title: Structural and biochemical analysis of the Hordeum vulgare L. HvGR-RBP1 protein, a glycine-rich RNA-binding protein involved in the regulation of barley plant development and stress response.
abstract: The timing of whole-plant senescence influences important agricultural traits such as yield and grain protein content. Post-transcriptional regulation by plant RNA-binding proteins is essential for proper control of gene expression, development, and stress responses. Here, we report the three-dimensional solution NMR structure and nucleic acid-binding properties of the barley glycine-rich RNA-binding protein HvGR-RBP1, whose transcript has been identified as being >45-fold up-regulated in early-as compared to late-senescing near-isogenic barley germplasm. NMR analysis reveals that HvGR-RBP1 is a multidomain protein comprising a well-folded N-terminal RNA Recognition Motif (RRM) and a structurally disordered C-terminal glycine-rich domain. Chemical shift differences observed in 2D (1)H-(15)N correlation (HSQC) NMR spectra of full-length HvGR-RBP1 and N-HvGR-RBP1 (RRM domain only) suggest that the two domains can interact both in-trans and intramolecularly, similar to what is observed in the tobacco NtGR-RBP1 protein. Further, we show that the RRM domain of HvGR-RBP1 binds single-stranded DNA nucleotide fragments containing the consensus nucleotide sequence 5'-TTCTGX-3' with low micromolar affinity in vitro. We also demonstrate that the C-terminal glycine-rich (HvGR) domain of Hv-GR-RBP1 can interact nonspecifically with ssRNA in vitro. Structural similarities with other plant glycine-rich RNA-binding proteins suggest that HvGR-RBP1 may be multifunctional. Based on gene expression analysis following cold stress in barley and E. coli growth studies following cold shock treatment, we conclude that HvGR-RBP1 functions in a manner similar to cold-shock proteins and harbors RNA chaperone activity. HvGR-RBP1 is therefore not only involved in the regulation of barley development including senescence, but also functions in plant responses to environmental stress.
authors.affiliation: Department of Chemistry and Biochemistry and §Department of Plant Sciences and Plant Pathology, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Tripet
authors.forename: Katelyn E
authors.initials: KE
authors.lastname: Mason
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Eilers
authors.forename: Jennifer
authors.initials: J
authors.lastname: Burns
authors.forename: Paul
authors.initials: P
authors.lastname: Powell
authors.forename: Andreas M
authors.initials: AM
authors.lastname: Fischer
authors.forename: Valérie
authors.initials: V
authors.lastname: Copié
mesh_terms: D058639:Cold-Shock Response / Q000502:physiology*; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001467:Hordeum / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018744:DNA, Plant; D004277:DNA, Single-Stranded; D010940:Plant Proteins; D018749:RNA, Plant; D016601:RNA-Binding Proteins; C105523:grp3 protein, Hordeum vulgare