Response mechanisms of different Saccharomyces cerevisiae strains to succinic acid.

BACKGROUND: The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance to low pH conditions, ease of genetic manipulation, and extensive application in industrial processes. However, when compared with bacterial producers, the SA titers and productivities achieved by engineered S. cerevisiae strains were relatively low. To develop efficient SA-producing strains, it's necessary to clearly understand how S. cerevisiae cells respond to SA. RESULTS: In this study, we cultivated five S. cerevisiae strains with different genetic backgrounds under different concentrations of SA. Among them, KF7 and NBRC1958 demonstrated high tolerance to SA, whereas NBRC2018 displayed the least tolerance. Therefore, these three strains were chosen to study how S. cerevisiae responds to SA. Under a concentration of 20 g/L SA, only a few differentially expressed genes were observed in three strains. At the higher concentration of 60 g/L SA, the response mechanisms of the three strains diverged notably. For KF7, genes involved in the glyoxylate cycle were significantly downregulated, whereas genes involved in gluconeogenesis, the pentose phosphate pathway, protein folding, and meiosis were significantly upregulated. For NBRC1958, genes related to the biosynthesis of vitamin B6, thiamin, and purine were significantly downregulated, whereas genes related to protein folding, toxin efflux, and cell wall remodeling were significantly upregulated. For NBRC2018, there was a significant upregulation of genes connected to the pentose phosphate pathway, gluconeogenesis, fatty acid utilization, and protein folding, except for the small heat shock protein gene HSP26. Overexpression of HSP26 and HSP42 notably enhanced the cell growth of NBRC1958 both in the presence and absence of SA. CONCLUSIONS: The inherent activities of small heat shock proteins, the levels of acetyl-CoA and the strains' potential capacity to consume SA all seem to affect the responses and tolerances of S. cerevisiae strains to SA. These factors should be taken into consideration when choosing host strains for SA production. This study provides a theoretical basis and identifies potential host strains for the development of robust and efficient SA-producing strains.

Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses.

BACKGROUND: Saccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses-which is needed for reduced energy consumption of ethanol distillation at industrial scale-is yet to be achieved. RESULTS: In the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present. CONCLUSION: Our results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.

Reversing the directionality of reactions between non-oxidative pentose phosphate pathway and glycolytic pathway boosts mycosporine-like amino acid production in Saccharomyces cerevisiae.

BACKGROUND: Mycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production. RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells. CONCLUSION: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

TIR predictor and optimizer: Web-tools for accurate prediction of translation initiation rate and precision gene design in Saccharomyces cerevisiae.

Translation initiation is the primary determinant of the rate of protein production. The variation in the rate with which this step occurs can cause up to three orders of magnitude differences in cellular protein levels. Several mRNA features, including mRNA stability in proximity to the start codon, coding sequence length, and presence of specific motifs in the mRNA molecule, have been shown to influence the translation initiation rate. These molecular factors acting at different strengths allow precise control of in vivo translation initiation rate and thus the rate of protein synthesis. However, despite the paramount importance of translation initiation rate in protein synthesis, accurate prediction of the absolute values of initiation rate remains a challenge. In fact, as of now, there is no available model for predicting the initiation rate in Saccharomyces cerevisiae. To address this, we train a machine learning model for predicting the in vivo initiation rate in S. cerevisiae transcripts. The model is trained using a diverse set of mRNA transcripts, enabling the comparison of initiation rates across different transcripts. Our model exhibited excellent accuracy in predicting the translation initiation rate and demonstrated its effectiveness with both endogenous and exogenous transcripts. Then, by combining the machine learning model with the Monte-Carlo search algorithm, we have also devised a method to optimize the nucleotide sequence of any gene to achieve a specific target initiation rate. The machine learning model we've developed for predicting translation initiation rates, along with the gene optimization method, are deployed as a web server. Both web servers are accessible for free at the following link: ajeetsharmalab.com/TIRPredictor. Thus, this research advances our fundamental understanding of translation initiation processes, with direct applications in biotechnology.

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

Truncating the spliceosomal 'rope protein' Prp45 results in Htz1 dependent phenotypes.

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.

