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Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases., (© 2023. The Author(s).)

RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male patients with RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy and investigate potential underlying mechanisms. In addition, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. We characterized an Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20 -KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart's ability to compensate for stress, we exposed Rbm20 -KO mice to acute catecholaminergic stimulation and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20 -R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients and show that these patients do not possess sex and gender differences in disease severity. Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands. NEW & NOTEWORTHY Optimal management of the cardiac disease is increasingly personalized, partly because of differences in outcomes between sexes. RBM20 cardiomyopathy has been described to be more severe in male patients, and this carries the risk that male patients are more scrutinized in the clinic than female patients. Our findings do not support this observation and suggest that treatment should not differ between male and female RBM20 cardiomyopathy patients, but instead should focus on the underlying disease mechanism.

Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context-including the adjacent transcriptional environment-also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency., (Copyright © 2020 Mueller et al.)

The cytoplasmic peptide:N-glycanase (Ngly1) is a de-N-glycosylating enzyme that cleaves N-glycans from misfolded glycoproteins and is involved in endoplasmic reticulum-associated degradation. The recent discovery of NGLY1-deficiency, which causes severe systemic symptoms, drew attention to the physiological function of Ngly1 in mammals. While several studies have been carried out to reveal the physiological necessity of Ngly1, the semi-lethal nature of Ngly1-deficient animals made it difficult to analyze its function in adults. In this study, we focus on the physiological function of Ngly1 in liver (hepatocyte)-specific Ngly1-deficient mice generated using the cre-loxP system. We found that hepatocyte-specific Ngly1-deficient mice showed abnormal hepatocyte nuclear size/morphology with aging but did not show other notable defects in unstressed conditions. This nuclear phenotype did not appear to be related to the function of the only gene currently reported to rescue Ngly1-deficient murine lethality so far, endo-β-N-acetylglucosaminidase. We also found that under a high fructose diet induced stress, the hepatocyte-specific Ngly1-deletion resulted in liver transaminases elevation and increased lipid droplet accumulation. We showed that the processing and localization of the transcription factor, nuclear factor erythroid 2-like 1 (Nfe2l1), was impaired in the Ngly1-deficient hepatocytes. Therefore, Nfe2l1, at least partially, contributes to the phenotypes observed in hepatocyte-specific Ngly1-deficient mice. Our results indicate that Ngly1 plays important roles in the adult liver impacting nuclear morphology and lipid metabolism. Hepatocyte-specific Ngly1-deficient mice could thus serve as a valuable animal model for assessing in vivo efficacy of drugs and/or treatment for NGLY1-deficiency., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.

Condensins are genome organisers that shape chromosomes and promote their accurate transmission. Several studies have also implicated condensins in gene expression, although any mechanisms have remained enigmatic. Here, we report on the role of condensin in gene expression in fission and budding yeasts. In contrast to previous studies, we provide compelling evidence that condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis. We further show that the changes in gene expression in post-mitotic fission yeast cells that result from condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite condensin inactivation. Thus, chromosome instability, rather than a direct role of condensin in the transcription process, changes gene expression. This knowledge challenges the concept of gene regulation by canonical condensin complexes., Competing Interests: CH, XR, LM, XS, CB, SC, ET, SC, LS, CH, SM, PB No competing interests declared, (© 2018, Hocquet et al.)

The conserved INO80 chromatin remodeling complex is involved in regulation of DNA damage repair, replication and transcription. It is commonly recruited to the transcription start region and contributes to the establishment of promoter-proximal nucleosomes. We find a substantial influence of INO80 on nucleosome dynamics and gene expression during stress induced transcription. Transcription induced by osmotic stress leads to genome-wide remodeling of promoter proximal nucleosomes. INO80 function is required for timely return of evicted nucleosomes to the 5΄ end of induced genes. Reduced INO80 function in Arp8-deficient cells leads to correlated prolonged transcription and nucleosome eviction. INO80 and the related complex SWR1 regulate incorporation of the H2A.Z isoform at promoter proximal nucleosomes. However, H2A.Z seems not to influence osmotic stress induced gene regulation. Furthermore, we show that high rates of transcription promote INO80 recruitment to promoter regions, suggesting a connection between active transcription and promoter proximal nucleosome remodeling. In addition, we find that absence of INO80 enhances bidirectional promoter activity at highly induced genes and expression of a number of stress induced transcripts. We suggest that INO80 has a direct repressive role via promoter proximal nucleosome remodeling to limit high levels of transcription in yeast., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genomes from yeast to humans are subject to pervasive transcription. A single round of pervasive transcription is sufficient to alter local chromatin conformation, nucleosome dynamics and gene expression, but is hard to distinguish from background signals. Size fractionated native elongating transcript sequencing (sfNET-Seq) was developed to precisely map nascent transcripts independent of expression levels. RNAPII-associated nascent transcripts are fractionation into different size ranges before library construction. When anchored to the transcription start sites (TSS) of annotated genes, the combined pattern of the output metagenes gives the expected reference pattern. Bioinformatic pattern matching to the reference pattern identified 9542 transcription units in Saccharomyces cerevisiae, of which 47% are coding and 53% are noncoding. In total, 3113 (33%) are unannotated noncoding transcription units. Anchoring all transcription units to the TSS or polyadenylation site (PAS) of annotated genes reveals distinctive architectures of linked pairs of divergent transcripts approximately 200nt apart. The Reb1 transcription factor is enriched 30nt downstream of the PAS only when an upstream (TSS -60nt with respect to PAS) noncoding transcription unit co-occurs with a downstream (TSS +150nt) coding transcription unit and acts to limit levels of upstream antisense transcripts. The potential for extensive transcriptional interference is evident from low abundance unannotated transcription units with variable TSS (median -240nt) initiating within a 500nt window upstream of, and transcribing over, the promoters of protein-coding genes. This study confirms a highly interleaved yeast genome with different types of transcription units altering the chromatin landscape in distinctive ways, with the potential to exert extensive regulatory control., (© 2024 The Authors. Yeast published by John Wiley & Sons Ltd.)

Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live-cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage-coat proteins that associate tightly with messenger RNA (mRNA) stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper-osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress-induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state., (© 2024 The Authors. Yeast published by John Wiley & Sons Ltd.)

The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The major genetic risk factor for Alzheimer's disease (AD), APOE4, accelerates beta-amyloid (Aβ) plaque formation, but whether this is caused by APOE expressed in microglia or astrocytes is debated. We express here the human APOE isoforms in astrocytes in an Apoe-deficient AD mouse model. This is not only sufficient to restore the amyloid plaque pathology but also induces the characteristic transcriptional pathological responses in Apoe-deficient microglia surrounding the plaques. We find that both APOE4 and the protective APOE2 from astrocytes increase fibrillar plaque deposition, but differentially affect soluble Aβ aggregates. Microglia and astrocytes show specific alterations in function of APOE genotype expressed in astrocytes. Our experiments indicate a central role of the astrocytes in APOE mediated amyloid plaque pathology and in the induction of associated microglia responses. Synopsis: In the early phase of Alzheimer's disease, astrocytes are the primary source of APOE in the brain. This study examined the effects of astrocyte-derived human APOE isoforms in APOE-deficient mouse brains on amyloid pathology and related cellular responses. Apoe-deficient mouse brains: Displayed no fibrillar amyloid plaques and had increased levels of soluble Aβ aggregates. Human APOE isoforms in astrocytes: Expression in astrocytes alone induced formation of fibrillar amyloid plaques (APOE4 > APOE2 > APOE3) and reduced soluble Aβ aggregates (APOE2 > APOE4 ≈ APOE3). Astrocyte transcriptomics: APOE isoforms caused differential changes, suggesting possible impairment of proteostasis and autophagy in APOE4 astrocytes. Microglial response: Apoe-deficient microglia exhibited clustering and a DAM-like response to amyloid pathology induced by astrocyte-secreted APOE. Astrocyte-derived APOE and microglia: Astrocyte-derived APOE could induce fibrillar amyloid plaques even without the presence of microglia, although to a lesser extent. In the early phase of Alzheimer's disease, astrocytes are the primary source of APOE in the brain. This study examined the effects of astrocyte-derived human APOE isoforms in APOE-deficient mouse brains on amyloid pathology and related cellular responses. [ABSTRACT FROM AUTHOR]

In recent years, the role of gut microbiota (GM) in bladder cancer has attracted significant attention. Research indicates that GM not only contributes to bladder carcinogenesis but also influences the efficacy of adjuvant therapies for bladder cancer. Despite this, interventions targeting GM have not been widely employed in the prevention and treatment of bladder cancer, mainly due to the incomplete understanding of the complex interactions between the host and gut flora. Simultaneously, aberrantly expressed non-coding RNAs (ncRNAs) have been frequently associated with bladder cancer, playing crucial roles in processes such as cell proliferation, invasion, and drug resistance. It is widely known that the regulation of GM-mediated host pathophysiological processes is partly regulated through epigenetic pathways. At the same time, ncRNAs are increasingly regarded as GM signaling molecules involved in GM-mediated epigenetic regulation. Accordingly, this review analyzes the ncRNAs that are closely related to the GM in the context of bladder cancer occurrence and treatment, and summarizes the role of their interaction with the GM in bladder cancer-related phenotypes. The aim is to delineate a regulatory network between GM and ncRNAs and provide a new perspective for the study and prevention of bladder cancer. [ABSTRACT FROM AUTHOR]

