Cytoplasmic switch of ARS2 isoforms promotes nonsense-mediated mRNA decay and arsenic sensitivity.

The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2's intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, and stress response.

Structure-Activity Relationship of Carbon Nitride Dots in Inhibiting Tau Aggregation.

Due to the numerous failed clinical trials of anti-amyloid drugs, microtubule associated protein tau (MAPT) now stands out as one of the most promising targets for AD therapy. In this study, we report for the first time the structure-dependent MAPT aggregation inhibition of carbon nitride dots (CNDs). CNDs have exhibited great promise as a potential treatment of Alzheimer's disease (AD) by inhibiting the aggregation of MAPT. In order to elucidate its structure-activity relationship, CNDs were separated via column chromatography and five fractions with different structures were obtained that were characterized by multiple spectroscopy methods. The increase of surface hydrophilic functional groups is consistent with the increase of polarity from fraction 1 to 5. Particle sizes (1-2 nm) and zeta potentials (~-20 mV) are similar among five fractions. With the increase of polarity from fraction 1 to 5, their MAPT aggregation inhibition capacity was weakened. This suggests hydrophobic interactions between CNDs and MAPT, validated via molecular dynamics simulations. With a zebrafish blood-brain barrier (BBB) model, CNDs were observed to cross the BBB through passive diffusion. CNDs were also found to inhibit the generation of multiple reactive oxygen species, which is an important contributor to AD pathogenesis.

Proceedings of the Dual Use Research of Concern Panel Discussion: challenges and perspectives.

To address real and perceived emerging risks originating from the ever-accelerating breakthroughs in life science research, the Dual Use Research of Concern (DURC) Panel Discussion, organized by Synbio Canada and the Alberta RNA Research and Training Institute (ARRTI), took place on June 23rd, 2021. It brought together six stakeholders from different levels of academic research, administration, governance, and science publishing to explore the current and future challenges in addressing DURC. Technological advancements within the life sciences, especially within the field of omics technology, make it difficult to apply a simple checklist for dual-use assessment and require continuous and integrated effort. Bottom-up approaches from within the scientific community are suggested by all stakeholders to enable efficient governance and address the true risks resulting from DURC, not just the alleged risks. To address such alleged risks, open and broadscale communication of DURC and its oversight policies may be required. At the same time, any form of open communication also contains the risk of information hazards, defined as potentially creating public fear or informing malicious actors. Here, an overview of the DURC panel and its outcomes is provided.

A novel histone H4 variant H4G regulates rDNA transcription in breast cancer.

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. We found that H4G is expressed in a variety of human cell lines and exhibit tumor-stage dependent overexpression in tissues from breast cancer patients. We found that H4G localized primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha-helix 3 of the histone fold motif with a histone chaperone, nucleophosmin 1. In addition, we found that modulating H4G expression affects rRNA expression levels, protein synthesis rates and cell-cycle progression. Our data suggest that H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

FKBP25 participates in DNA double-strand break repair.

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification.

Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability.

FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.

The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module.

Prolyl isomerases are defined by a catalytic domain that facilitates the cis-trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.

Environmental influences on the epigenomes of herpetofauna and fish.

Herpetofauna (amphibians and reptiles) and fish represent important sentinel and indicator species for environmental and ecosystem health. It is widely accepted that the epigenome plays an important role in gene expression regulation. Environmental stimuli, including temperature and pollutants, influence gene activity, and there is growing evidence demonstrating that an important mechanism is through modulation of the epigenome. This has been primarily studied in human and mammalian models; relatively little is known about the impact of environmental conditions or pollutants on herpetofauna or fish epigenomes and the regulatory consequences of these changes on gene expression. Herein we review recent studies that have begun to address this deficiency, which have mainly focused on limited specific epigenetic marks and individual genes or large-scale global changes in DNA methylation, owing to the comparative ease of measurement. Greater understanding of the epigenetic influences of these environmental factors will depend on increased availability of relevant species-specific genomic sequence information to facilitate chromatin immunoprecipitation and DNA methylation experiments.

The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit.

Peptidyl-proline isomerases of the FK506-binding protein (FKBP) family belong to a class of enzymes that catalyze the cis-trans isomerization of prolyl-peptide bonds in proteins. A handful of FKBPs are found in the nucleus, implying that the isomerization of proline in nuclear proteins is enzymatically controlled. FKBP25 is a nuclear protein that has been shown to associate with chromatin modifiers and transcription factors. In this study, we performed the first proteomic characterization of FKBP25 and found that it interacts with numerous ribosomal proteins, ribosomal processing factors, and a small selection of chromatin modifiers. In agreement with previous reports, we found that nucleolin is a major FKBP25-interacting protein and demonstrated that this interaction is dependent on rRNA. FKBP25 interacts with the immature large ribosomal subunit in nuclear extract but does not associate with mature ribosomes, implicating this FKBP's action in ribosome biogenesis. Despite engaging nascent 60S ribosomes, FKBP25 does not affect steady-state levels of rRNAs or its pre-rRNA intermediates. We conclude that FKBP25 is likely recruited to preribosomes to chaperone one of the protein components of the ribosome large subunit.