Saccharomyces cerevisiae oral immunization in mice using multi-antigen of the African swine fever virus elicits a robust immune response.

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens-KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L- induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV.

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories.

BACKGROUND: Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis. RESULTS: We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L. CONCLUSIONS: We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.

Hypothesis: evidence that the PRS gene products of Saccharomyces cerevisiae support both PRPP synthesis and maintenance of cell wall integrity.

The gene products of PRS1-PRS5 in Saccharomyces cerevisiae are responsible for the production of PRPP (5-phospho-D-ribosyl-α-1-pyrophosphate). However, it has been demonstrated that they are also involved in the cell wall integrity (CWI) signalling pathway as shown by protein-protein interactions (PPIs) with, for example Slt2, the MAP kinase of the CWI pathway. The following databases: SGD, BioGRID and Hit Predict, which collate PPIs from various research papers, have been scrutinized for evidence of PPIs between Prs1-Prs5 and components of the CWI pathway. The level of certainty in PPIs was verified by interaction scores available in the Hit Predict database revealing that well-documented interactions correspond with higher interaction scores and can be graded as high confidence interactions based on a score > 0.28, an annotation score ≥ 0.5 and a method-based high confidence score level of ≥ 0.485. Each of the Prs1-Prs5 polypeptides shows some degree of interaction with the CWI pathway. However, Prs5 has a vital role in the expression of FKS2 and Rlm1, previously only documented by reporter assay studies. This report emphasizes the importance of investigating interactions using more than one approach since every method has its limitations and the use of different methods, as described herein, provides complementary experimental and statistical data, thereby corroborating PPIs. Since the experimental data described so far are consistent with a link between PRPP synthetase and the CWI pathway, our aim was to demonstrate that these data are also supported by high-throughput bioinformatic analyses promoting our hypothesis that two of the five PRS-encoding genes contain information required for the maintenance of CWI by combining data from our targeted approach with relevant, unbiased data from high-throughput analyses.

New insight into protective effect against oxidative stress and biosynthesis of exopolysaccharides produced by Lacticaseibacillus paracasei NC4 from fermented eggplant.

Fermented eggplant is a traditional fermented food, however lactic acid bacteria capable of producing exopolysaccharide (EPS) have not yet been exploited. The present study focused on the production and protective effects against oxidative stress of an EPS produced by Lacticaseibacillus paracasei NC4 (NC4-EPS), in addition to deciphering its genomic features and EPS biosynthesis pathway. Among 54 isolates tested, strain NC4 showed the highest EPS yield and antioxidant activity. The maximum EPS production (2.04 ± 0.11 g/L) was achieved by culturing in MRS medium containing 60 g/L sucrose at 37 °C for 48 h. Under 2 mM H2O2 stress, the survival of a yeast model Saccharomyces cerevisiae treated with 0.4 mg/mL NC4-EPS was 2.4-fold better than non-treated cells, which was in agreement with the catalase and superoxide dismutase activities measured from cell lysates. The complete genome of NC4 composed of a circular chromosome of 2,888,896 bp and 3 circular plasmids. The NC4 genome comprises more genes with annotated function in nitrogen metabolism, phosphorus metabolism, cell division and cell cycle, and iron acquisition and metabolism as compared to other reported L. paracasei. Of note, the eps gene cluster is not conserved across L. paracasei. Pathways of sugar metabolism for EPS biosynthesis were proposed for the first time, in which gdp pathway only present in few plant-derived bacteria was identified. These findings shed new light on the cell-protective activity and biosynthesis of EPS produced by L. paracasei, paving the way for future efforts to enhance yield and tailor-made EPS production for food and pharmaceutical industries.

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

Structural basis for the distinct roles of non-conserved Pro116 and conserved Tyr124 of BCH domain of yeast p50RhoGAP.

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.

Uncharacterized yeast gene YBR238C, an effector of TORC1 signaling in a mitochondrial feedback loop, accelerates cellular aging via HAP4- and RMD9-dependent mechanisms.