Cryptic transcription refers to the unintended expression of non-canonical sites within the genome, producing aberrant RNA and proteins that may disrupt cellular functions. In this opinion piece, I will explore the role of histone modifications in modulating cryptic transcription and its implications for gene expression and cellular integrity, particularly with a focus on H3K36 and H3K4 methylation marks. H3K36 tri-methylation plays a crucial role in maintaining chromatin integrity by facilitating the recruitment of the Rpd3S histone deacetylase (HDAC) complex, which helps restore closed chromatin states following transcription and prevents cryptic initiation within gene bodies. In parallel, crosstalk between H3K4 di-methylation and histone ubiquitylation and sumoylation is critical for recruiting the Set3 HDAC complex, which maintains low histone acetylation levels in gene bodies and further suppresses cryptic transcription. Therefore, by elucidating these regulatory mechanisms, this opinion highlights the intricate interplay of histone modifications in preserving transcriptional fidelity and suggests potential pathways for future research to develop novel therapies for age-related disorders and other diseases associated with dysregulated gene expression. [ABSTRACT FROM AUTHOR]

Efficient transcriptional regulation of the stress response is critical for microorganism survival. In yeast, stress-related gene expression, particularly for antioxidant enzymes like catalases, mitigates reactive oxygen species such as hydrogen peroxide (H2O2), preventing cell damage. The halotolerant yeast Debaryomyces hansenii shows oxidative stress tolerance, largely due to high catalase activity from DhCTA and DhCTT genes. This study evaluates D. hansenii's response to oxidative stress caused by H2O2 under saline conditions, focusing on cell viability, gene expression, and catalase activity. Chromatin organization in the promoter of DhCTA and DhCTT was analyzed, revealing low nucleosome occupancy in promoter regions, correlating with active gene expression. Stress-related motifs for transcription factors like Msn2/4 and Sko1 were found, suggesting regulation by the DhHog1 MAP kinase. Analysis of a Dhhog1Δ mutant showed DhHog1's role in DhCTA expression under H2O2 or NaCl conditions. These findings highlight DhHog1's critical role in regulating the stress response in D. hansenii, offering insights for enhancing stress tolerance in halotolerant yeasts, particularly for industrial applications in saline wastewater management. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, oxidative phosphorylation involves multisubunit complexes of mixed genetic origin. Assembling these complexes requires an organelle-independent synchronizing system for the proper expression of nuclear and mitochondrial genes. Here we show that proper expression of the F1FO ATP synthase (complex V) depends on a cytosolic complex (AME) made of two aminoacyl-tRNA synthetases (cERS and cMRS) attached to an anchor protein, Arc1p. When yeast cells adapt to respiration the Snf1/4 glucose-sensing pathway inhibits ARC1 expression triggering simultaneous release of cERS and cMRS. Free cMRS and cERS relocate to the nucleus and mitochondria, respectively, to synchronize nuclear transcription and mitochondrial translation of ATP synthase genes. Strains releasing asynchronously the two aminoacyl-tRNA synthetases display aberrant expression of nuclear and mitochondrial genes encoding subunits of complex V resulting in severe defects of the oxidative phosphorylation mechanism. This work shows that the AME complex coordinates expression of enzymes that require intergenomic control., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed.

Recent research has uncovered extensive variability in the boundaries of transcript isoforms, yet the functional consequences of this variation remain largely unexplored. Here, we systematically discriminate between the molecular phenotypes of overlapping coding and non-coding transcriptional events from each genic locus using a novel genome-wide, nucleotide-resolution technique to quantify the half-lives of 3' transcript isoforms in yeast. Our results reveal widespread differences in stability among isoforms for hundreds of genes in a single condition, and that variation of even a single nucleotide in the 3' untranslated region (UTR) can affect transcript stability. While previous instances of negative associations between 3' UTR length and transcript stability have been reported, here, we find that shorter isoforms are not necessarily more stable. We demonstrate the role of RNA-protein interactions in conditioning isoform-specific stability, showing that PUF3 binds and destabilizes specific polyadenylation isoforms. Our findings indicate that although the functional elements of a gene are encoded in DNA sequence, the selective incorporation of these elements into RNA through transcript boundary variation allows a single gene to have diverse functional consequences.

