Driving forces behind phase separation of the carboxy-terminal domain of RNA polymerase II.

Eukaryotic gene regulation and pre-mRNA transcription depend on the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II. Due to its highly repetitive, intrinsically disordered sequence, the CTD enables clustering and phase separation of Pol II. The molecular interactions that drive CTD phase separation and Pol II clustering are unclear. Here, we show that multivalent interactions involving tyrosine impart temperature- and concentration-dependent self-coacervation of the CTD. NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations demonstrate the presence of diverse tyrosine-engaging interactions, including tyrosine-proline contacts, in condensed states of human CTD and other low-complexity proteins. We further show that the network of multivalent interactions involving tyrosine is responsible for the co-recruitment of the human Mediator complex and CTD during phase separation. Our work advances the understanding of the driving forces of CTD phase separation and thus provides the basis to better understand CTD-mediated Pol II clustering in eukaryotic gene transcription.

Stress-induced nuclear condensation of NELF drives transcriptional downregulation.

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.

Mechanisms for Active Regulation of Biomolecular Condensates.

Liquid-liquid phase separation is a key organizational principle in eukaryotic cells, on par with intracellular membranes. It allows cells to concentrate specific proteins into condensates, increasing reaction rates and achieving switch-like regulation. We propose two active mechanisms that can explain how cells regulate condensate formation and size. In both, the cell regulates the activity of an enzyme, often a kinase, that adds post-translational modifications to condensate proteins. In enrichment inhibition, the enzyme enriches in the condensate and weakens interactions, as seen in stress granules (SGs), Cajal bodies, and P granules. In localization-induction, condensates form around immobilized enzymes that strengthen interactions, as observed in DNA repair, transmembrane signaling, and microtubule assembly. These models can guide studies into the many emerging roles of biomolecular condensates.

Functional Metagenomics Reveals an Overlooked Diversity and Novel Features of Soil-Derived Bacterial Phosphatases and Phytases.

Phosphatases, including phytases, play a major role in cell metabolism, phosphorus cycle, biotechnology, and pathogenic processes. Nevertheless, their discovery by functional metagenomics is challenging. Here, soil metagenomic libraries were successfully screened for genes encoding phosphatase activity. In this context, we report the largest number and diversity of phosphatase genes derived from functional metagenome analysis. Two of the detected gene products carry domains which have never been associated with phosphatase activity before. One of these domains, the SNARE-associated domain DedA, harbors a so-far-overlooked motif present in numerous bacterial SNARE-associated proteins. Our analysis revealed a previously unreported phytase activity of the alkaline phosphatase and sulfatase superfamily (cl23718) and of purple acid phosphatases from nonvegetal origin. This suggests that the classical concept comprising four classes of phytases should be modified and indicates high performance of our screening method for retrieving novel types of phosphatases/phytases hidden in metagenomes of complex environments.IMPORTANCE Phosphorus (P) is a key element involved in numerous cellular processes and essential to meet global food demand. Phosphatases play a major role in cell metabolism and contribute to control the release of P from phosphorylated organic compounds, including phytate. Apart from the relationship with pathogenesis and the enormous economic relevance, phosphatases/phytases are also important for reduction of phosphorus pollution. Almost all known functional phosphatases/phytases are derived from cultured individual microorganisms. We demonstrate here for the first time the potential of functional metagenomics to exploit the phosphatase/phytase pools hidden in environmental soil samples. The recovered diversity of phosphatases/phytases comprises new types and proteins exhibiting largely unknown characteristics, demonstrating the potential of the screening method for retrieving novel target enzymes. The insights gained into the unknown diversity of genes involved in the P cycle highlight the power of function-based metagenomic screening strategies to study Earth's phosphatase pools.

RNA polymerase II clustering through carboxy-terminal domain phase separation.

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.

Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.