Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions.

Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation.

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

Histone deacetylation promotes transcriptional silencing at facultative heterochromatin.

It is important to accurately regulate the expression of genes involved in development and environmental response. In the fission yeast Schizosaccharomyces pombe, meiotic genes are tightly repressed during vegetative growth. Despite being embedded in heterochromatin these genes are transcribed and believed to be repressed primarily at the level of RNA. However, the mechanism of facultative heterochromatin formation and the interplay with transcription regulation is not understood. We show genome-wide that HDAC-dependent histone deacetylation is a major determinant in transcriptional silencing of facultative heterochromatin domains. Indeed, mutation of class I/II HDACs leads to increased transcription of meiotic genes and accumulation of their mRNAs. Mechanistic dissection of the pho1 gene where, in response to phosphate, transient facultative heterochromatin is established by overlapping lncRNA transcription shows that the Clr3 HDAC contributes to silencing independently of SHREC, but in an lncRNA-dependent manner. We propose that HDACs promote facultative heterochromatin by establishing alternative transcriptional silencing.

Influence of spine surgery on the ability to perform an emergency stop while driving a car.

BACKGROUND: Spinal surgeries have strongly increased in number over the past decade. The question of when it is safe to resume driving is thereby one the most frequently asked questions that patients ask of their treating physician. OBJECTIVE: The aim of this study was to assess braking performance before and after spine surgery. METHODS: Reaction time, foot transfer time (together brake response time [BRT]), and brake force (BF) were evaluated in a drive simulator. A longitudinal patient cohort (n= 27) was tested preoperatively and at the first follow-up. A cross-sectional cohort (n= 27) was tested at > 1 year postoperatively. The values from these groups were compared with a healthy age-matched control group of 24 volunteers. RESULTS: No significant improvement in BRT was seen in lumbar fusion three months postoperatively (p= 0.597); BF was even weaker than it was preoperatively (p= 0.044). In comparison to the control group (median BRT 479 ms), preoperative BRT was already impaired in lumbar fusion patients (median 560 ms), representing an increased braking distance of 2.25 m at 100 km/h. CONCLUSION: Although most patients performed adequately, about one third presented critical braking performance. Risk factors for impaired braking may include scheduled multisegmental fusion surgery, female sex, and pain.

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination.

The regulation and functions of the nuclear RNA exosome complex.

The RNA exosome complex is the most versatile RNA-degradation machine in eukaryotes. The exosome has a central role in several aspects of RNA biogenesis, including RNA maturation and surveillance. Moreover, it is emerging as an important player in regulating the expression levels of specific mRNAs in response to environmental cues and during cell differentiation and development. Although the mechanisms by which RNA is targeted to (or escapes from) the exosome are still not fully understood, general principles have begun to emerge, which we discuss in this Review. In addition, we introduce and discuss novel, previously unappreciated functions of the nuclear exosome, including in transcription regulation and in the maintenance of genome stability.

Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1.

In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these "decay-promoting" introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

lncRNA recruits RNAi and the exosome to dynamically regulate pho1 expression in response to phosphate levels in fission yeast.

Numerous noncoding transcripts of unknown function have recently been identified. In this study, we report a novel mechanism that relies on transcription of noncoding RNA prt (pho1-repressing transcript) regulating expression of the pho1 gene. A product of this gene, Pho1, is a major secreted phosphatase needed for uptake of extracellular phosphate in fission yeast. prt is produced from the promoter located upstream of the pho1 gene in response to phosphate, and its transcription leads to deposition of RNAi-dependent H3K9me2 across the pho1 locus. In contrast, phosphate starvation leads to loss of H3K9me2 and pho1 induction. Strikingly, deletion of Clr4, a H3K9 methyltransferase, results in faster pho1 induction in response to phosphate starvation. We propose a new role for noncoding transcription in establishing transient heterochromatin to mediate an effective transcriptional response to environmental stimuli. RNAi recruitment to prt depends on the RNA-binding protein Mmi1. Importantly, we found that the exosome complex and Mmi1 are required for transcription termination and the subsequent degradation of prt but not pho1 mRNA. Moreover, in mitotic cells, transcription termination of meiotic RNAs also relies on this mechanism. We propose that exosome-dependent termination constitutes a specialized system that primes transcripts for degradation to ensure their efficient elimination.