MKT1 is a pleiotropic stress response gene identified by several quantitative trait studies with MKT1 89G as a causal variant, contributing to growth advantage in multiple stress environments. MKT1 has been shown to regulate HO endonuclease posttranscriptionally via the Pbp1-Pab1 complex. RNA-binding protein Puf3 modulates a set of nuclear-encoded mitochondrial transcripts whose expression was found to be affected by MKT1 alleles. This study attempts to relate the MKT1 allele-derived growth advantage with the stability of Puf3 targets during stress and elucidate the roles of Pbp1 and Puf3 in this mechanism. Our results showed that the growth advantage of the MKT1 89G allele in cycloheximide and H 2 O 2 was PBP1-dependent, whereas in 4-nitroquinoline 1-oxide, the growth advantage was dependent on both PUF3 and PBP1. We compared the messenger RNA decay kinetics of a set of Puf3 targets in multiple stress environments to understand the allele-specific regulation by MKT1. In oxidative stress, the MKT1 89G allele modulated the differential expression of nuclear-encoded mitochondrial genes in a PBP1- and PUF3-dependent manner. Additionally, MKT1 89G stabilised Puf3 targets, namely, COX17, MRS1 and RDL2, in an allele and stress-specific manner. Our results showed that COX17, MRS1 and RDL2 had a stress-specific response in stress environments, with the MKT1 89G allele contributing to better growth; this response was both PBP1- and PUF3-dependent. Our results indicate that the common allele, MKT1 89G , regulates stress responses by differentially stabilising Puf3-target mitochondrial genes, which allows for the strain's better growth in stress environments., (© 2023 John Wiley & Sons Ltd.)

Understanding the relationship between DNA sequence variation and phenotypic variation in complex or quantitative traits is one of the major challenges in modern biology. We are witnessing a deluge of DNA sequence information and association studies of genetic polymorphisms with phenotypes of interest in families and populations. In addition, it has become clear that large portions of eukaryotic genomes beyond protein-coding genes are transcribed, generating numerous noncoding RNA (ncRNA) molecules whose functions remain mostly unknown.DNA oligonucleotide microarrays constitute a powerful technology for studying the expression of genes in different organisms. The Saccharomyces cerevisiae tiling array presents a significant advance over previous array-based platforms. It has a high density of overlapping probes that start on average every 8 bp along each strand of the genome, enabling precise definition of transcript structure. Furthermore, the array includes probes specific for the polymorphic positions of another, distantly related yeast strain, allowing accurate measurement of allele-specific expression in a hybrid of the two strains. This technology thus allows high-resolution, quantitative, strand- and allele-specific measurements of transcription from a full eukaryotic genome. In this chapter, we describe the methods for extracting RNA, synthesizing first-strand cDNA, fragmenting, and labeling of samples for hybridization to the tiling array. Combining genome-wide information on variation in DNA sequence with variation in transcript structure and levels promises to increase our understanding of the genotype-to-phenotype relationship.

The Set3 histone deacetylase complex (Set3C) binds histone H3 dimethylated at lysine 4 (H3K4me2) to mediate deacetylation of histones in 5'-transcribed regions. To discern how Set3C affects gene expression, genome-wide transcription was analyzed in yeast undergoing a series of carbon source shifts. Deleting SET3 primarily caused changes during transition periods, as genes were induced or repressed. Surprisingly, a majority of Set3-affected genes are overlapped by noncoding RNA (ncRNA) transcription. Many Set3-repressed genes have H3K4me2 instead of me3 over promoter regions, due to either reduced H3K4me3 or ncRNA transcription from distal or antisense promoters. Set3C also represses internal cryptic promoters, but in different regions of genes than the Set2/Rpd3S pathway. Finally, Set3C stimulates some genes by repressing an overlapping antagonistic antisense transcript. These results show that overlapping noncoding transcription can fine-tune gene expression, not via the ncRNA but by depositing H3K4me2 to recruit the Set3C deacetylase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms., Competing Interests: The authors have declared that no competing interests exist.

Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 5' leader of pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in composition, as bacterial RNase P contains a catalytic RNA core and one protein subunit, while eukaryotic nuclear RNase P retains the catalytic RNA but has at least nine protein subunits. The additional eukaryotic protein subunits most likely provide additional functionality to RNase P, with one possibility being additional RNA recognition capabilities. To investigate the possible range of additional RNase P substrates in vivo, a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting that nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turn over pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively nonspecific capacity to bind and cleave mixed sequence RNAs, these data suggest that nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.

Genome-wide transcription profiling has revealed extensive expression of non-coding RNAs antisense to genes, yet their functions, if any, remain to be understood. In this study, we perform a systematic analysis of sense-antisense expression in response to genetic and environmental changes in yeast. We find that antisense expression is associated with genes of larger expression variability. This is characterized by more 'switching off' at low levels of expression for genes with antisense compared to genes without, yet similar expression at maximal induction. By disrupting antisense transcription, we demonstrate that antisense expression confers an on-off switch on gene regulation for the SUR7 gene. Consistent with this, genes that must respond in a switch-like manner, such as stress-response and environment-specific genes, are enriched for antisense expression. In addition, our data provide evidence that antisense expression initiated from bidirectional promoters enables the spreading of regulatory signals from one locus to neighbouring genes. These results indicate a general regulatory effect of antisense expression on sense genes and emphasize the importance of antisense-initiating regions downstream of genes in models of gene regulation.

