Project description:Lysine methylation is a critical post-translational modification (PTM) involved in diverse physiological and pathological processes. Interferon regulatory factor 3 (IRF3) plays a pivotal role in antitumor immunity; however, the regulatory mechanisms and functional impact of IRF3 methylation within the tumor microenvironment remain incompletely understood. This study demonstrates that monomethylation of IRF3 at lysine 193 (K193) suppresses its phosphorylation-dependent activation. Mass spectrometry-based protein interactome analysis identified lysine methyltransferase 5A (KMT5A) as the key enzyme responsible for IRF3 K193 monomethylation. In colorectal cancer (CRC), aberrantly high expression of KMT5A impaired in vivo antitumor immune responses. Mechanistically, KMT5A catalyzes IRF3 monomethylation at K193, which impedes IRF3 phosphorylation and subsequent activation, thereby suppressing the production of type I interferons (IFN-I). Collectively, these findings elucidate KMT5A-mediated IRF3 K193 methylation as a critical regulatory axis promoting tumor immune evasion and progression. Furthermore, IRF3 K193 methylation represents a promising therapeutic target for CRC intervention.
Project description:We report our newly developed method for high-throughput sequencing of 2'-O methylation sites using positive signal and site specific approach. The pilot experiment using MiSeq generated ~2.5 million reads per sample. The 2'-O methylation site identification produced a promising distribution pattern matching known sites. For accurate and sensitive base call, >10 million reads are required per sample as evidenced in the NextSeq experiment.
Project description:Human genome encodes nine protein arginine methyltransferases (PRMT1–9), which catalyze three types of arginine methylation: monomethylation (MMA), asymmetric dimethylation (ADMA), and symmetric dimethylation (SDMA). These modifications can alter protein-protein and protein-nucleic acid interactions and play critical roles in transcription regulation and RNA metabolism. A few years ago, we characterized the newest member of the PRMT family–PRMT9 as a SDMA modifying enzyme and identified the splicing factor SF3B2 as its methylation substrate, linking its function to pre-mRNA splicing. However, the biological function of PRMT9 and the molecular mechanism by which PRMT9-catalyzed SF3B2 arginine methylation regulates pre-mRNA splicing remain largely unknown. Here, by charactering an intellectual disability patient-derived PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncovered an important function of PRMT9 in neuronal development. We found that G189R mutation completely abolishes PRMT9 methyltransferase activity and destabilizes the protein by promoting its ubiquitination and proteasome degradation. PRMT9 loss in hippocampal neurons alters RNA splicing of ~1800 transcripts, which likely account for the abnormal synapse development and impaired learning and memory observed in the Prmt9 cKO mouse. Mechanistically, we discovered a critical protein-RNA interaction between the arginine 508 (R508) of SF3B2, the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element located upstream of the branch point sequence (BPS). Additionally, we provide strong evidence that supports SF3B2 being the major and likely only substrate of PRMT9, thus highlighting the conserved function of PRMT9/SF3B2 axis in pre-mRNA splicing regulation.
Project description:The interferon regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to influenza A virus infections in Irf3-/-, Irf7-/- and Irf3-/-Irf7-/- knock-out mice.
Project description:IRF3 is one of the most critical transcription factor in down stream of pattern recognition receptors (such as toll-like receptor and RIG-I-like receptor) signalling pathway. IRF3 is known to induce the expression of type I IFN gene upon virus infection. To furter examine the role of IRF3 in virus-induced gene expression, we preformed microarray analysis in IRF3-/- peritoneal macrophages infected with VSV, and found that IRF3 suppresses the expression of Il12b gene. Peritoneal macrophages from WT of IRF3-/- B6 mice were infected with VSV(1 M.O.I. ) for 6 hous, and then subjected to microarray analysis.