The interactome of Cryptococcus neoformans Rmt5 reveals multiple regulatory points in fungal cell biology and pathogenesis

Protein arginine methylation is a key post-translational modification in eukaryotes that modulates core cellular processes, including translation, morphology, transcription, and RNA fate. However, this has not been explored in Cryptococcus neoformans, a human-pathogenic basidiomycetous encapsulated fungus. We characterized the five protein arginine methyltransferases in C. neoformans and highlight Rmt5 as critical regulator of cryptococcal morphology and virulence. An rmt5∆ mutant was defective in thermotolerance, had a remodeled cell wall, and exhibited enhanced growth in an elevated carbon dioxide atmosphere and in chemically induced hypoxia. We revealed that Rmt5 interacts with post-transcriptional gene regulators, such as RNA-binding proteins and translation factors. Further investigation of the rmt5∆ mutant showed that Rmt5 is critical for the homeostasis of eIF2α and its phosphorylation state following 3-amino-1,2,4-triazole-induced ribosome stalling. RNA sequencing of one rmt5∆ clone revealed stable chromosome 9 aneuploidy that was ameliorated by complementation but did not impact the rmt5∆ phenotype. As a result of these diverse interactions and functions, loss of RMT5 enhanced phagocytosis by murine macrophages and attenuated disease progression in mice. Taken together, our findings link arginine methylation to critical cryptococcal cellular processes that impact pathogenesis, including post-transcriptional gene regulation by RNA- binding proteins.

Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

PRMT6 physically associates with nuclear factor Y to regulate photoperiodic flowering in Arabidopsis

The timing of floral transition is critical for reproductive success in flowering plants. In long-day (LD) plant Arabidopsis, the floral regulator gene FLOWERING LOCUS T (FT) is a major component of the mobile florigen. FT expression is rhythmically activated by CONSTANS (CO), and specifically accumulated at dusk of LDs. However, the underlying mechanism of adequate regulation of FT transcription in response to day-length cues to warrant flowering time still remains to be investigated. Here, we identify a homolog of human protein arginine methyltransferases 6 (HsPRMT6) in Arabidopsis, and confirm AtPRMT6 physically interacts with three positive regulators of flowering Nuclear Factors YC3 (NF-YC3), NF-YC9, and NF-YB3. Further investigations find that AtPRMT6 and its encoding protein accumulate at dusk of LDs. PRMT6-mediated H3R2me2a modification enhances the promotion of NF-YCs on FT transcription in response to inductive LD signals. Moreover, AtPRMT6 and its homologues proteins AtPRMT4a and AtPRMT4b coordinately inhibit the expression of FLOWERING LOCUS C, a suppressor of FT. Taken together, our study reveals the role of arginine methylation in photoperiodic pathway and how the PRMT6-mediating H3R2me2a system interacts with NF-CO module to dynamically control FT expression and facilitate flowering time.

Structure, Activity and Function of the PRMT2 Protein Arginine Methyltransferase

PRMT2 belongs to the protein arginine methyltransferase (PRMT) family, which catalyzes the arginine methylation of target proteins. As a type I enzyme, PRMT2 produces asymmetric dimethyl arginine and has been shown to have weak methyltransferase activity on histone substrates in vitro, suggesting that its authentic substrates have not yet been found. PRMT2 contains the canonical PRMT methylation core and a unique Src homology 3 domain. Studies have demonstrated its clear implication in many different cellular processes. PRMT2 acts as a coactivator of several nuclear hormone receptors and is known to interact with a multitude of splicing-related proteins. Furthermore, PRMT2 is aberrantly expressed in several cancer types, including breast cancer and glioblastoma. These reports highlight the crucial role played by PRMT2 and the need for a better characterization of its activity and cellular functions.

