Conformation-dependent ligand hot spots in the spliceosomal RNA helicase BRR2.

The conversion of hits to leads in drug discovery involves the elaboration of chemical core structures to increase their potency. In fragment-based drug discovery, low-molecular-weight compounds are tested for protein binding and are subsequently modified, with the tacit assumption that the binding mode of the original hit will be conserved among the derivatives. However, deviations from binding mode conservation are rather frequently observed, but potential causes of these alterations remain incompletely understood. Here, two crystal forms of the spliceosomal RNA helicase BRR2 were employed as a test case to explore the consequences of conformational changes in the target protein on the binding behaviour of fragment derivatives. The initial fragment, sulfaguanidine, bound at the interface between the two helicase cassettes of BRR2 in one crystal form. Second-generation compounds devised by structure-guided docking were probed for their binding to BRR2 in a second crystal form, in which the original fragment-binding site was altered due to a conformational change. While some of the second-generation compounds retained binding to parts of the original site, others changed to different binding pockets of the protein. A structural bioinformatics analysis revealed that the fragment-binding sites correspond to predicted binding hot spots, which strongly depend on the protein conformation. This case study offers an example of extensive binding-mode changes during hit derivatization, which are likely to occur as a consequence of multiple binding hot spots, some of which are sensitive to the flexibility of the protein.

Recruitment of a splicing factor to the nuclear lamina for its inactivation.

Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments.

Long-range allostery mediates cooperative adenine nucleotide binding by the Ski2-like RNA helicase Brr2.

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing.

The inactive C-terminal cassette of the dual-cassette RNA helicase BRR2 both stimulates and inhibits the activity of the N-terminal helicase unit.

The RNA helicase bad response to refrigeration 2 homolog (BRR2) is required for the activation of the spliceosome before the first catalytic step of RNA splicing. BRR2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the N-terminal cassette of BRR2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudoenzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N- and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of BRR2 in complex with an activating domain of the spliceosomal Prp8 protein at 2.4 Å resolution compared with the crystal structure of BRR2 alone. Likewise, inspection of BRR2 structures within spliceosomal complexes revealed that the cassettes occupy different relative positions and engage in different intercassette contacts during different splicing stages. Engineered disulfide bridges that locked the cassettes in two different relative orientations had opposite effects on the RNA-unwinding activity of the N-terminal cassette, with one configuration enhancing and the other configuration inhibiting RNA unwinding compared with the unconstrained protein. Moreover, we found that differences in relative positioning of the cassettes strongly influence RNA-stimulated ATP hydrolysis by the N-terminal cassette. Our results indicate that the inactive C-terminal cassette of BRR2 can both positively and negatively affect the activity of the N-terminal helicase unit from a distance.

RNAi-mediated knockdown of the Rhau helicase preferentially depletes proteins with a Guanine-quadruplex motif in the 5'-UTR of their mRNA.

Guanine-quadruplex (G-quadruplex) structures in mRNAs have been shown to modulate gene expression. However, the overall biological relevance of this process is under debate, as cellular helicases unwind G-quadruplex structures. The helicase Rhau (encoded by the DHX36 gene) was reported to be the major source of RNA G-quadruplex resolving activity in lysates of human cells. In the current study, we depleted Rhau by RNAi-mediated silencing and analyzed the effect on proteins whose mRNAs harbor a G-quadruplex motif in their 5'-UTRs. A targeted investigation of the proto-oncogenes Bcl-2 and NRAS, which are well-known examples for the translational repression of G-quadruplex structures, did not reveal effects caused by Rhau silencing. We therefore carried out a global analysis of changes in protein levels by label-free quantification using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Following Rhau knockdown, of all the identified proteins, only 1.9% were significantly downregulated to at least 70%. According to a bioinformatic analysis with the QGRS mapper, 33% of the downregulated proteins were predicted to harbor a G-quadruplex motif in the 5'-UTR of their respective mRNAs, compared to only 11% in the complete dataset. This indicates that in an unexpectedly small set of genes, in which G-quadruplex motifs are unusually common in the 5'-UTR of their mRNAs, Rhau helicase is responsible for the regulation of their expression.