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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.
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Cyp33 binds AU-rich RNA motifs via an extended interface that competitively disrupts the gene repressive Cyp33-MLL1 interaction in vitro
Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif ( AAUAAUAA ) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.
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- Award ID(s):
- 1716425
- PAR ID:
- 10284905
- Editor(s):
- Millet, Oscar
- Date Published:
- Journal Name:
- PLOS ONE
- Volume:
- 16
- Issue:
- 2
- ISSN:
- 1932-6203
- Page Range / eLocation ID:
- e0237956
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0237956
