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Cold stress is one of the major abiotic stress factors affecting rice growth and development, leading to significant yield loss in the context of global climate change. Exploring natural variants that confer cold resistance and the underlying molecular mechanism responsible for this is the major strategy to breed cold-tolerant rice varieties. Here, we show that natural variations of a SIMILAR to RCD ONE (SRO) gene, OsSRO1c , confer cold tolerance in rice at both seedling and booting stages. Our in vivo and in vitro experiments demonstrated that Os SRO1c possesses intrinsic liquid–liquid phase-separation ability and recruits Os DREB2B, an AP2/ERF transcription factor that functions as a positive regulator of cold stress, into its biomolecular condensates in the nucleus, resulting in elevated transcriptional activity of Os DREB2B. We found that the Os SRO1c- Os DREB2B complex directly responds to low temperature through dynamic phase transitions and regulates key cold-response genes, including COLD1. Furthermore, we showed that introgression of an elite haplotype of OsSRO1c into a cold-susceptible indica rice could significantly increase its cold resistance. Collectively, our work reveals a novel cold-tolerance regulatory module in rice and provides promising genetic targets for molecular breeding of cold-tolerant rice varieties. This study reveals that OsSRO1c possesses intrinsic liquid–liquid phase-separation properties and can enhance rice cold tolerance by interacting with the transcription factor OsDREB2B. The OsSRO1c-OsDREB2B complex undergoes cold-dependent phase transitions to upregulate the expression of key cold-response genes such as COLD1. Introducing an elite OsSRO1c haplotype into susceptible rice varieties significantly improves cold resistance, providing valuable targets for breeding cold-tolerant rice varieties. [ABSTRACT FROM AUTHOR]

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface. Author summary: The 2022 outbreak of mpox is caused by monkeypox virus closely related to the best-studied model, vaccinia virus. Genome replication, which takes place largely autonomously in the cytosol of the infected cell, is still not really understood. Viral DNA synthesis involves a DNA repair enzyme, the uracil-DNA glycosylase D4 linked to the structural protein A20 forming the processivity factor, which in turn binds to the DNA polymerase E9 forming the complex required for processive DNA synthesis. Here we present the 3.8 Å structure of the apo form of vaccinia virus polymerase holoenzyme E9-A20-D4 obtained by cryo-electron microscopy. Several recent structures from monkeypox virus, with or without bound DNA, and our structure provide static views of the complex revealing a previously undescribed contact between E9 and D4. Our small-angle X-ray scattering data show that other, more open conformations without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Based on site-directed mutagenesis and binding studies, we show that the contact between E9 and D4, which serves to encircle the template strand for processive DNA synthesis, is essential, although the interaction is weak and transient in the absence of DNA. [ABSTRACT FROM AUTHOR]

This document provides a list of references for scientific articles related to stress memory and phase separation in plants. The articles cover various topics, including the molecular mechanisms of stress memory, the formation of liquid-liquid phase separation in cells, and the role of specific proteins in stress adaptation. These references serve as a valuable resource for further research on the subject. [Extracted from the article]

Several viruses express factors to silence host gene expression via widespread mRNA degradation. This phenotype is the result of the coordinated activity of the viral endonuclease SOX and the cellular RNA degradation enzyme Xrn1 during lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection. While most cellular transcripts are highly downregulated, a subset of host mRNA escapes turnover via unknown mechanisms. One of the most prominent escapees is the interleukin 6 (IL-6) mRNA, which accumulates robustly during KSHV lytic infection and is not subjected to SOX-induced degradation. Here we reveal that the IL-6 mRNA contains a dominant, cis-acting ∼100-nucleotide element within its 3' untranslated region (UTR) that renders it directly refractory to cleavage by SOX. This element specifically interacts with a cellular protein complex both in SOX-transfected cells and in KSHV-infected B cells. Using a directed RNA pulldown approach, we identified two components of this complex to be the AU-rich element (ARE) binding proteins AUF1 and HuR. Depletion of these proteins significantly reduced the protective capacity of the IL-6 RNA element in SOX-expressing cells. These findings suggest that SOX activity may be directly counteracted by select RNA regulatory complexes and reveal a novel mechanism contributing to the robust expression of IL-6 during KSHV replication.

The article discusses the phosphorylation of phyB by GSK3s as a key mechanism that brings temperature sensors together in plants. It highlights the intricate crosstalk between light and temperature signals, which influence plant architecture, growth rate, and developmental transitions. The authors demonstrate that BIN2 phosphorylates phyB, contributing to photobody formation and stabilization, revealing a new mechanism in the regulation of phyB photobodies and temperature sensing. The research sheds light on the complex signaling networks plants use to optimize their developmental programs under different temperature regimes. [Extracted from the article]

The identification of genome-wide transcription factor binding sites (TFBS) is a critical step in deciphering gene and transcriptional regulatory networks. However, determining the genome-wide binding of specific TFs or TF complexes remains a technical challenge. DNA affinity purification sequencing (DAP-seq) and modifications such as sequential DAP-seq (seq-DAP-seq) are robust in vitro methods for mapping individual TF or TF complex binding sites in a genome-wide manner. DAP-seq protocols use a genomic DNA (gDNA) library from any target organism with or without amplification, allowing the determination of TF binding on naked or endogenously modified DNA, respectively. As a first step, the gDNA is fragmented to ~200 bp, end-repaired, and sequencing adaptors are added. This gDNA library can be used directly or an amplification step may be performed to remove DNA modifications such as cytosine methylation. DNA libraries are then incubated with an affinity-tagged TF or TF- complex immobilized on magnetic beads. The TF or TF complex of interest is generally produced using recombinant protein expression and purified prior to DNA affinity purification. After incubation of the DNA library with the immobilized TF of interest, multiple wash steps are performed to reduce non-specific DNA binding and the TF-DNA complexes eluted. The eluted DNA is PCR-amplified and sequenced using next-generation sequencing. The resulting sequence reads are mapped to the corresponding reference genome, identifying direct potential bound regions and binding sites of the TF or TF complex of interest. Predictive TFBS models are generated from the bound regions using downstream bioinformatics analysis pipelines. Here, we present a detailed protocol outlining the steps required for seq-DAP-seq of a heterooligomeric TF complex (Fig. 1) and briefly describe the downstream bioinformatics pipeline used to develop a robust TFBS model from sequencing data generated from a DAP-seq experiment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.