A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry

Abstract

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high‐resolution mass spectrometry, a total of 147 symmetric dimethyl‐arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH‐type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.

Proteomic identification of arginine-methylated proteins in colon cancer cells and comparison of messenger RNA expression between colorectal cancer and adjacent normal tissues

“…These results strongly support that CRC tumors and adjacent noncancerous tissues are a good source for further identification and quantification of arginine methylation in proteins. Recently, we reported a number of MMA, ADMA, and SDMA sites in CRC tissues at the proteome level using immunoenrichment of arginine-methylated peptides combined with high-resolution liquid chromatography-MS/MS [20,21]. There was a significant presence of methyl-arginine residues on nucleic acid binding proteins, protein complexes involved in transcription, and enzymes [21,21].…”

“…Recently, we reported a number of MMA, ADMA, and SDMA sites in CRC tissues at the proteome level using immunoenrichment of arginine-methylated peptides combined with high-resolution liquid chromatography-MS/MS [20,21]. There was a significant presence of methyl-arginine residues on nucleic acid binding proteins, protein complexes involved in transcription, and enzymes [21,21]. Among the 7 proteins identified in the present study, HSPA8 and CCT7 were also identified as MMA-containing proteins in CRC tissues [20,21].…”

“…Recently, we reported that CRC tissue maintains significantly higher level of symmetric arginine dimethylation compared to matched noncancerous tissue by quantitative analysis [21]. Among the proteins showing elevated symmetric arginine modification, several SDMA-containing proteins were overexpressed in CRC tissues relative to paired noncancerous tissues, indicating that elevated arginine methylation in CRC tissues was mainly due to overexpression of the substrate proteins [21]. Using 3 independent microarray datasets from the NCBI GEO database, we compared the mRNA expression of the 7 methyl-accepting proteins between CRC and adjacent normal tissues.…”

“…Using this approach, we identified 455 MMA sites in 272 proteins and 314 ADMA sites in 155 proteins from CRC tissues acquired from patients [20]. In addition, we identified 147 SDMA sites in 94 proteins, and quantitative analysis comparing CRC and normal tissues revealed a significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of 4 arginine sites in 4 proteins [21]. However, this approach requires large amount of protein (10 mg per 1 type of arginine modification).…”

Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

“…Therefore, unveiling the full scope of their substrates is key to understanding their functions and underlying molecular mechanisms. Among the members of the PRMT family, PRMT1 and PRMT5 have been suggested to contribute to the formation of most of aDMA and sDMA in human cells, respectively 27,28 . The global identification of the substrates of PRMT1, PRMT4, and PRMT5 has been reported in several studies, thus greatly increasing our understanding of arginine methylation and its cellular functions [6][7][8] .…”