In the last decade, it became clear that transcription goes far beyond that of protein-coding genes. Most RNA molecules are transcribed from intergenic regions or introns and exhibit much variability in size, expression level, secondary structure, and evolutionary conservation. While for several types of non-coding RNAs some cellular functions have been reported, like for micro-RNAs and small nucleolar RNAs, for most others no indications of function or regulation have so far been found. Therefore, the RNA population inside a cell is diverse and cryptic and, thus, demands powerful methods to study its composition, abundance, and structure. DNA oligonucleotide microarrays have proven to be of great utility to study transcription of genes in various organisms. Recently, due to advancement in microarray technology, tiling microarrays that extend transcription measurement to genomic regions beyond protein-coding genes were designed for several species. The Saccharomyces cerevisiae yeast tiling array contains overlapping probes across the full genomic sequence, with consecutive probes starting every 8 bp on average on each strand, enabling strand-specific measurement of transcription from a full eukaryotic genome. Here, we describe the methods used to extract yeast RNA, convert it into first-strand cDNA, fragment, and label it for hybridization to the tiling array. This protocol will enable researchers not only to study which genes are expressed and to what levels, but also to identify non-coding RNAs and to study the structure of transcripts including their untranslated regions, alternative start, stop, and processing sites. This information will allow understanding their roles inside cells.

Genome-wide pervasive transcription has been reported in many eukaryotic organisms, revealing a highly interleaved transcriptome organization that involves hundreds of previously unknown non-coding RNAs. These recently identified transcripts either exist stably in cells (stable unannotated transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway (cryptic unstable transcripts, CUTs). One characteristic of pervasive transcription is the extensive overlap of SUTs and CUTs with previously annotated features, which prompts questions regarding how these transcripts are generated, and whether they exert function. Single-gene studies have shown that transcription of SUTs and CUTs can be functional, through mechanisms involving the generated RNAs or their generation itself. So far, a complete transcriptome architecture including SUTs and CUTs has not been described in any organism. Knowledge about the position and genome-wide arrangement of these transcripts will be instrumental in understanding their function. Here we provide a comprehensive analysis of these transcripts in the context of multiple conditions, a mutant of the exosome machinery and different strain backgrounds of Saccharomyces cerevisiae. We show that both SUTs and CUTs display distinct patterns of distribution at specific locations. Most of the newly identified transcripts initiate from nucleosome-free regions (NFRs) associated with the promoters of other transcripts (mostly protein-coding genes), or from NFRs at the 3' ends of protein-coding genes. Likewise, about half of all coding transcripts initiate from NFRs associated with promoters of other transcripts. These data change our view of how a genome is transcribed, indicating that bidirectionality is an inherent feature of promoters. Such an arrangement of divergent and overlapping transcripts may provide a mechanism for local spreading of regulatory signals-that is, coupling the transcriptional regulation of neighbouring genes by means of transcriptional interference or histone modification.

Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.

Recent transcription profiling studies have revealed an unanticipatedly large proportion of antisense transcription across eukaryotic and bacterial genomes. However, the extent and significance of antisense transcripts is controversial partly because experimental artifacts are suspected. Here, we present a method to generate clean genome-wide transcriptome profiles, using actinomycin D (ActD) during reverse transcription. We show that antisense artifacts appear to be triggered by spurious synthesis of second-strand cDNA during reverse transcription reactions. Strand-specific hybridization signals obtained from Saccharomyces cerevisiae tiling arrays were compared between samples prepared with and without ActD. Use of ActD removed about half of the detectable antisense transcripts, consistent with their being artifacts, while sense expression levels and about 200 antisense transcripts were not affected. Our findings thus facilitate a more accurate assessment of the extent and position of antisense transcription, towards a better understanding of its role in cells.

