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Arginine methylation of hnRNPUL1 regulates interaction with NBS1 and recruitment to sites of DNA damage.

Gurunathan G, Yu Z, Coulombe Y, Masson JY, Richard S - Sci Rep (2015)

Bottom Line: Arginine methylation is a post-translational modification required for the maintenance of genomic integrity.Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo.The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1(RK). hnRNPUL1(RK) was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Molecular Oncology Group and Segal Cancer Center, Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research and Departments of Oncology and Medicine, McGill University, Montréal, Québec, Canada H3T 1E2.

ABSTRACT
Arginine methylation is a post-translational modification required for the maintenance of genomic integrity. Cells deficient in protein arginine methyltransferase 1 (PRMT1) have DNA damage signaling defects, defective checkpoint activation and extensive genomic instability. Herein we identify the DNA damage protein and RNA binding protein, hnRNPUL1, to be a substrate of PRMT1. We identify the dimethylation of R584, R618, R620, R645, and R656, as well as the monomethylation of R661 R685 and R690 within hnRNPUL1 in U2OS cells by mass spectrometry. Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo. The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1(RK). hnRNPUL1(RK) was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1. Co-immunoprecipitation studies showed that hnRNPUL1(RK) had impaired ability to associate with the DNA damage protein NBS1. Moreover, hnRNPUL1(RK) was not recruited to sites of DNA damage, unlike wild type hnRNPUL1, in the presence of transcriptional inhibitors. These findings define a role for arginine methylation during the DNA damage response to regulate protein-protein interactions for the recruitment at sites of damage.

No MeSH data available.


Related in: MedlinePlus

RGG/RG motif regulates GFP-hnRNPUL1 recruitment at breaks.A) U2OS cells were transfected with wild type GFP-hnRNPUL1 and GFP-hnRNPUL1RK and subjected to laser micro-irradiation treatment (forms a DNA damage ‘track’; white box). Exclusion of GFP-UL1 proteins from laser damage sites (2 min post-laser scissor damage) was monitored via live imaging (exclusion) or recruitment in the presence of DRB (inclusion) B) Quantification of exclusion and inclusion patterns of GFP-UL1 proteins are indicated as a percentage.
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f4: RGG/RG motif regulates GFP-hnRNPUL1 recruitment at breaks.A) U2OS cells were transfected with wild type GFP-hnRNPUL1 and GFP-hnRNPUL1RK and subjected to laser micro-irradiation treatment (forms a DNA damage ‘track’; white box). Exclusion of GFP-UL1 proteins from laser damage sites (2 min post-laser scissor damage) was monitored via live imaging (exclusion) or recruitment in the presence of DRB (inclusion) B) Quantification of exclusion and inclusion patterns of GFP-UL1 proteins are indicated as a percentage.

Mentions: hnRNPUL1 exhibits two types of dynamics at DNA damage sites depending on the presence or absence of RNA28. hnRNPUL1 is rapidly excluded from laser microirradiation sites, but in the presence of transcription inhibitors, it is effectively recruited at DNA damage tracks28. To assess the role that the RGG/RG motif in recruitment following DNA damage, we transfected U2OS cells with GFP-hnRNPUL1 or GFP- hnRNPUL1RK. The cells were examined for recruitment to laser microirradiated nuclear regions in the absence and presence of the transcriptional inhibitor, 5,6 dichloro-β-D-ribofuranosylbenzimidazole (DRB). Both GFP-hnRNPUL1 and hnRNPUL1RK were excluded from laser microirradiated nuclear regions in U2OS cells without DRB (Fig. 4). GFP-hnRNPUL1 was effectively recruited at sites of DNA damage, while GFP-hnRNPUL1RK was not recruited at these DNA damage sites with DRB treatment (Fig. 4). These findings suggest that arginine methylation of the RGG/RG motifs is required for the recruitment at sites of DNA damage in the presence of transcription inhibitors, but are not required for the exclusion of hnRNPUL1 from sites of DNA damage.


Arginine methylation of hnRNPUL1 regulates interaction with NBS1 and recruitment to sites of DNA damage.

