Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca 2+ release activated Ca 2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca 2+ sensor required for activation of CRAC current (I CRAC ) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca 2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I CRAC . In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I CRAC . The results show that cells expressing low Orai1 : STIM1 ratios produce I CRAC with strong fast Ca 2+ -dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I CRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca 2+ , Ba 2+ and Sr 2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca 2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Graphical AbstractHighlights d RB specifically binds to p65, but not other NF-kB/Rel family proteins d RB-p65 interaction relies on CDK4/6 S249/T252 phosphorylation of RB d S249/T252-phosphorylated RB inhibits NF-kB activity and PD-L1 expression d S249/T252 phospho-mimetic peptide promotes cancer immunity via PD-L1 suppression SUMMARY Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor kB (NF-kB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-kB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-kB activity and PD-L1 expression and suggest that the RB-NF-kB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.