Changes in Conformational Dynamics of mRNA upon AtGRP7 Binding Studied by Fluorescence Correlation Spectroscopy

Abstract

The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

Functional diversity of the plant glycine-rich proteins superfamily

“…This is the case for the cysteine-rich domain of AtGRP3 responsible for interaction with AtWAK1, 19 and the RNA-binding sequences of Class IV GRPs. [21][22][23][24][25][26][27][28][32][33][34] On the other hand, Class I GRPs that present no additional characterized domain besides a signal peptide presented functional properties. PvGRP1.8 and AtGRP5 are composed of nothing but a glycinerich domain suggesting that this domain is responsible for their function.…”

“…AtRBG7 preferentially binds to ssRNA if it is fully stretched rather than to molecules presenting a stable secondary structure. 26 AtRBG7 binds to its own pre-mRNA generating an alternativelly spliced form that is rapidly degraded. A mutation on a conserved arginine of the RRM motif of AtRBG7 prevents this autoregulation.…”

Structural basis of nucleic acid binding byNicotiana tabacumglycine-rich RNA-binding protein: implications for its RNA chaperone function

“…Interaction between Nt RRM and ssDNA or RNA was studied using a 6-nt probe (5′-TTCTGG-3′ for DNA and 5′-UUCUGG-3′ for RNA; Eurofins MWG Operon) that was previously identified as a minimum binding sequence for homologue At GR-RBP7 (34). The RNA and DNA probes were either ordered as HPLC purified (RNA) or purified over a G10 column (DNA), lyophilized and dissolved in NMR buffer to a stock concentration of 0.18 mM (RNA) or 1 mM (DNA).…”

Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules

“…Studying interactions between a RNA-binding protein (Arabidopsis thaliana glycine-rich RNA-binding protein, AtGRP7) and specific RNA sequences, we demonstrated that changes in conformational dynamics of the RNA upon protein binding can be observed and analyzed as reporter for the binding process. [93] Such experiments yield structural information about various binding modes but also serve as diagnostic tool to detect protein-nucleotide [93] as well as protein-protein [94] interactions.…”