Gurunathan G, Yu Z, Coulombe Y, Masson JY, Richard S - Sci Rep (2015)

RGG/RG motif regulates GFP-hnRNPUL1 recruitment at breaks.A) U2OS cells were transfected with wild type GFP-hnRNPUL1 and GFP-hnRNPUL1RK and subjected to laser micro-irradiation treatment (forms a DNA damage ‘track’; white box). Exclusion of GFP-UL1 proteins from laser damage sites (2 min post-laser scissor damage) was monitored via live imaging (exclusion) or recruitment in the presence of DRB (inclusion) B) Quantification of exclusion and inclusion patterns of GFP-UL1 proteins are indicated as a percentage.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4447065&req=5

f4: RGG/RG motif regulates GFP-hnRNPUL1 recruitment at breaks.A) U2OS cells were transfected with wild type GFP-hnRNPUL1 and GFP-hnRNPUL1RK and subjected to laser micro-irradiation treatment (forms a DNA damage ‘track’; white box). Exclusion of GFP-UL1 proteins from laser damage sites (2 min post-laser scissor damage) was monitored via live imaging (exclusion) or recruitment in the presence of DRB (inclusion) B) Quantification of exclusion and inclusion patterns of GFP-UL1 proteins are indicated as a percentage.
Mentions: hnRNPUL1 exhibits two types of dynamics at DNA damage sites depending on the presence or absence of RNA28. hnRNPUL1 is rapidly excluded from laser microirradiation sites, but in the presence of transcription inhibitors, it is effectively recruited at DNA damage tracks28. To assess the role that the RGG/RG motif in recruitment following DNA damage, we transfected U2OS cells with GFP-hnRNPUL1 or GFP- hnRNPUL1RK. The cells were examined for recruitment to laser microirradiated nuclear regions in the absence and presence of the transcriptional inhibitor, 5,6 dichloro-β-D-ribofuranosylbenzimidazole (DRB). Both GFP-hnRNPUL1 and hnRNPUL1RK were excluded from laser microirradiated nuclear regions in U2OS cells without DRB (Fig. 4). GFP-hnRNPUL1 was effectively recruited at sites of DNA damage, while GFP-hnRNPUL1RK was not recruited at these DNA damage sites with DRB treatment (Fig. 4). These findings suggest that arginine methylation of the RGG/RG motifs is required for the recruitment at sites of DNA damage in the presence of transcription inhibitors, but are not required for the exclusion of hnRNPUL1 from sites of DNA damage.

Bottom Line: Arginine methylation is a post-translational modification required for the maintenance of genomic integrity.Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo.The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1(RK). hnRNPUL1(RK) was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1.

View Article: PubMed Central - PubMed

Affiliation: Terry Fox Molecular Oncology Group and Segal Cancer Center, Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research and Departments of Oncology and Medicine, McGill University, Montréal, Québec, Canada H3T 1E2.

ABSTRACT
Arginine methylation is a post-translational modification required for the maintenance of genomic integrity. Cells deficient in protein arginine methyltransferase 1 (PRMT1) have DNA damage signaling defects, defective checkpoint activation and extensive genomic instability. Herein we identify the DNA damage protein and RNA binding protein, hnRNPUL1, to be a substrate of PRMT1. We identify the dimethylation of R584, R618, R620, R645, and R656, as well as the monomethylation of R661 R685 and R690 within hnRNPUL1 in U2OS cells by mass spectrometry. Moreover, we define the arginines within the RGG/RG motifs as the site of methylation by PRMT1 both in vitro and in vivo. The arginines 612, 618, 620, 639, 645, 656 and 661 within the human hnRNPUL1 RGG/RG motifs were substituted with lysines to generate hnRNPUL1(RK). hnRNPUL1(RK) was hypomethylated and lacked the ability to interact with PRMT1, unlike wild type hnRNPUL1. Co-immunoprecipitation studies showed that hnRNPUL1(RK) had impaired ability to associate with the DNA damage protein NBS1. Moreover, hnRNPUL1(RK) was not recruited to sites of DNA damage, unlike wild type hnRNPUL1, in the presence of transcriptional inhibitors. These findings define a role for arginine methylation during the DNA damage response to regulate protein-protein interactions for the recruitment at sites of damage.

No MeSH data available.


Related in: MedlinePlus