Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

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SUMMARYNF‐Y transcription factor comprises three subunits: NF‐YA, NF‐YB and NF‐YC. NF‐YB and NF‐YC dimerize through their histone fold domain (HFD), which can bind DNA in a non‐sequence‐specific fashion while serving as a scaffold for NF‐YA trimerization. Upon trimerization, NF‐YA specifically recognizes the CCAAT box sequence on promoters and enhancers. In plants, each NF‐Y subunit is encoded by several genes giving rise to hundreds of potential heterotrimeric combinations. In addition, plant NF‐YBs and NF‐YCs interact with other protein partners to recognize a plethora of genomic motifs, as the CCT protein family that binds CORE sites. The NF‐Y subunit organization and its DNA‐binding properties, together with the NF‐Y HFD capacity to adapt different protein modules, represent plant‐specific features that play a key role in development, growth and reproduction. Despite their relevance, these features are still poorly understood at the molecular level. Here, we present the structures of Arabidopsis and rice NF‐YB/NF‐YC dimers, and of an Arabidopsis NF‐Y trimer in complex with the FT CCAAT box, together with biochemical data on NF‐Y mutants. The dimeric structures identify the key residues for NF‐Y HFD stabilization. The NF‐Y/DNA structure and the mutation experiments shed light on HFD trimerization interface properties and the NF‐YA sequence appetite for the bases flanking the CCAAT motif. These data explain the logic of plant NF‐Y gene expansion: the trimerization adaptability and the flexible DNA‐binding rules serve the scopes of accommodating the large number of NF‐YAs, CCTs and possibly other NF‐Y HFD binding partners and a diverse audience of genomic motifs.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure–function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1—the largest protein in the complex—as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.