Arginine Methyltransferase PRMT5 Methylates and Destabilizes Mxi1 to Confer Radioresistance in Lung Cancer

BackgroundRadioresistance is regarded as the main cause of local recurrence and distant metastasis in lung cancer. However, the underlying mechanisms of radioresistance remain incompletely understood. This study investigates the roles and regulatory mechanisms of arginine methyltransferase PRMT5 in lung cancer radioresistance.MethodsImmunoprecipitation assay and GST pulldown were used to detect the protein-protein interaction. The methylation of Mxi1 was determined by in vivo and in vitro arginine methylation assays. In vivo ubiquitination and CHX chase assays were performed to examine the stability of Mxi1. The biological effects of PRMT5 and its specific inhibitor EPZ015666 in lung cancer were evaluated both in vitro and in vivo.ResultsWe show that the arginine methyltransferase PRMT5 interacts with and methylates Mxi1, which promotes the binding of the β-Trcp ligase to Mxi1, facilitating the ubiquitination and degradation of Mxi1 in lung cancer. Furthermore, genetic blockade of PRMT5 impairs DNA damage repair and enhances lung cancer radiosensitivity in vitro and in vivo, and these phenotypes are partially reversed by Mxi1 silencing. More importantly, pharmacological inhibition of PRMT5 with the specific inhibitor EPZ015666 leads to extraordinary radiosensitization in vitro and in vivo in lung cancer.ConclusionsOur data indicate that PRMT5 methylates and destabilizes Mxi1 to confer radioresistance, suggesting that PRMT5 may be a promising radiosensitization target in lung cancer.

Targeting PRMT9 Suppresses Acute Myeloid Leukemia Maintenance

AML is a heterogenous disease in which prognosis and treatment is determined by recurrent genetic mutations and chromosomal abnormalities. Some genetic alterations result in aberrant RNA translation, which is exploited by leukemia stem cells (LSCs) to produce short-lived oncoproteins (c-Myc, Mcl-1) to promote cell survival. Since LSCs are considered as the source of treatment failure and relapse, it is critical to understand how LSCs hijack the translational machinery in order to develop effective therapeutics in AML. Arginine methylation catalyzed by protein arginine methyltransferases 1-9 (PRMT1-9) regulates various activities, including RNA translation. We revealed that PRMT1 over activation contributes to leukemia maintenance (Blood, 2019; Blood, 2019). Unlike PRMT1, the recently described PRMT9, is not defined in leukemia. Follow up of AML patients from existing datasets (GSE12417, TARGET) confirms that patients with higher levels of PRMT9 correlates with decreased overall survival. In our AML cohort, we observed significantly increased PRMT9 mRNA levels in AML CD34 + cells relative to normal counterparts (Fig. 1A), spurring our interest in evaluating its function in AML. We thus developed a Prmt9 conditional KO mouse (Mx1-Cre/Prmt9 f/f) and crossed it with an MLL-AF9 (MA9) knock-in mouse to generate Prmt9-KO/MA9 mice for further transplant assay. As a result, Prmt9-KO significantly delayed leukemogenesis in recipient mice carrying MA9 transplants evidenced by prolonged survival (Fig. 1B). Prmt9-KO impaired leukemia-initiating activity evidenced by secondary transplantation that significant less residual CD45.2 + MA9 leukemia cells were found in BM of secondary recipients receiving Prmt9-KO cells relative to those of Prmt9-WT controls. We next validated PRMT9 function in human AML. PRMT9-KD potentially impaired survival of primary AML CD34 + cells, whereas large sparing normal counterparts (Fig. 1C). Moreover, we designed a WT PRMT9 construct and corresponding catalytically dead mutant, both resistant to shPRMT9 (WT-R and MUT-R). Unlike MUT-R, WT-R expression rescued survival effects seen following shPRMT9 treatment, indicating that PRMT9 catalytic activity is required for AML survival (Fig. 1D). To define PRMT9 downstream effectors, we searched for methylated substrates in Molm13 with inducible PRMT9-KD or corresponding controls by performing SILAC analysis coupled with quantitative Mass-Spec (MS) (Fig. 1E). Complete analyses of normalized methyl peptides SILAC ratios revealed more prominent down-regulation of R-methylation in PRMT9-KD cells, with 49 methyl sites downregulated. Notably, methylated PAPB1 C-terminus peptide containing R493 was most significantly depleted upon PRMT9-KD. PABP1 potentiates translation initiation by binding to the poly(A) mRNA tail and interacting with factors like eIF4G. We thus generated an anti-R493 methylation antibody. Indeed, PRMT9 was confirmed to catalyze R493 methylation through in-vitro and cellular methylation assays (Fig. 1F, G). To define the function of R493 methylation, we ectopically expressed either WT PABP1 or the R493K mutant in Molm13 cells and then engineered those cells to express shPABP1 to KD endogenous PABP1. Notably, unlike WT PABP1, expression of R493K suppressed protein synthesis and increased apoptosis after endogenous PABP1-KD, suggesting that PRMT9 mediated PABP1-R493 methylation promotes AML viability (Fig. 1H, I). To discover PRMT9 inhibitors, we conducted a structure-based virtual screen of 960,000 compounds from NCI and Zinc compound libraries (Fig. 1J). We requested the top 300 candidates to assess their anti-leukemia activity in Molm13 cells (Fig. 1K). The top 20 most effective compounds were further analyzed via PRMT9 methylation assay (R493 antibody detection). NSC641396 exhibited the most potent PRMT9 inhibitory effects, as evidenced by decreased PABP1 R493 methylation levels at a dose <1 µΜ (Fig. 1L). NSC641396 treatment on AML CD34 + cells altered polysome profiling, decreased protein biosynthesis and reduced levels of short-lived proteins, suggesting translation inhibition (Fig. 1M). Taken together, our results suggest that PRMT9 plays an oncogenic role in AML or LSC maintenance, by promoting PABP1 methylation mediated translation activity. Further studies are needed to explore if targeting PRMT9 with newly identified lead compound ablates LSCs activity. Figure 1 Figure 1. Disclosures Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair

Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.

PRMT7: A Pivotal Arginine Methyltransferase in Stem Cells and Development

Protein arginine methylation is a posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs), which play critical roles in many biological processes. To date, nine PRMT family members, namely, PRMT1, 2, 3, 4, 5, 6, 7, 8, and 9, have been identified in mammals. Among them, PRMT7 is a type III PRMT that can only catalyze the formation of monomethylarginine and plays pivotal roles in several kinds of stem cells. It has been reported that PRMT7 is closely associated with embryonic stem cells, induced pluripotent stem cells, muscle stem cells, and human cancer stem cells. PRMT7 deficiency or mutation led to severe developmental delay in mice and humans, which is possibly due to its crucial functions in stem cells. Here, we surveyed and summarized the studies on PRMT7 in stem cells and development in mice and humans and herein provide a discussion of the underlying molecular mechanisms. Furthermore, we also discuss the roles of PRMT7 in cancer, adipogenesis, male reproduction, cellular stress, and cellular senescence, as well as the future perspectives of PRMT7-related studies. Overall, PRMT7 mediates the proliferation and differentiation of stem cells. Deficiency or mutation of PRMT7 causes developmental delay, including defects in skeletal muscle, bone, adipose tissues, neuron, and male reproduction. A better understanding of the roles of PRMT7 in stem cells and development as well as the underlying mechanisms will provide information for the development of strategies for in-depth research of PRMT7 and stem cells as well as their applications in life sciences and medicine.

PRMT5 in T cells drives Th17 responses, mixed granulocytic inflammation and severe allergic airway inflammation

Severe asthma is characterized by steroid insensitivity and poor symptom control, and is responsible for the majority of asthma-related hospital costs. Therapeutic options remain limited, in part due to a lack of mechanisms driving severe asthma phenotypes. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in asthmatic lungs. Here, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and granulocytic lung inflammation, pathology, airway remodeling and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, ROR-γt and Th17 responses, with PRMT5-dependent increases in ROR-γt agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.