title: Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids.
abstract: Abstract Analysis of arginine methylation, which affects specific protein interactions in eukaryotic cells, requires access to methylated protein for biophysical and biochemical studies. Methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon co-expression with protein arginine methyltransferase 1 in E. coli was monitored by mass spectrometry and found to be identical to the modification of hnRNP K purified from mammalian cells. Recombinant non-methylated and arginine-methylated hnRNP K (MethnRNP K) were used to characterize self-aggregation and nucleic acid binding. Analytical ultracentrifugation and static light scattering experiments revealed that hnRNP K methylation does not impact reversible self-aggregation, which can be prevented by high ionic strength and organic additives. Filter binding assays were used to compare the binding of non-methylated and MethnRNP K to the pyrimidine repeat-containing differentiation control element (DICE) of reticulocyte 15-lipoxygenase mRNA 3' UTR. No affinity differences were detected for both hnRNP K variants. A series of oligonucleotides carrying various numbers of C4 motifs at different positions was used in steady state competition assays with fluorescently-labeled functional differentiation control element (2R). Quantitative evaluation indicated that all hnRNP K homology domains of hnRNP K contribute differentially to RNA binding, with KH1-KH2 acting as a tandem domain and KH3 as an individual binding domain.
authors.forename: Bodo
authors.initials: B
authors.lastname: Moritz
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.forename: Isabel S
authors.initials: IS
authors.lastname: Naarmann-de Vries
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Elmar
authors.initials: E
authors.lastname: Wahle
authors.forename: Antje
authors.initials: A
authors.lastname: Ostareck-Lederer
authors.forename: Dirk H
authors.initials: DH
authors.lastname: Ostareck
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D004926:Escherichia coli / Q000201:enzymology; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008745:Methylation; D051379:Mice; D009696:Nucleic Acids / Q000378:metabolism*; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry / Q000378:metabolism
chemical_list: D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D009696:Nucleic Acids; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases