Human RNA‐binding motif 3 protein (RBM3) is a cold‐shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N‐terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a βαββαβ topology, which is found in many RNA‐binding proteins. NMR‐monitored titration experiments and molecular dynamic simulations show that the beta‐sheet and two loops form the RNA‐binding interface. Hydrogen bond, pi–pi and pi–cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross‐linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold‐shock protein. Temperature‐dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature‐dependent oligomerization.
The RNA recognition motif (RRM) occurs widely in RNA-binding proteins, but does not always by itself support full binding. For example, it is known that binding of SL1 RNA to the protein U1-70K in the U1 spliceosomal particle is reduced when a region flanking the RRM is truncated. How the RRM flanking regions that together with the RRM make up an ‘extended RRM’ (eRRM) contribute to complex stability and structural organization is unknown. We study the U1-70K eRRM bound to SL1 RNA by thermal dissociation and laser temperature jump kinetics; long-time molecular dynamics simulations interpret the experiments with atomistic resolution. Truncation of the helix flanking the RRM on its N-terminal side, ‘N-helix,’ strongly reduces overall binding, which is further weakened under higher salt and temperature conditions. Truncating the disordered region flanking the RRM on the C-terminal side, ‘C-IDR’, affects the local binding site. Surprisingly, all-atom simulations show that protein truncation enhances base stacking interactions in the binding site and leaves the overall number of hydrogen bonds intact. Instead, the flanking regions of the eRRM act in a distributed fashion via collective interactions with the RNA when external stresses such as temperature or high salt mimicking osmotic imbalance are applied.
more » « less- PAR ID:
- 10369198
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 14
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 8193-8206
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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