T-cell receptor activation decreases excitability of cortical interneurons by inhibiting α7 nicotinic receptors.

Many proteins in the immune system are also expressed in the brain. One such class of immune proteins are T-cell receptors (TCRs), whose functions in T lymphocytes in adaptive immunity are well characterized. In the brain, TCRs are confined to neocortical neurons, but their functional role has not been determined. In mouse layer 1 neocortical neurons, TCR activation inhibited α7 nicotinic currents. TCRs modulated α7 currents via tyrosine phosphorylation of α7 nicotinic receptors (nAChRs) through src tyrosine kinases because eliminating lck kinase expression, coexpressing fyn kinase dead, or mutating tyrosine to alanine in α7 blocked the effect of TCR activation. We found that TCR stimulation decreased surface α7 nAChRs and reduced single-channel conductance. These results reveal that TCRs play a major role in the modulation of cholinergic neurotransmission in the brain mediated by α7 nAChRs and that this has a profound effect on regulating neuronal excitability.

Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization.

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

Intrinsic disorder of a nucleoplasmin-like histone chaperone specifies its discrete nuclear and nucleolar functions.

Nucleoplasmin (NPM) histone chaperones regulate distinct processes in the nucleus and nucleolus. While intrinsically disordered regions (IDRs) are hallmarks of NPMs, it is not clear whether all NPM functions require these unstructured features. We assessed the importance of IDRs in a yeast NPM-like protein and found that regulation of rDNA copy number and genetic interactions with the nucleolar RNA surveillance machinery require the highly conserved FKBP prolyl isomerase domain, but not the NPM domain or IDRs. By contrast, transcriptional repression in the nucleus requires IDRs. Furthermore, multiple lysines in polyacidic serine/lysine motifs of IDRs are required for both lysine polyphosphorylation and NPM-mediated transcriptional repression. These results demonstrate that this NPM-like protein relies on IDRs only for some of its chromatin-related functions.

Resolving the functions of peptidylprolyl isomerases: insights from the mutagenesis of the nuclear FKBP25 enzyme.

Peptidylprolyl isomerases have been implicated in chromatin regulation through their association with histones, chromatin-modifying enzymes and DNA-binding transcription factors. As with other post-translational modifications to proteins, a mechanistic understanding of the regulation of biological processes is fostered by loss-of-function studies both in vitro and in vivo. For peptidylprolyl isomerases, this can be accomplished with small-molecule inhibitors with high affinity for the isomerase active site or by mutation of amino acid residues that contribute to catalysis. In the present article, we review caveats to each of these approaches, and place emphasis on the thorough characterization of loss-of-function mutations in FKBPs (FK506-binding proteins). Using a case study of mutagenesis of the nuclear FKBP25 peptidylprolyl isomerase enzyme, we demonstrate that certain mutations generate a loss-of-function phenotype because they induce a complete loss of the FKBP domain fold, whereas other mutations are 'surgical' in that they ablate catalytic isomerase activity, while maintaining domain structure. Peptidylprolyl isomerases are thought to have both catalytic and non-catalytic functions, but differentiating between these mechanisms has proved to be challenging. The domain-destabilizing and surgical mutants described will facilitate the characterization of these two reported functions of peptidylprolyl isomerases.

Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides.

Alkylated guanidinium compounds exhibit microbiocidal activity in marine environments, yet the mode of action of these compounds has not been defined. A comprehensive chemical-genetic approach in budding yeast was used to define the biological processes affected by these compounds. N-Butyl-N'-decylguanidinium and N-hexyl-N'-(3-hydroxypropyl)-N''-octylguanidinium chlorides were shown to prevent yeast growth in a dose-dependent manner. All non-essential genes required for tolerance of sub-lethal amounts of these biocides were identified. These unbiased and systematic screens reveal the two related guanidinium compounds have a non-overlapping spectrum of targets in vivo. A functional tryptophan biosynthetic pathway is essential for tolerance of both biocides, which identifies tryptophan amino acid import as one process affected by these compounds. Further analysis of hypersensitive gene lists demonstrates that the substitutions on alkylated guanidiums confer important functional differences in vivo: one derivative renders the ability to generate acidic vacuoles essential, while the other is synthetically lethal with mutants in the transcriptional response to chemical stress. Altogether the results define the common and distinct biological processes affected by biocidal alkylated guanidinium salts.

