evident, that represent alternatively spliced transcripts, confirmed by subcloning and Sanger sequencing (lower panels). These include transcripts, which through omission of exon 7, or exons 6, 7 and 8, retain an open reading frame and result in protein products with small internal deletions. Loading control, GAPDH. (b) TRAIP protein levels are reduced in patients with Arg185* and the Arg18Cys mutations. Immunoblotting with an affinity purified rabbit anti-TRAIP antibody raised against recombinant TRAIP protein demonstrates reduced levels of the 53 kDa TRAIP protein in P3, and marked depletion in P1 and P2 where protein is only detectable on prolonged exposure (Supplementary Fig. 2). Loading controls, actin and vinculin.
Mentions: We obtained cell lines from all three patients and characterized these to confirm the deleterious effect of mutations on the TRAIP protein. The Arg185* mutation introduces a premature stop codon in exon 7 and would be expected to cause nonsense-mediated decay of the TRAIP transcript. RT-PCR was therefore performed on RNA extracted from patient cells to assess transcript levels. This demonstrated marked reduction in TRAIP mRNA both in the immortalized lymphoblastoid cell line from P1 and a primary fibroblast cell line from P2 (Fig. 2a). Notably, low levels of residual full-length mRNA as well as alternatively spliced transcripts were evident. Cloning and sequencing of these RT-PCR products demonstrated that the latter included transcripts that are predicted to produce in-frame deletions of the TRAIP protein of 37 or 99 amino acids (Fig. 2a).

Bottom Line: Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism.TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes.Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

View Article: PubMed Central - PubMed

Affiliation: MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, UK.

ABSTRACT
DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Show MeSH
Related in: MedlinePlus