Many distantly related organisms have convergently evolved traits and lifestyles that enable them to live in similar ecological environments. However, the extent of phenotypic convergence evolving through the same or distinct genetic trajectories remains an open question. Here, we leverage a comprehensive dataset of genomic and phenotypic data from 1,049 yeast species in the subphylum Saccharomycotina (Kingdom Fungi, Phylum Ascomycota) to explore signatures of convergent evolution in cactophilic yeasts, ecological specialists associated with cacti. We inferred that the ecological association of yeasts with cacti arose independently approximately 17 times. Using a machine learning–based approach, we further found that cactophily can be predicted with 76% accuracy from both functional genomic and phenotypic data. The most informative feature for predicting cactophily was thermotolerance, which we found to be likely associated with altered evolutionary rates of genes impacting the cell envelope in several cactophilic lineages. We also identified horizontal gene transfer and duplication events of plant cell wall–degrading enzymes in distantly related cactophilic clades, suggesting that putatively adaptive traits evolved independently through disparate molecular mechanisms. Notably, we found that multiple cactophilic species and their close relatives have been reported as emerging human opportunistic pathogens, suggesting that the cactophilic lifestyle—and perhaps more generally lifestyles favoring thermotolerance—might preadapt yeasts to cause human disease. This work underscores the potential of a multifaceted approach involving high-throughput genomic and phenotypic data to shed light onto ecological adaptation and highlights how convergent evolution to wild environments could facilitate the transition to human pathogenicity. Many distantly related organisms have convergently evolved traits and lifestyles that enable them to live in similar ecological environments. This study of 1,049 yeast species reveals that across 17 convergent transitions to cactus-association (cactophily), putatively adaptive traits evolved independently through disparate molecular mechanisms. [ABSTRACT FROM AUTHOR]

Cytoplasmic poly(A)-binding protein (PABPC; Pab1 in yeast) is thought to be involved in multiple steps of post-transcriptional control, including translation initiation, translation termination, and mRNA decay. To understand both the direct and indirect roles of PABPC in more detail, we have employed mass spectrometry to assess the abundance of the components of the yeast proteome, as well as RNA-Seq and Ribo-Seq to analyze changes in the abundance and translation of the yeast transcriptome, in cells lacking the PAB1 gene. We find that pab1Δ cells manifest drastic changes in the proteome and transcriptome, as well as defects in translation initiation and termination. Defects in translation initiation and the stabilization of specific classes of mRNAs in pab1Δ cells appear to be partly indirect consequences of reduced levels of specific initiation factors, decapping activators, and components of the deadenylation complex in addition to the general loss of Pab1's direct role in these processes. Cells devoid of Pab1 also manifested a nonsense codon readthrough phenotype indicative of a defect in translation termination. Collectively, our results indicate that, unlike the loss of simpler regulatory proteins, elimination of cellular Pab1 is profoundly pleiotropic and disruptive to numerous aspects of post-transcriptional regulation. Author summary: Many human diseases are caused by having too much or too little of certain cellular proteins. The amount of an individual protein is influenced by the level of its messenger mRNA (mRNA) and the efficiency of translation of the mRNA into a polypeptide chain by the ribosomes. Cytoplasmic poly(A)-binding protein (PABPC) plays numerous roles in the regulation of this multi-staged process, but understanding its specific role has been challenging because it is sometimes unclear whether experimental results are related to PABPC's direct role in a specific biochemical process or to indirect effects of its other roles, leading to conflicting models of PABPC's functions between studies. Here, we characterized defects in different stages of protein synthesis in response to loss of PABPC in yeast cells by measuring whole-cell levels of mRNAs, ribosome-associated mRNAs, and proteins. We demonstrated that defects in most steps of protein synthesis other than the last can be explained by reduced levels of mRNAs that code for proteins important for that step in addition to loss of PABPC's direct role on that step. Our data and analyses serve as resources aiding interpretation of experiments in which PABPC levels have been manipulated and generally point to the resulting complexity of such manipulations. [ABSTRACT FROM AUTHOR]

Genomic rearrangements play a crucial role in shaping biological phenotypic diversity and driving species evolution. Synthetic chromosome rearrangement and modification by LoxP-mediated evolution (SCRaMbLE) has been applied to explore large-scale genomic rearrangements, yet it has been observed that these rearrangements occur exclusively in genomic regions containing loxPsym sites. Here, we found that SCRaMbLE of synthetic yeast harboring synthetic chromosome V and X can generate a variety of synthetic segment insertions into wild-type chromosomes, ranging from 1 to 300 kb. Furthermore, it was revealed that the novel insertions impacted the transcriptional level of neighboring regions and affected the production of exemplar pathway of zeaxanthin. Collectively, our results improve the understanding of the ability of SCRaMbLE to generate complex structural variations in nonsynthetic regions and provide a potential model to explore genomic transposable events. [ABSTRACT FROM AUTHOR]

During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. We analyzed the genome-wide transcriptional response to activation of the JNK pathway in the Drosophila embryo by serial analysis of gene expression (SAGE) and identified loci encoding cell adhesion molecules and cytoskeletal regulators as JNK responsive genes. The role of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, in embryogenesis was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly.