The roles of peptidyl-proline isomerases in gene regulation.

The post-translational modification of proteins and enzymes provides a dynamic and reversible means to control protein function and transmit biological signals. While covalent modifications such as phosphorylation and acetylation have drawn much attention, in the past decade the involvement of peptidyl-proline isomerases (PPIs) in signaling and post-translational modification of protein function has become increasingly apparent. Three distinct families of PPI enzymes (parvulins, cyclophilins, and FK506-binding proteins (FKBPs)) each have the capacity to catalyze cis-trans proline isomerization in substrate proteins, and this modification can regulate both structure and function. In eukaryotic cells, a subset of these enzymes is localized to the nucleus, where they regulate gene expression at multiple control points. Here we summarize this body of work that together establishes a clear role of these enzymes as evolutionarily conserved players in the control of both transcription of mRNAs and the assembly of chromatin.

Ligand-induced disorder-to-order transitions characterized by structural proteomics and molecular dynamics simulations.

For disordered proteins, ligand binding can be a critical event that changes their structural dynamics. The ability to characterize such changes would facilitate the development of drugs designed to stabilize disordered proteins, whose mis-folding is important for a number of pathologies, including neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. In this study, we used hydrogen/deuterium exchange, differential crosslinking, differential surface modification, and molecular dynamics (MD) simulations to characterize the structural changes in disordered proteins that result from ligand binding. We show here that both an ATP-independent protein chaperone, Spy L32P, and the FK506 binding domain of a prolyl isomerase, FKBP-25 F145A/I223P, are disordered, yet exhibit structures that are distinct from chemically denatured unfolded states in solution, and that they undergo transitions to a more structured state upon ligand binding. These systems may serve as models for the characterization of ligand-induced disorder-to-order transitions in proteins using structural proteomics approaches. SIGNIFICANCE: In this study, we used hydrogen/deuterium exchange, differential crosslinking, differential surface modification, and molecular-dynamics simulations to characterize the structural changes in disordered proteins that result from ligand binding. The protein-ligand systems studied here (the ATP-independent protein chaperone, Spy L32P, and the FK506 binding domain of a prolyl isomerase, FKBP-25 F145A/I223P) may serve as models for understanding ligand-induced disorder-to-order transitions in proteins. Additionally, the structural proteomic techniques demonstrated here are shown to be effective tools for the characterization of disorder-to-order transitions and can be used to facilitate study of other systems in which this class of structural transition can be used for modulating major pathological features of disease, such as the abnormal protein aggregation that occurs with Parkinson's disease and Alzheimer's disease.

Advantages of next-generation sequencing versus the microarray in epigenetic research.

Several recent studies from the field of epigenetics have combined chromatin-immunoprecipitation (ChIP) with next-generation high-throughput sequencing technologies to describe the locations of histone post-translational modifications (PTM) and DNA methylation genome-wide. While these reports begin to quench the chromatin biologists thirst for visualizing where in the genome epigenetic marks are placed, they also illustrate several advantages of sequencing based genomics compared to microarray analysis. Accordingly, next-generation sequencing (NGS) technologies are now challenging microarrays as the tool of choice for genome analysis. The increased affordability of comprehensive sequence-based genomic analysis will enable new questions to be addressed in many areas of biology. It is inevitable that massively-parallel sequencing platforms will supercede the microarray for many applications, however, there are niches for microarrays to fill and interestingly we may very well witness a symbiotic relationship between microarrays and high-throughput sequencing in the future.

Histone Chaperone Paralogs Have Redundant, Cooperative, and Divergent Functions in Yeast.

Gene duplications increase organismal robustness by providing freedom for gene divergence or by increasing gene dosage. The yeast histone chaperones Fpr3 and Fpr4 are paralogs that can assemble nucleosomes in vitro; however, the genomic locations they target and their functional relationship is poorly understood. We refined the yeast synthetic genetic array approach to enable the functional dissection of gene paralogs. Applying this method to Fpr3 and Fpr4 uncovered redundant, cooperative, and divergent functions. While Fpr3 is uniquely involved in chromosome segregation, Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. We find that the TRAMP5 RNA exosome is critical for fitness in Δfpr3Δfpr4 yeast and leverage this information to identify an important role for Fpr4 at the 5' ends of protein coding genes. Additionally, Fpr4 and TRAMP5 negatively regulate RNAs from the nontranscribed spacers of ribosomal DNA. Yeast lacking Fpr3 and Fpr4 exhibit a genome instability phenotype at the ribosomal DNA, which implies that these histone chaperones regulate chromatin structure and DNA access at this location. Taken together. we provide genetic and transcriptomic evidence that Fpr3 and Fpr4 operate separately, cooperatively, and redundantly to regulate a variety of chromatin environments.