NEW YORK (GenomeWeb) – University of Copenhagen researchers have completed one of the largest characterizations of arginine methylation to date.
In work published last week in Science Signaling, the researchers identified more than 8,000 arginine methylation sites across 3,000-plus human proteins, suggesting the modification occurs with similar frequency as more commonly studied post-translational modifications like phosphorylation and ubiquitination.
The study also found a more extensive role for the modification in various RNA-related processes than had previously been identified, Michael Nielsen, a University of Copenhagen researcher and senior author on the paper, told GenomeWeb. He added that, given the role of somatic mutations of RNA-associated proteins in various cancers, this suggests arginine methylation could play a regulatory role in certain forms of the disease.
Past research has identified roles for the modification in various protein-protein, protein-RNA, and protein-DNA interactions. However, Nielsen said, technical limitations have hindered large-scale identification of arginine methylation sites.
Among the key challenges, he noted, is the fact that "the most abundant arginine methylation sites belong to abundant RNA-binding proteins." The high abundance of these proteins makes it difficult to detect arginine methylation on lower-abundance proteins.
To access these lower-abundance modified proteins, Nielsen and his colleagues used a combination of high-pH prefractionation with immunoenrichment of arginine monomethylated peptides. This enrichment process allowed the researchers to focus their efforts on arginine-methylated peptides only, upping the sensitivity of their analysis.
Improvements in mass spec instrumentation also helped the researchers expand their coverage. "Parts of the reported improvements are certainly due to the overall improvements in speed and sensitivity of the latest generations of mass spectrometers, which in turn allows for a deeper and broader identification of any investigated PTM," Nielsen said.
His team used a Thermo Fisher Scientific Q Exactive HF for the study, running each of the antibody-enriched fractions on a 77-minute LC gradient, allowing for analysis of the entire sample in roughly 22 hours.
Looking at HEK293 cells, the researchers identified 8,030 arginine methylation sits on 3,300 proteins. Previous analyses had topped out at around 1,000 modification sites, Neilsen said, adding that the researchers estimated their characterization covered around half of the human arginine methylome.
"Further improvements in technology and methodology may still be required to completely characterize every single arginine methylated protein in a human cell," he said.
In addition to improved depth, the analysis offered good reliability, with 73 percent of sites identified in at least two independent replications. More than 80 percent of the identified sites were novel, based on comparisons to publicly available databases. Roughly 50 percent of modified proteins identified in the analysis had more than one arginine methylation site, while 3.7 percent contained more than 10 sites.
Comparing the presence of arginine methylation to that of other common modifications, the researchers found that, within the 3,300 modified proteins they identified, approximately 7 percent of all arginines were methylated. The authors noted that this is similar to phosphorylation, where roughly 9 percent of all serines and 3 percent of all threonines are phosphorylated according to figures from public phosphoproteomes databases; and ubiquitination, where around 7 percent of lysine residues are ubiquitylated.
Generally speaking, the findings indicate arginine methylation is involved in a broader range of cellular processes than had been previously demonstrated, Nielsen said. While the modification has long been implicated in various RNA processes, he and his colleagues also found it present on "proteins involved in other cellular processes such as endocytosis, DNA replication, and insulin signaling," he said.
And within RNA-related processes, the analysis found a wider role for the modification than was previously understood.
"It has been known for many years that arginine methylation plays a functional role in various RNA processes," Nielsen said. "However, the cellular extent of the modification in RNA-associated processes has not fully been appreciated until now."
He noted that he and his colleagues found that proteins linked to the spliceosome, RNA degradation, and RNA transportation were highly modified by arginine methylation. Additionally, the researchers identified arginine methylation modifying several RNA processing complexes that had not previously been linked to the modification.
One notable example was the nuclear pore complex, which aids the transport of molecules across the nuclear envelope. The study authors found that this complex had high levels of arginine methylation, a finding that, they noted, "supports a yet-uncharacterized function for arginine methylation in regulating shuttling of RNA and proteins between the cytoplasm and nucleus."
Additionally, they found arginine methylation of tRNA synthetases, which had not been previously observed, Nielsen said, adding that this suggests the modification "may play a functional role in this process."
The researchers also analyzed the frequency of somatic mutations at arginine methylation sites. Arginine residues, they noted, are more frequently targeted by somatic mutations compared to any other amino acid, according to data from the Catalogue of Somatic Mutations in Cancer.
The Science Signaling study found that these mutations targeted methylated arginines less frequently than unmodified arginines, though these modified sites were still targeted at a higher rate than other amino acids. Looking at the different roles of the targeted proteins, they found that proteins targeted by mutations at arginine methylation sites were enriched for gene expression and RNA metabolism functions.
"With RNA-associated proteins known to be targeted by somatic mutations in various types of cancers, our data supports an imperative regulatory function of arginine methylation in cancer," Nielsen said.
Nielsen added that he and his colleagues are continuing to investigate the modification across a range of projects. One current aim is to move beyond their HEK293 cell analysis to study the tissue specificity of arginine methylation in a variety of different mammalian cells.
The researchers are working to better understand the functional role of arginine methylation in the various cellular processes where they observed its influence. Additionally, they are looking at the different roles and functional consequences of the nine protein arginine methyltransferases that catalyze the modification.