When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67–Mtr2 in the yeast Saccharomyces cerevisiae (TAP–p15 in humans)1,2. Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm3. Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.Antisense RNAs boost gene expression by accelerating the export of mRNA from the nucleus to the cytoplasm through the Dbp2-mediated formation of double-stranded RNAs, which might explain the prevalence of antisense RNAs. [ABSTRACT FROM AUTHOR]

The primary functions of mitochondria are to produce energy and participate in the apoptosis of cells, with them being highly conserved among eukaryotes. However, the composition of mitochondrial genomes, mitochondrial DNA (mtDNA) replication, and mitochondrial inheritance varies significantly among animals, plants, and fungi. Especially in fungi, there exists a rich diversity of mitochondrial genomes, as well as various replication and inheritance mechanisms. Therefore, a comprehensive understanding of fungal mitochondria is crucial for unraveling the evolutionary history of mitochondria in eukaryotes. In this review, we have organized existing reports to systematically describe and summarize the composition of yeast-like fungal mitochondrial genomes from three perspectives: mitochondrial genome structure, encoded genes, and mobile elements. We have also provided a systematic overview of the mechanisms in mtDNA replication and mitochondrial inheritance during bisexual mating. Additionally, we have discussed and proposed open questions that require further investigation for clarification. [ABSTRACT FROM AUTHOR]

BACKGROUND: Per- and polyfluoroalkyl Substances (PFAS) are synthetic chemicals widely detected in humans and the environment. Exposure to perfluorooctanesulfonic acid (PFOS) or perfluorohexanesulfonic acid (PFHxS) was previously shown to cause dark-phase hyperactivity in larval zebrafish. OBJECTIVES: The objective of this study was to elucidate the mechanism by which PFOS or PFHxS exposure caused hyperactivity in larval zebrafish. METHODS: Swimming behavior was assessed in 5-d postfertilization (dpf) larvae following developmental (1-4 dpf) or acute (5 dpf) exposure to 0.43-7.86μM PFOS, 7.87-120μ M PFHxS, or 0.4% dimethyl sulfoxide (DMSO). After developmental exposure and chemical washout at 4 dpf, behavior was also assessed at 5-8 dpf. RNA sequencing was used to identify differences in global gene expression to perform transcriptomic benchmark concentration-response (BMCT) modeling, and predict upstream regulators in PFOS- or PFHxS-exposed larvae. CRISPR/Cas9-based gene editing was used to knockdown peroxisome proliferator-activated receptors (ppars) pparaa/ab, pparda/db, or pparg at day 0. Knockdown crispants were exposed to 7.86μM PFOS or 0.4% DMSO from 1-4 dpf and behavior was assessed at 5 dpf. Coexposure with the ppard antagonist GSK3787 and PFOS was also performed RESULTS: Transient dark-phase hyperactivity occurred following developmental or acute exposure to PFOS or PFHxS, relative to the DMSO control. In contrast, visual startle response (VSR) hyperactivity only occurred following developmental exposure and was irreversible up to 8 dpf. Similar global transcriptomic profiles, BMCT estimates, and enriched functions were observed in PFOS- and PFHxS-exposed larvae, and ppars were identified as putative upstream regulators. Knockdown of pparda/db, but not pparaa/ab or pparg, blunted PFOS-dependent VSR hyperactivity to control levels. This finding was confirmed via antagonism of ppard in PFOS-exposed larvae. DISCUSSION: This work identifies a novel adverse outcome pathway for VSR hyperactivity in larval zebrafish. We demonstrate that developmental, but not acute, exposure to PFOS triggered persistent VSR hyperactivity that required ppard function. [ABSTRACT FROM AUTHOR]

The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments of human cancer cells. We develop a model that reliably quantifies mRNA-specific synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3'UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation. Author summary: In our work, we aim to quantify two fundamental processes in the life cycle of a eukaryotic messenger RNA (mRNA), namely export from the nuclear compartment to the cytosol, and degradation. To enable a genome-wide evaluation, we have implemented an experimental-bioinformatics approach. We use a chemical moiety, 4-thiouridine (4sU), which is incorporated into RNA molecules when added to the solution, to mark RNA which is recently synthesized and distinguish it from already existing RNA. We take a time series of recent and already existing RNA within both the nuclear and cytoplasmic cellular compartments. We have devised a computational model that can reliably quantify mRNA nuclear export and cytosolic degradation rates from this data. We revealed that the export of mRNA from the nucleus to the cytoplasm emerges as a comparatively tardy event. Subsequently, mRNA molecules within the cytoplasm experience swift degradation. This identifies the nucleus as the predominant residence for the greater portion of an mRNA's lifecycle. Yet, we have also found notable exceptions from this scheme which are immediately exported after transcription without delay, suggesting the plausible existence of alternative mRNA export pathways. In sum, our research underscores the pivotal role of nuclear mRNA export as a determining factor in the orchestration of mRNA metabolism and the regulation of gene expression within eukaryotic cells. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR–Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR–Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR–Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR–Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end. [ABSTRACT FROM AUTHOR]

