We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Biophysical and biochemical analysis of hnRNP K: arginine methylation, reversible aggregation and combinatorial binding to nucleic acids.
- Authors
Moritz, Bodo; Lilie, Hauke; Naarmann-de Vries, Isabel S.; Urlaub, Henning; Wahle, Elmar; Ostareck-Lederer, Antje; Ostareck, Dirk H.
- 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.
- Subjects
BIOPHYSICS; BIOCHEMISTRY; ARGININE; METHYLATION; BIOLOGICAL aggregation; NUCLEIC acids; EUKARYOTIC cells; PROTEIN-protein interactions
- Publication
Biological Chemistry, 2014, Vol 395, Issue 7/8, p837
- ISSN
1431-6730
- Publication type
Article
- DOI
10.1515/hsz-2014-0146