Project description:Protein arginine methylation, catalyzed by the protein arginine methyltransferase (PRMT) family, is recognized as a widespread post-translational modification (PTM) with implications in a plethora of biological processes in eukaryotes. PRMT proteins were classified into three types, type I, II and III, according to the final methyl-arginine products generated. Despite that thousands of substrates have been identified for Type I and II PRMTs, a full scope of arginine methylation catalyzed by the only type III PRMT, PRMT7, as well as its connection with that of type I and II PRMTs remain unknown, limiting our understanding of the network of arginine methylation and its functions in cells. In this study, global profiling of PRMT4 (type I), PRMT5 (type II) and PRMT7 (type III) substrates (also referred as methylome) revealed that PRMT7 methylated a GAR (glycine and arginine) motif similar as PRMT5, while PRMT4 uniquely methylated a motif in which proline was highly enriched. PRMT4, 5 and 7-methylome were all enriched with proteins functioning in mRNA splicing, and knockdown of PRMT4, 5 or 7 led to a global change of alternative splicing events, among which a cohort with implications in cancer development was co-regulated by all three PRMTs. PRMT4, 5 and 7-mediated arginine methylation on splicing factors such as hnRNPA1, though at different status, were shown to enhance RNA binding in general and participate in the regulation of PRMT4, 5 and 7 co-regulated alternative splicing events.The clinical significance of PRMT4, 5 and 7-mediated arginine methylation was underscored by the fact that these three PRMTs as well as hnRNPA1 arginine methylation were over-represented in multiple types of cancers, which were associated with aberrant gene alternative splicing. Consistently, silencing or pharmacological inhibition of PRMT4, 5 and 7 altered gene alternative splicing and suppressed cancer cell growth, and co-treatment exhibited synergistic effects. Taken together, our study provided an integrative view of substrates for the three types of PRMTs, revealing the important function of arginine methylation in RNA splicing and cancer.

Project description:Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine/glycine-dependent creatine biosynthesis. RNA was extracted by RNAeasy kits (Qiagen) from the MCF7 or PC3 cells which exposed to the control full DMEM or deprived one (or all) amino acid media for 24 or 48 hours.

Project description:Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine/glycine-dependent creatine biosynthesis.

			Action	DRS
	P20160929-04_FTMSms2hcd.RAW	Raw
	P20160929-05_FTMSms2hcd.RAW	Raw
	P20160929-06_FTMSms2hcd.RAW	Raw
	P20160929-08_FTMSms2hcd.RAW	Raw
	P20160929-09_FTMSms2hcd.RAW	Raw

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The GAR Domain Determines Subcellular Localization of the Growth Controlling RNA Binding Protein Nucleolin

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