Newsletter

Applications 2022

• 129 internal applications
• 79 external applications (Czech)
• 27 applications from foreign users

You can find more information here.

Application overview in total

a, Kinome tree representation of the selectivity of OTS964 (at 1 µM concentration) in the Eurofins panel of 412 human kinases. The size of the red circles shows the percentage of inhibition of kinase activity. The green circle corresponds to CDK11. The percentage of CDK11 inhibition was derived from IVKAs presented in b and Fig. 3f The blue circle corresponds to TOPK. The percentage of TOPK inhibition was estimated from the published IC₅₀ = 353 nM and Extended Data Fig. 1b. b, Immunoblots of IVKAs of Flag-tagged (F) CDK11 WT, G579S and kinase dead (KD) mutants (left), or Flag-tagged CDK9 WT and KD mutant (right) phosphorylation of glutathione-S-transferase (GST)-tagged RNAPII^CTD substrate with the indicated concentrations of OTS964. c, The percentage of normalized NanoBRET ratio for CDK11/cyclin L2 or CDK9/cyclin T1 after OTS964 or control dinaciclib treatment. n = 2 biologically independent replicates; a representative replicate is shown. d, Immunoblot analysis of proteins after treatment of cells with OTS964 for 4 h. Short and long indicate short and long exposures of the film.

Dalibor Blažek Research Group

Significance

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA–protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex^. Splicing factor 3B subunit 1 (SF3B1) protein—a subunit of the U2 small nuclear ribonucleoprotein—is phosphorylated during spliceosome activation, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the B^act complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex B^act and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Hluchý, M., Gajdušková, P., Ruiz de los Mozos, I., Rájecký, M., Kluge, M., Berger, B-T., Slabá, Z., Potěšil, D., Weiß, E., Ule, J., Zdráhal, Z., Knapp, S., Paruch, K., Friedel, C.C., and Blažek, D.:

CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, Nature. (2022) 609, 829-834. https://doi.org/10.1038/s41586-022-05204-z

a)Ribbon diagram of the NKR-P1 CTLD. Secondary structure elements are labeled in different colors: helix α1 is red, helix α2 is yellow, and β-strands and loops are cyan. b)Comparison between NKR-P1 dimers formed by the glycosylated (cyan), deglycosylated free (green), and LLT1-bound (blue) forms of NKR-P1. c) Comparison between helices α1- and α2-centered dimerization of murine dectin-1 (magenta) and human LLT1 (green), respectively; helices α1 and α2 are shown in red and yellow. Structural alignments of dectin-1 and NKR-P1 homodimers and LLT1 and NKR-P1 homodimers, prepared by aligning only one monomer from each dimer, are shown on the right-hand side. Although the CTLD fold is conserved in each pair of the aligned monomers, the helix α1- and helix α2-centered dimers show inverse arrangement.

Ondřej Vaněk Research Group

Significance

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Blaha, J., Skalova, T. Kalouskova, B., Skorepa, O., Cmunt, D., Grobarova, V.,Pazicky, S., Polachova, E., Abreu, C., Stransky, J., Koval, T., Duskova, J.,Zhao, Y.,Harlos, K., Hasek, J. Dohnalek, J., and Vanek, O.:

Structure of the human NK cell NKR-P1:LLT1 receptor: ligand complex reveals clustering in the immune synapse, Nature Comm. (2022)13:5022, https://doi.org/10.1038/s41467-022-32577-6