Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1145927 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30175125_1.fastq.gz | Fastqsanger.gz | |||
| SRR30175125_2.fastq.gz | Fastqsanger.gz | |||
| SRR30175126_1.fastq.gz | Fastqsanger.gz | |||
| SRR30175126_2.fastq.gz | Fastqsanger.gz | |||
| SRR30175127_1.fastq.gz | Fastqsanger.gz |
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Project description:Protein arginine methylations are crucial post-translational modifications (PTMs) in eukaryotes, playing a significant regulatory role in diverse biological processes. Different forms of arginine methylations are known to co-exist within densely modified arginine-rich protein regions. However, pinpointing the precise pattern of arginine methylation and their functional implications have been challenging. In this study, we employed high-resolution mass spectrometry and in vitro enzyme reactions to uncover that the RGG/RG motifs located in the C-terminal region of METTL14 undergo both asymmetric and symmetric dimethylations (aDMA and sDMA). Specifically, PRMT5 catalyses the symmetric dimethylation of residues R425 and R445 within the extensively methylated region of METTL14, whereas PRMT1 demonstrates a broader substrate specificity. Our results indicate that mutating R425 and R445 to lysines reduces the catalytic efficiency of the METTL3:METTL14 complex, implying that sDMA modifications at these residues are important for MELL14’s function. Moreover, our findings underscore the role of sDMA modifications on METTL14 in regulating m6A-specific gene expression, including BRD4, SP1, and c-MYC, in acute myeloid leukaemia (AML) cell lines. Finally, the proliferation of AML cells is more sensitive to the combined inhibition of METTL3 and PRMT5 activity than inhibiting either alone. In summary, our study delineates the methylation landscape of the C terminal region of METTL14 and shows that sDMA modifications at residues R425 and R445 modulate the m6A deposition activity of the methyltransferase complex. These insights suggest that sDMA modifications in METTL14 could represent a promising therapeutic target for the treatment of AML.
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Project description:PRMT5 is the major enzyme that catalyzes the symmetric dimethylation of arginine (sDMA) in mammalian cells. Its activity is crucial for many biological processes including transcriptional regulation, mRNA processing, as well as DNA damage and repair. However, the protein substrates identified for PRMT5 have yet been limited in numbers, hindering the understanding of PRMT5 functions in normal as well as diseased cells. Herein we developed an optimized strategy for proteomic profiling of cellular sDMA and apply it to the discovery of novel PRMT5 substrates. Our results show that a combination of proteolytic digestion by the metalloprotease ulilysin and using electron-transfer dissociation with supplemental activation as the mass spectrometry method significantly increased sDMA site identification and localization after immuno-affinity enrichment. By employing SILAC-based differential quantitative proteomic approach and a PRMT5 specific inhibitor, we identified 325 sDMA sites being regulated by PRMT5, 220 being novel sites, which greatly expanded the number of PRMT5 substrates. Biochemical studies confirmed the newly discovered PRMT5 substrate, Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1) and revealed that the RGG/RG motif in the central region of SERBP1 contains both PRMT5-catalyzed sDMA and Type I PRMT-catalyzed asymmetric dimethylation of arginine (aDMA). Unexpectedly, using the optimized methylarginine profiling strategy, we also discovered arginine trimethylation, a previously unknown type of modification, on the central RGG/RG region of SERBP1. By mutational studies we demonstrated that the trimethyl modification on R172 of SERBP1 regulates its recruitment to stress granule (SG) under oxidative stress, indicating a functional role of arginine trimethylation in the cell. Overall, the optimized profiling strategy not only enabled the identification of extensive, non-overlapping sDMA sites and novel PRMT5 substrates, but also revealed a potentially important new PTM, arginine trimethylation. The work lays the foundation for further investigations on the functional interplay of different methylation modifications on arginines, especially in the heavily methylated RGG/RG motif of many RNA-binding proteins.
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