Cells must change their properties in order to adapt to a constantly changing environment. Most of the cellular sensing and regulatory mechanisms described so far are based on proteins that serve as sensors, signal transducers, and effectors of signalling pathways, resulting in altered cell physiology. In recent years, however, remarkable examples of the critical role of non-coding RNAs in some of these regulatory pathways have been described in various organisms. In this review, we focus on all classes of non-coding RNAs that play regulatory roles during stress response, starvation, and ageing in different yeast species as well as in structured yeast populations. Such regulation can occur, for example, by modulating the amount and functional state of tRNAs, rRNAs, or snRNAs that are directly involved in the processes of translation and splicing. In addition, long non-coding RNAs and microRNA-like molecules are bona fide regulators of the expression of their target genes. Non-coding RNAs thus represent an additional level of cellular regulation that is gradually being uncovered. [ABSTRACT FROM AUTHOR]

The mitochondria contain their own genome derived from an alphaproteobacterial endosymbiont. From thousands of protein-coding genes originally encoded by their ancestor, only between 1 and about 70 are encoded on extant mitochondrial genomes (mitogenomes). Thanks to a dramatically increasing number of sequenced and annotated mitogenomes a coherent picture of why some genes were lost, or relocated to the nucleus, is emerging. In this review, we describe the characteristics of mitochondria-to-nucleus gene transfer and the resulting varied content of mitogenomes across eukaryotes. We introduce a 'burst-upon-drift' model to best explain nuclear-mitochondrial population genetics with flares of transfer due to genetic drift. [ABSTRACT FROM AUTHOR]

RNA pol II assembly occurs in the cytoplasm before translocation of the enzyme to the nucleus. Affecting this assembly influences mRNA transcription in the nucleus and mRNA decay in the cytoplasm. However, very little is known about the consequences on ncRNA synthesis. In this work, we show that impairment of RNA pol II assembly leads to a decrease in cryptic non-coding RNAs (preferentially CUTs and SUTs). This alteration is partially restored upon overcoming the assembly defect. Notably, this drop in ncRNAs is only partially dependent on the nuclear exosome, which suggests a major specific effect of enzyme assembly. Our data also point out a defect in transcription termination, which leads us to propose that CTD phosphatase Rtr1 could be involved in this process. [ABSTRACT FROM AUTHOR]

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono‐, di‐, and tri‐methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE‐SPECIFIC DEMETHYLASE 1‐LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, we show that LDL3‐mediated H3K4me2 demethylation depends on the transcription elongation factor Paf1C and phosphorylation of the C‐terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is significantly attenuated in the ldl3 mutant, demonstrating the genome‐wide impacts of the transcription‐driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 demethylation in plants as chromatin records of transcriptional activity, which ensures robust gene control. Synopsis: In plants, H3K4me2 chromatin modification is negatively correlated with transcription. This study shows that this depends on the histone demethylase LDL3 co‐transcriptionally removing H3K4me2 from gene bodies. Defects in RNAPII phosphorylation and transcriptional elongation mimic loss of LDL3 function.LDL3 binds to CTD‐phosphorylated RNAPII in vivo.Transcriptional elongation factor Paf1C regulates the localization of LDL3 and affects H3K4me2.The negative correlation between H3K4me2 and transcription is attenuated in the ldl3 mutant. [ABSTRACT FROM AUTHOR]

Environmental pollution is a growing threat to natural ecosystems and one of the world's most pressing concerns. The increasing worldwide use of pharmaceuticals has elevated their status as significant emerging contaminants. Pharmaceuticals enter aquatic environments through multiple pathways related to anthropogenic activity. Their high consumption, insufficient waste treatment, and the incapacity of organisms to completely metabolize them contribute to their accumulation in aquatic environments, posing a threat to all life forms. Various analytical methods have been used to quantify pharmaceuticals. Biotechnology advancements based on next-generation sequencing (NGS) techniques, like eDNA metabarcoding, have enabled the development of new methods for assessing and monitoring the ecotoxicological effects of pharmaceuticals. eDNA metabarcoding is a valuable biomonitoring tool for pharmaceutical pollution because it (a) provides an efficient method to assess and predict pollution status, (b) identifies pollution sources, (c) tracks changes in pharmaceutical pollution levels over time, (d) assesses the ecological impact of pharmaceutical pollution, (e) helps prioritize cleanup and mitigation efforts, and (f) offers insights into the diversity and composition of microbial and other bioindicator communities. This review highlights the issue of aquatic pharmaceutical pollution while emphasizing the importance of using modern NGS-based biomonitoring actions to assess its environmental effects more consistently and effectively. [ABSTRACT FROM AUTHOR]

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion. [ABSTRACT FROM AUTHOR]