Uncovering the regulators of cellular aging will unravel the complexity of aging biology and identify potential therapeutic interventions to delay the onset and progress of chronic, aging-related diseases. In this work, we systematically compared genesets involved in regulating the lifespan of Saccharomyces cerevisiae (a powerful model organism to study the cellular aging of humans) and those with expression changes under rapamycin treatment. Among the functionally uncharacterized genes in the overlap set, YBR238C stood out as the only one downregulated by rapamycin and with an increased chronological and replicative lifespan upon deletion. We show that YBR238C and its paralog RMD9 oppositely affect mitochondria and aging. YBR238C deletion increases the cellular lifespan by enhancing mitochondrial function. Its overexpression accelerates cellular aging via mitochondrial dysfunction. We find that the phenotypic effect of YBR238C is largely explained by HAP4- and RMD9-dependent mechanisms. Furthermore, we find that genetic- or chemical-based induction of mitochondrial dysfunction increases TORC1 (Target of Rapamycin Complex 1) activity that, subsequently, accelerates cellular aging. Notably, TORC1 inhibition by rapamycin (or deletion of YBR238C) improves the shortened lifespan under these mitochondrial dysfunction conditions in yeast and human cells. The growth of mutant cells (a proxy of TORC1 activity) with enhanced mitochondrial function is sensitive to rapamycin whereas the growth of defective mitochondrial mutants is largely resistant to rapamycin compared to wild type. Our findings demonstrate a feedback loop between TORC1 and mitochondria (the TORC1-MItochondria-TORC1 (TOMITO) signaling process) that regulates cellular aging processes. Hereby, YBR238C is an effector of TORC1 modulating mitochondrial function.

Nonsense-mediated mRNA decay of metal-binding activator MAC1 is dependent on copper levels and 3'-UTR length in Saccharomyces cerevisiae.

The nonsense-mediated mRNA decay (NMD) pathway was initially identified as a surveillance pathway that degrades mRNAs containing premature termination codons (PTCs). NMD is now also recognized as a post-transcriptional regulatory pathway that regulates the expression of natural mRNAs. Earlier studies demonstrated that regulation of functionally related natural mRNAs by NMD can be differential and condition-specific in Saccharomyces cerevisiae. Here, we investigated the regulation of MAC1 mRNAs by NMD in response to copper as well as the role the MAC1 3'-UTR plays in this regulation. MAC1 is a copper-sensing transcription factor that regulates the high-affinity copper uptake system. MAC1 expression is activated upon copper deprivation. We found that MAC1 mRNAs are regulated by NMD under complete minimal (CM) but escaped NMD under low and high copper conditions. Mac1 protein regulated gene, CTR1 is not regulated by NMD in conditions where MAC1 mRNAs are NMD sensitive. We also found that the MAC1 3'-UTR is the NMD targeting feature on the mRNAs, and that MAC1 mRNAs lacking 3'-UTRs were stabilized during copper deprivation. Our results demonstrate a mechanism of regulation for a metal-sensing transcription factor, at both the post-transcriptional and post-translational levels, where MAC1 mRNA levels are regulated by NMD and copper, while the activity of Mac1p is controlled by copper levels.

Using Dimensionality Reduction to Visualize Phenotypic Changes in High-Throughput Microscopy.

High-throughput microscopy has enabled screening of cell phenotypes at unprecedented scale. Systematic identification of cell phenotype changes (such as cell morphology and protein localization changes) is a major analysis goal. Because cell phenotypes are high-dimensional, unbiased approaches to detect and visualize the changes in phenotypes are still needed. Here, we suggest that changes in cellular phenotype can be visualized in reduced dimensionality representations of the image feature space. We describe a freely available analysis pipeline to visualize changes in protein localization in feature spaces obtained from deep learning. As an example, we use the pipeline to identify changes in subcellular localization after the yeast GFP collection was treated with hydroxyurea.

On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

Swd2/Cps35 determines H3K4 tri-methylation via interactions with Set1 and Rad6.

BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.

General regulatory factors exert differential effects on nucleosome sliding activity of the ISW1a complex.

BACKGROUND: Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown. RESULTS: Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome. CONCLUSIONS: Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.

The 6-kilodalton peptide 1 in plant viruses of the family Potyviridae is a viroporin.

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.