Cryo-EM structure and functional analysis of the chromatin remodeler RSF.

The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.

Cryo-EM structures of Thogoto virus polymerase reveal unique RNA transcription and replication mechanisms among orthomyxoviruses.

Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5' end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses' broad-infectivity range.

Shotgun lipidomics combined targeted MRM reveals sphingolipid signatures of coronary artery disease.

Sphingolipids (SPLs) are bioactive lipids that manifest structural diversity and complexity in eukaryotes. However, the distributions and functions of these molecules in mammalian tissues/cells have not been systematically investigated. Herein, we integrated shotgun lipidomics with targeted LC-MRM/MS approach to comprehensively analyze SPL species in various biological samples with high accuracy. Preliminarily, 1311 SPL molecules were identified in 18 kinds of mammalian samples, including 3 groups of human sera, 10 mouse tissues and 5 cell lines via 26 sphingoid long-chain bases scanning. The sphingolipidome compositions and distributions were systematically characterized and distinct qualitative and quantitative profiles were clearly exhibited in various samples, indicating unique biological functions of the sphingolipidomes. Next, targeted SPLs analysis by LC-MRM/MS with critical criteria monitoring two characteristic fragments of one precursor was applied to human serum samples from 24 coronary artery disease (CAD) patients and 12 healthy controls, which successfully quantified 170 SPL molecules. Ten novel SPL molecules were discovered as a potential diagnostic panel for CAD patients via multivariate exploratory receiver operating characteristic curve-based biomarker analysis. The diagnostic panel with the 10 SPL molecules achieved 97.2% accuracy, with a favorable auxiliary diagnostic value (AUC = 1.000), for the detection of CAD. These results clearly support the sphingolipidomic approach in application to discovering disease biomarker panel as well as deep investigation of biological functions of complex SPLs in mammalian samples.

CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 is an RNA-binding protein controlling plant immunity via an RNA processing complex.

Plant innate immunity is capable of combating diverse and ever evolving pathogens. The plasticity of innate immunity could be boosted by RNA processing. Arabidopsis thaliana CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5), a key negative immune regulator, is a component of the nuclear pore complex. Here we further identified CPR5 as a component of RNA processing complexes. Through genetic screening, we found that RNA splicing activator NineTeen Complex and RNA polyadenylation factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR, coordinately function downstream of CPR5 to activate plant immunity. CPR5 and these two regulators form a complex that is localized in nuclear speckles, an RNA processing organelle. Intriguingly, we found that CPR5 is an RNA-binding protein belonging to the Transformer 2 (Tra2) subfamily of the serine/arginine-rich family. The RNA recognition motif of CPR5 protein binds the Tra2-targeted RNA sequence in vitro and is functionally replaceable by those of Tra2 subfamily proteins. In planta, it binds RNAs of CPR5-regulated alternatively spliced genes (ASGs) identified by RNA-seq. ARGONAUTE 1 (AGO1) is one of the ASGs and, consistent with this, the ago1 mutant suppresses the cpr5 phenotype. These findings reveal that CPR5 is an RNA-binding protein linking RNA processing with plant immunity.

Altered DNA methylation pattern reveals epigenetic regulation of Hox genes in thoracic aortic dissection and serves as a biomarker in disease diagnosis.

BACKGROUND: Thoracic aortic dissection (TAD) is a severe disease with limited understandings in its pathogenesis. Altered DNA methylation has been revealed to be involved in many diseases etiology. Few studies have examined the role of DNA methylation in the development of TAD. This study explored alterations of the DNA methylation landscape in TAD and examined the potential role of cell-free DNA (cfDNA) methylation as a biomarker in TAD diagnosis. RESULTS: Ascending aortic tissues from TAD patients (Stanford type A; n = 6) and healthy controls (n = 6) were first examined via whole-genome bisulfite sequencing (WGBS). While no obvious global methylation shift was observed, numerous differentially methylated regions (DMRs) were identified, with associated genes enriched in the areas of vasculature and heart development. We further confirmed the methylation and expression changes in homeobox (Hox) clusters with 10 independent samples using bisulfite pyrosequencing and quantitative real-time PCR (qPCR). Among these, HOXA5, HOXB6 and HOXC6 were significantly down-regulated in TAD samples relative to controls. To evaluate cfDNA methylation pattern as a biomarker in TAD diagnosis, cfDNA from TAD patients (Stanford type A; n = 7) and healthy controls (n = 4) were examined by WGBS. A prediction model was built using DMRs identified previously from aortic tissues on methylation data from cfDNA. Both high sensitivity (86%) and specificity (75%) were achieved in patient classification (AUC = 0.96). CONCLUSIONS: These findings showed an altered epigenetic regulation in TAD patients. This altered epigenetic regulation and subsequent altered expression of genes associated with vasculature and heart development, such as Hox family genes, may contribute to the loss of aortic integrity and TAD pathogenesis. Additionally, the cfDNA methylation in TAD was highly disease specific, which can be used as a non-invasive biomarker for disease prediction.

