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Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680 S PRRA R↓SV 687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif R R xR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert S PRRAR↓ S V can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin’s ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress. The BLM helicase interacts with the topoisomerase TOP3A and RMI1 to form the BTR complex. Here, the authors reveal that this complex contains multiple binding sites for the single-stranded DNA-binding complex RPA, and that RPA-binding stimulates BLM recruitment to stalled replication forks to promote their restart after replication stress.

CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9—Sc++ and a higher-fidelity mutant HiFi-Sc++—that have simultaneously broad 5′-NNG-3′ PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc++ and HiFi-Sc++ extend the use of CRISPR editing for diverse applications.A hybrid CRISPR–Cas9 variant is able to specifically and efficiently target a larger proportion of the genome.

Background SWEET ( MtN3_saliva ) domain proteins, a recently identified group of efflux transporters, play an indispensable role in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development. The SWEET gene family is predominantly studied in Arabidopsis and members of the family are being investigated in rice. To date, no transcriptome or genomics analysis of soybean SWEET genes has been reported. Results In the present investigation, we explored the evolutionary aspect of the SWEET gene family in diverse plant species including primitive single cell algae to angiosperms with a major emphasis on Glycine max . Evolutionary features showed expansion and duplication of the SWEET gene family in land plants. Homology searches with BLAST tools and Hidden Markov Model-directed sequence alignments identified 52 SWEET genes that were mapped to 15 chromosomes in the soybean genome as tandem duplication events. Soybean SWEET ( GmSWEET ) genes showed a wide range of expression profiles in different tissues and developmental stages. Analysis of public transcriptome data and expression profiling using quantitative real time PCR (qRT-PCR) showed that a majority of the GmSWEET genes were confined to reproductive tissue development. Several natural genetic variants (non-synonymous SNPs, premature stop codons and haplotype) were identified in the GmSWEET genes using whole genome re-sequencing data analysis of 106 soybean genotypes. A significant association was observed between SNP-haplogroup and seed sucrose content in three gene clusters on chromosome 6. Conclusion Present investigation utilized comparative genomics, transcriptome profiling and whole genome re-sequencing approaches and provided a systematic description of soybean SWEET genes and identified putative candidates with probable roles in the reproductive tissue development. Gene expression profiling at different developmental stages and genomic variation data will aid as an important resource for the soybean research community and can be extremely valuable for understanding sink unloading and enhancing carbohydrate delivery to developing seeds for improving yield.

Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

The mechanisms by which genetic variation affects transcription regulation and phenotypes at the nucleotide level are incompletely understood. Here we use natural genetic variation as an in vivo mutagenesis screen to assess the genome-wide effects of sequence variation on lineage-determining and signal-specific transcription factor binding, epigenomics and transcriptional outcomes in primary macrophages from different mouse strains. We find substantial genetic evidence to support the concept that lineage-determining transcription factors define epigenetic and transcriptomic states by selecting enhancer-like regions in the genome in a collaborative fashion and facilitating binding of signal-dependent factors. This hierarchical model of transcription factor function suggests that limited sets of genomic data for lineage-determining transcription factors and informative histone modifications can be used for the prioritization of disease-associated regulatory variants. Naturally occurring genetic variation between inbred mouse strains is used as a mutagenesis strategy to investigate mechanisms responsible for the selection and function of cis -regulatory elements in macrophages; lineage-determining transcription factors are proposed to select enhancer-like regions in the genome in a collaborative fashion and facilitate the binding of signal-dependent factors. Genetic variation and the transcriptome The DNA sequence determinants that guide binding of lineage-determining transcription factors (LDTFs) are poorly understood. Here, Christopher Glass and colleagues exploit the naturally occurring genetic variation between inbred mouse strains as a mutagenesis strategy to investigate molecular mechanisms responsible for the selection and function of cis -regulatory elements in macrophages. By integrating data sets on transcription factor binding, histone marks and gene expression they develop a hierarchical model of LDTF function in selecting enhancer-like regions in a collaborative fashion and facilitating binding of signal-dependent factors. This model can be used to predict functional and potentially disease-causing regulatory variants.

Intrinsically disordered regions (IDRs) are often fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function. TDP43 regulates RNA processing and undergoes liquid-liquid phase separation. Here, the authors combine bioinformatic analysis and quantitative measurements of phase properties in living cells to uncover the rules that link the amino acid sequence of TDP43 with its phase properties and find that unexpectedly, mutants of TDP43 that are deficient in phase separation can still function to mediate splicing.

Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.

Ruminants are a diverse group of mammals that includes families containing well-known taxa such as deer, cows, and goats. However, their evolutionary relationships have been contentious, as have the origins of their distinctive digestive systems and headgear, including antlers and horns (see the Perspective by Ker and Yang). To understand the relationships among ruminants, L. Chen et al. sequenced 44 species representing 6 families and performed a phylogenetic analysis. From this analysis, they were able to resolve the phylogeny of many genera and document incomplete lineage sorting among major clades. Interestingly, they found evidence for large population reductions among many taxa starting at approximately 100,000 years ago, coinciding with the migration of humans out of Africa. Examining the bony appendages on the head—the so-called headgear—Wang et al. describe specific evolutionary changes in the ruminants and identify selection on cancer-related genes that may function in antler development in deer. Finally, Lin et al. take a close look at the reindeer genome and identify the genetic basis of adaptations that allow reindeer to survive in the harsh conditions of the Arctic. Science , this issue p. eaav6202 , p. eaav6335 , p. eaav6312 ; see also p. 1130 The genetics of distinctive reindeer characteristics are identified from their genome. The reindeer is an Arctic species that exhibits distinctive biological characteristics, for which the underlying genetic basis remains largely unknown. We compared the genomes of reindeer against those of other ruminants and nonruminant mammals to reveal the genetic basis of light arrhythmicity, high vitamin D metabolic efficiency, the antler growth trait of females, and docility. We validate that two reindeer vitamin D metabolic genes ( CYP27B1 and POR ) show signs of positive selection and exhibit higher catalytic activity than those of other ruminants. A mutation upstream of the reindeer CCND1 gene endows an extra functional binding motif of the androgen receptor and thereby may result in female antlers. Furthermore, a mutation (proline-1172→threonine) in reindeer PER2 results in loss of binding ability with CRY1, which may explain circadian arrhythmicity in reindeer.