16 research outputs found
A cryptic RNA-binding domain mediates Syncrip recognition and exosomal partitioning of miRNA targets
Exosomal miRNA transfer is a mechanism for cell-cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip's amino-terminal domain, which was previously thought to mediate protein-protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip's RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5Ⲡto this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences. Š 2018 The Author(s)
A cryptic RNA-binding domain mediates Syncrip recognition and exosomal partitioning of miRNA targets.
Exosomal miRNA transfer is a mechanism for cellâcell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncripâs amino-terminal domain, which was previously thought to mediate proteinâprotein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncripâs RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5Ⲡto this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences
IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties
Understanding p300-transcription factor interactions using sequence variation and hybridization
Lin28a uses distinct mechanisms of binding to RNA and affects positively and negatively miRNA levels
Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3â˛-5Ⲡexoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing
Activityâdirected synthesis of inhibitors of the p53/hDM2 proteinâprotein interaction
Proteinâprotein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structuralâdiverse starting points for discovery of PPI inhibitors remains a significant challenge. Activityâdirected synthesis (ADS), a functionâdriven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined smallâmolecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors
Identification of β-Strand Mediated Protein-Protein Interaction Inhibitors Using Ligand-Directed Fragment Ligation
β-Strand mediated
protein-protein interactions (PPIs) represent underexploited targets for
chemical probe development despite representing a significant proportion of
known and therapeutically relevant PPI targets. β-strand mimicry is challenging
given that both amino acid
side-chains and backbone hydrogen-bonds are typically required for
molecular recognition, yet these are oriented along perpendicular vectors. This
paper describes an alternative approach using GKAP/SHANK1 PDZ as a model and
dynamic ligation screening to identify small-molecule replacements for tranches
of peptide sequence. A peptide truncation of GKAP functionalized at the N- and
C-termini with acylhydrazone groups was used as an anchor. Reversible
acylhydrazone bond exchange with a library of aldehyde fragments in the
presence of the protein as template and in
situ screening using a fluorescence anisotropy (FA) assay identified
peptide hybrid hits with comparable affinity to the GKAP peptide binding
sequence. Identified hits were validated using FA, ITC, NMR and X-ray
crystallography to confirm selective inhibition of the target PDZ-mediated PPI
and mode of binding. These analyses together with molecular dynamics
simulations demonstrated the ligands make transient interactions with an
unoccupied basic patch through electrostatic interactions, establishing
proof-of-concept that this unbiased approach to ligand discovery represents a
powerful addition to the armory of tools that can be used to identify PPI
modulators
Predicting and Experimentally Validating Hot-Spot Residues at Protein-Protein Interfaces
Here we describe a comparative analysis of multiple CAS methods, which highlights effective approaches to improve the accuracy of predicting hot-spot residues. Alongside this, we introduce a new method, BUDE Alanine Scanning, which can be applied to single structures from crystallography, and to structural ensembles from NMR or molecular dynamics data. The comparative analyses facilitate accurate prediction of hot-spots that we validate experimentally with three diverse targets: NOXA-B/MCL-1 (an ι helix-mediated PPI), SIMS/SUMO and GKAP/SHANK-PDZ (both β strand-mediated interactions). Finally, the approach is applied to the accurate prediction of hot-residues at a topographically novel Affimer/BCL-xL protein-protein interface