Novel tool to quantify cell wall porosity relates wall structure to cell growth and drug uptake.

Even though cell walls have essential functions for bacteria, fungi, and plants, tools to investigate their dynamic structure in living cells have been missing. Here, it is shown that changes in the intensity of the plasma membrane dye FM4-64 in response to extracellular quenchers depend on the nano-scale porosity of cell walls. The correlation of quenching efficiency and cell wall porosity is supported by tests on various cell types, application of differently sized quenchers, and comparison of results with confocal, electron, and atomic force microscopy images. The quenching assay was used to investigate how changes in cell wall porosity affect the capability for extension growth in the model plant Arabidopsis thaliana Results suggest that increased porosity is not a precondition but a result of cell extension, thereby providing new insight on the mechanism plant organ growth. Furthermore, it was shown that higher cell wall porosity can facilitate the action of antifungal drugs in Saccharomyces cerevisiae, presumably by facilitating uptake.

Anisotropic Cell Expansion Is Affected through the Bidirectional Mobility of Cellulose Synthase Complexes and Phosphorylation at Two Critical Residues on CESA3.

Here we report that phosphorylation status of S211 and T212 of the CESA3 component of Arabidopsis (Arabidopsis thaliana) cellulose synthase impacts the regulation of anisotropic cell expansion as well as cellulose synthesis and deposition and microtubule-dependent bidirectional mobility of CESA complexes. Mutation of S211 to Ala caused a significant decrease in the length of etiolated hypocotyls and primary roots, while root hairs were not significantly affected. By contrast, the S211E mutation stunted the growth of root hairs, but primary roots were not significantly affected. Similarly, T212E caused a decrease in the length of root hairs but not root length. However, T212E stunted the growth of etiolated hypocotyls. Live-cell imaging of fluorescently labeled CESA showed that the rate of movement of CESA particles was directionally asymmetric in etiolated hypocotyls of S211A and T212E mutants, while similar bidirectional velocities were observed with the wild-type control and S211E and T212A mutant lines. Analysis of cell wall composition and the innermost layer of cell wall suggests a role for phosphorylation of CESA3 S211 and T212 in cellulose aggregation into fibrillar bundles. These results suggest that microtubule-guided bidirectional mobility of CESA complexes is fine-tuned by phosphorylation of CESA3 S211 and T212, which may, in turn, modulate cellulose synthesis and organization, resulting in or contributing to the observed defects of anisotropic cell expansion.

MTHFR promotes heterochromatin maintenance.

Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in the folate cycle, catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. Methionine serves as the precursor of the active methyl donor S-adenosylmethionine, which provides methyl groups for many biological methylations. It has been reported that MTHFR is highly phosphorylated under unperturbed conditions and T34 is the priming phosphorylation site. In this report, we generated a phospho-specific antibody that recognized T34-phosphorylated form of MTHFR and revealed that MTHFR was phosphorylated at T34 in vivo and this phosphorylation peaked during mitosis. We further demonstrated that the cyclin-dependent kinase 1 (CDK1)/Cyclin B1 complex is the kinase that mediates MTHFR phosphorylation at T34 and the MTHFR immunocomplex purified from mitotic cells exhibited lower enzymatic activity. Inhibition of MTHFR expression resulted in a decrease of H3K9me3 levels, and an increase of transcription of the centromeric heterochromatin markers. Taken together, our results demonstrated that CDK1/Cyclin B1 phosphorylates MTHFR on T34 and MTHFR plays a role in the heterochromatin maintenance at the centromeric region.