Protein Arginine Methyltransferase 5 (PRMT5) Mutations in Cancer Cells

Abstract

Arginine methylation is a form of posttranslational modification that regulates many cellular functions such as development, DNA damage repair, inflammatory response, splicing, and signal transduction, among others. Protein arginine methyltransferase 5 (PRMT5) is one of nine identified methyltransferases, and it can methylate both histone and non-histone targets. It has pleiotropic functions, including recruitment of repair machinery to a chromosomal DNA double strand break (DSB) and coordinating the interplay between repair and checkpoint activation. Thus, PRMT5 has been actively studied as a cancer treatment target, and small molecule inhibitors of its enzymatic activity have already been developed. In this report, we analyzed all reported PRMT5 mutations appearing in cancer cells using data from the Catalogue of Somatic Mutations in Cancers (COSMIC). Our goal is to classify mutations as either drivers or passengers to understand which ones are likely to promote cellular transformation. Using gold standard artificial intelligence algorithms, we uncovered several key driver mutations in the active site of the enzyme (D306H, L315P, and N318K). In silico protein modeling shows that these mutations may affect the affinity of PRMT5 for S-adenosylmethionine (SAM), which is required as a methyl donor. Electrostatic analysis of the enzyme active site shows that one of these mutations creates a tunnel in the vicinity of the SAM binding site, which may allow interfering molecules to enter the enzyme active site and decrease its activity. We also identified several non-coding mutations that appear to affect PRMT5 splicing. Our analyses provide insights into the role of PRMT5 mutations in cancer cells. Additionally, since PRMT5 single molecule inhibitors have already been developed, this work may uncover future directions in how mutations can affect targeted inhibition.

Keywords: arginine methylation; cancer; mutation; post-translational modification.

Figure 1

PRMT5 mutation distribution in human cancers. (A) Structure of PRMT5 protein showing conservation between S. pombe and H. sapiens. Residue conservation between the two species was determined using the NCBI Protein-BLAST tool. (B) Distribution of different types of PRMT5 mutations in human cancers. (C) Frequency of PRMT5 mutations occurring in the various protein regions. A one-sample Kolmogorov-Smirnov (K-S) test for uniformity was performed using SPSS. The Monte Carlo two-sided significance value is 0.645, indicating that mutations distribute uniformly over the PRMT5 region.

Figure 2

Mutation bias in the PRMT5 coding region. (A) The percent of substitutions for each amino acid that produce pathogenic mutations. (B) PRMT5 amino acid changes that have a statistically significant deviation from rates of change in human cancers. Chi-square test between PRMT5 and values reported by Anoosha et al. [45]. (C) Base pair substitutions in PRMT5. (D) Mutation spectrum of PRMT5 base pair substitutions in cancer cells. C/G > T/A transitions dominate the PRMT5 mutation landscape. (E) C > T transitions are the most prominent form of mutation. (F) The percent substitution of the four nucleotides occurring in the 1st, 2nd, or 3rd codon position. (G) C > T and G > A substitutions are more likely to occur in the first codon position.

Figure 3

Significant PRMT5 mutations occurring in cancer cells. (A) Mutations with a significant VEST4 value (red) or significant VEST4 and CHASM values (blue) (Supplementary Table S1). (B) Truncating PRMT5 mutations (*) that are likely to affect the function of the gene. (C) PRMT5 locus structural variations. The data extracted from COSMIC using the CONNAN function.

Figure 4

PRMT5 mutations were placed in their respective binding pockets. (A) Mutation of D306H (green to cyan) in the substrate-binding pocket. (B) Mutation of L315P (green to magenta) in a SAM binding pocket with SAM bound (green sticks). (C) Mutation of N318K (green to red) in a SAM binding pocket with SAM bound (green sticks). Figure was made by aligning the wild-type structure (green) of PRMT5 (PDB ID: 4GQB) [36] to the mutant structures using PyMOL.

Figure 5

Electrostatic comparisons between wild-type (left panels) and mutated PRMT5 (right panels) residues. Comparisons between (A) D306H wild type and mutant, (B) L315P wild type and mutant, and (C) N318K wild type and mutant. Electrostatic surface potentials were calculated and displayed in PyMOL using the APBS plug-in. This is shown using a surface representation where red is a negative charge, blue is a positive charge, and white is neutral. The electrostatic scale is shown at the bottom, ranging from −5 to +5 kT/e.

Figure 6

Change in the CASTp calculated binding pocket of wild-type PRMT5 versus mutants. (A) Wild type (left) and D306H mutant (right). (B) Wild type (left) and L315P mutant (right). Comparison of the available binding pocket (red) between the wild-type (left) and the mutant (right). The site of mutation + available binding pocket shift is indicated by the yellow ring. Protein structure is illustrated by the gray ribbon. Structure and pocket visualization were directly pulled from the CASTp server.