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XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction., (© 2024 Button et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Huntington's disease arises from a toxic gain of function in the huntingtin ( HTT ) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological, and plasma metabolite levels. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic β-catenin function., (© 2023 Bragg et al.)

RNA-protein interactions are important in development and disease, but identification of novel RNA-protein interactions remains challenging. Here, we describe an updated capture method to identify direct and specific RNA-protein interactions. First, RNA and protein are covalently cross-linked in living cells by treatment with UV light at 254 nanometers wavelength. The antisense purification approach is dependent upon nucleic acid hybridization between biotinylated DNA probes and a target RNA. Target protein:RNA:DNA complexes are enriched by capture on streptavidin magnetic beads and purified through several denaturing washes that remove nonspecific protein and nucleic acid interactors. Mass spectrometry is used to identify proteins that are specifically enriched in the target RNA capture. This method has been applied to discover the protein interactions of noncoding RNAs but can be used to capture any RNA where the target sequence is known., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of cytosine base editing causes significant increases in C•G to A•T "byproduct" introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle., Competing Interests: AK is a member of the SAB of Pairwise Plants, is an equity holder for Pairwise Plants and Beam Therapeutics, and receives royalties from Pairwise Plants, Beam Therapeutics, and Editas Medicine via patents licensed from Harvard University. GY is co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Locana and Eclipse BioInnovations. GY is a visiting professor at the National University of Singapore. AK’s and GY’s interests have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Burnett, Wong, Vasquez, McHugh, Yeo and Komor.)

Objective: To understand the enablers and barriers to implementing a set of adaptive processes aimed at supporting secondary schools to reflect on and subsequently address how they could adjust school practices, culture and the environment to create a whole school approach to promoting healthy lifestyles., Study Design: A qualitative, comparative case study., Methods: Two in depth case studies were created of two purposefully selected schools in low socio-economic areas of South West England. Data were collected via meetings, observations, field notes, interviews and audit. Interviews were transcribed verbatim. Individual thematic analyses were conducted for each school and a comparative analysis approach was used to understand the barriers and enablers across both cases., Results: Schools were supported to use a health-promoting lens and identify feasible improvements through an adaptive and context specific process. The school environment and ethos were identified as the areas where schools could conceive the most adjustments to enhance the promotion of healthy lifestyle choices. With the lack of government policy for health promotion in schools (HPS), the Head teacher's approach to health was key to making meaningful changes., Conclusions: Health promoting school approaches need to be adaptive to local context, actively involve community partners and link to local initiatives where possible, with support from Head teachers and business managers. Starting with what teachers, pupils and parents see as the barriers to health can create a whole school ethos for broad reaching and sustainable HPS programmes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

RNA molecules interact with proteins to perform a variety of functions in living cells. The binding partners of many RNAs, in particular the newly discovered class of long noncoding RNAs (lncRNAs), remain largely unknown. RNA antisense purification coupled with mass spectrometry (RAP-MS) is a method that enables the identification of direct and specific protein interaction partners of a specific RNA molecule. Because RAP-MS uses direct RNA-protein cross-linking methods coupled along with highly denaturing purification conditions, RAP-MS provides a short list of high confidence protein interactors.

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins--SHARP, SAF-A and LBR--are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome.

Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration., (© 2014 The Authors.)

The importance of RNA-protein interactions in controlling mRNA regulation and non-coding RNA function is increasingly appreciated. A variety of methods exist to comprehensively define RNA-protein interactions. We describe these methods and the considerations required for designing and interpreting these experiments.

ADMS and AERMOD are the two most widely used dispersion models for regulatory purposes. It is, therefore, important to understand the differences in the predictions of the models and the causes of these differences. The treatment by the models of flat terrain has been discussed previously; in this paper the focus is on their treatment of complex terrain. The paper includes a discussion of the impacts of complex terrain on airflow and dispersion and how these are treated in ADMS and AERMOD, followed by calculations for two distinct cases: (i) sources above a deep valley within a relatively flat plateau area (Clifty Creek power station, USA); (ii) sources in a valley in hilly terrain where the terrain rises well above the stack tops (Ribblesdale cement works, England). In both cases the model predictions are markedly different. At Clifty Creek, ADMS suggests that the terrain markedly increases maximum surface concentrations, whereas the AERMOD complex terrain module has little impact. At Ribblesdale, AERMOD predicts very large increases (a factor of 18) in the maximum hourly average surface concentrations due to plume impaction onto the neighboring hill; although plume impaction is predicted by ADMS, the increases in concentration are much less marked as the airflow model in ADMS predicts some lateral deviation of the streamlines around the hill.

Bactofilins are fibre-forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic 'crooked' cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637-4635 (BacN-P) has no obvious phenotype. In M. xanthus, BacM exists as a 150-amino-acid full-length version and as a version cleaved before Ser28. In the cell, native BacM forms 3 nm wide fibres, which assemble into bundles forming helix-like cytoplasmic cables throughout the cell, and in a subset of cells additionally a polarly arranged lateral rod-like structure. Isolated fibres consist almost completely of the N-terminally truncated version, suggesting that the proteolytic cleavage occurs before or during fibre formation. Fusion of BacM to mCherry perturbs BacM function and cellular fibre arrangement, resulting for example in the formation of one prominent polar corkscrew-like structure per cell. Immunofluorescence staining of BacM and MreB shows that their cellular distributions are not matching. Taken together, these data suggest that rod-shaped bacteria like M. xanthus use bactofilin fibres to achieve and maintain their characteristic cell morphology and cell wall stability., (© 2011 Blackwell Publishing Ltd.)

Cell differentiation is widespread during the development of multicellular organisms, but rarely observed in prokaryotes. One example of prokaryotic differentiation is the gram-negative bacterium Myxococcus xanthus. In response to starvation, this gliding bacterium initiates a complex developmental programme that results in the formation of spore-filled fruiting bodies. How the cells metabolically support the necessary complex cellular differentiation from rod-shaped vegetative cells into spherical spores is unknown. Here, we present evidence that intracellular lipid bodies provide the necessary metabolic fuel for the development of spores. Formed at the onset of starvation, these lipid bodies gradually disappear until they are completely used up by the time the cells have become mature spores. Moreover, it appears that lipid body formation in M. xanthus is an important initial step indicating cell fate during differentiation. Upon starvation, two subpopulations of cells occur: cells that form lipid bodies invariably develop into spores, while cells that do not form lipid bodies end up becoming peripheral rods, which are cells that lack signs of morphological differentiation and stay in a vegetative-like state. These data indicate that lipid bodies not only fuel cellular differentiation but that their formation represents the first known morphological sign indicating cell fate during differentiation.

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.

Background: Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions., Results: We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a DeltaH of -13 +/- 2 kJ/mol and a DeltaS of -0.04 +/- 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site., Conclusion: We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.