Eukaryotic Gene Research Articles

The BAF complex enhances transcription through interaction with H3K56ac in the histone globular domain

Histone post-translational modifications play pivotal roles in eukaryotic gene expression. To date, most studies have focused on modifications in unstructured histone N-terminal tail domains and their binding proteins. However, transcriptional regulation by chromatin-effector proteins that directly recognize modifications in histone globular domains has yet to be clearly demonstrated, despite the richness of their multiple modifications. Here, we show that the ATP-dependent chromatin-remodeling BAF complex stimulates p53-dependent transcription through direct interaction with H3K56ac located on the lateral surface of the histone globular domain. Mechanistically, the BAF complex recognizes nucleosomal H3K56ac via the DPF domain in the DPF2 subunit and exhibits enhanced nucleosome-remodeling activity in the presence of H3K56ac. We further demonstrate that a defect in H3K56ac–BAF complex interaction leads to impaired p53-dependent gene expression and DNA damage responses. Our study provides direct evidence that histone globular domain modifications participate in the regulation of gene expression.

The North Pacific Eukaryotic Gene Catalog of metatranscriptome assemblies and annotations

Marine microbial eukaryotes (protists) perform essential metabolic functions in oceanic ecosystems. The diversity of protist functions remains poorly understood as few species have been isolated in laboratory settings. Metatranscriptomes provide an invaluable tool for exploring protist diversity and genetic capacities within their natural habitats. Here, we introduce the North Pacific Eukaryotic Gene Catalog, a compilation of metatranscriptome data derived from a total of 261 metatranscriptomes: 169 metatranscriptomes were derived from samples collected on three meridional surface transects along 158°W, each spanning ~20 degrees of latitude from the North Pacific Subtropical Gyre (NPSG) to the North Pacific Transition Zone (NPTZ); 92 metatranscriptomes were derived from two diel-resolved field studies, one in the NPSG at 157°W, 23°N, one in the NPTZ at 158°W, 41°N. The metatranscriptome sequences were de novo assembled into 175 assemblies and pooled into five datasets each containing between 22 M and 49 M contigs clustered at 99% protein identity. Assemblies were annotated by taxonomy and function, and enumerated by short read alignment. All data are available in the Zenodo repository, with underlying code available on github.

Structure of a step II catalytically activated spliceosome from Chlamydomonas reinhardtii.

Pre-mRNA splicing, a fundamental step in eukaryotic gene expression, is executed by the spliceosomes. While there is extensive knowledge of the composition and structure of spliceosomes in yeasts and humans, the structural diversity of spliceosomes in non-canonical organisms remains unclear. Here, we present a cryo-EM structure of a step II catalytically activated spliceosome (C* complex) derived from the unicellular green alga Chlamydomonas reinhardtii at 2.6 Å resolution. This Chlamydomonas C* complex comprises 29 proteins and four RNA elements, creating a dynamic assembly that shares a similar overall architecture with yeast and human counterparts but also has unique features of its own. Distinctive structural characteristics include variations in protein compositions as well as some noteworthy RNA features. The splicing factor Prp17, with four fragments and a WD40 domain, is engaged in intricate interactions with multiple protein and RNA components. The structural elucidation of Chlamydomonas C* complex provides insights into the molecular mechanism of RNA splicing in plants and understanding splicing evolution in eukaryotes.

Candidatus Desulforudis audaxviator dominates a 975 m deep groundwater community in central Sweden

The continental bedrock contains groundwater-bearing fractures that are home to microbial populations that are vital in mediating the Earth’s biogeochemical cycles. However, their diversity is poorly understood due to the difficulty of obtaining samples from this environment. Here, a groundwater-bearing fracture at 975 m depth was isolated by employing packers in order to characterize the microbial community via metagenomes combined with prokaryotic and eukaryotic marker genes (16S and 18S ribosomal RNA gene). Genome-resolved analyses revealed a community dominated by sulfate-reducing Bacillota, predominantly represented by Candidatus Desulforudis audaxviator and with Wood-Ljungdahl as the most prevalent pathway for inorganic carbon fixation. Moreover, the eukaryotic community had a considerable diversity and was comprised of mainly flatworms, chlorophytes, crustaceans, ochrophytes, and fungi. These findings support the important role of the Bacillota, with the sulfate reducer Candidatus Desulforudis audaxviator as its main representative, as primary producers in the often energy-limited groundwaters of the continental subsurface.

A fine kinetic balance of interactions directs transcription factor hubs to genes.

Eukaryotic gene regulation relies on the binding of sequence-specific transcription factors (TFs). TFs bind chromatin transiently yet occupy their target sites by forming high-local concentration microenvironments (hubs and condensates) that increase the frequency of binding. Despite their ubiquity, such microenvironments are difficult to study in endogenous contexts due to technical limitations. Here, we use live embryo light-sheet imaging, single-molecule tracking, and genomics to overcome these limitations and investigate how hubs are localized to target genes to drive TF occupancy and transcription. By examining mutants of a hub-forming TF, Zelda, in Drosophila embryos, we find that hub formation propensity, spatial distributions, and temporal stabilities are differentially regulated by DNA binding and disordered protein domains. We show that hub localization to genomic targets is driven by a finely-tuned kinetic balance of interactions between proteins and chromatin, and hubs can be redirected to new genomic sites when this balance is perturbed.

National-scale distribution of protists associated with sorghum leaves and roots.

Protists, as integral constituents of the plant microbiome, are posited to confer substantial benefits to plant health and performance. Despite their significance, protists have received considerably less attention compared to other constituents of the plant microbiome, such as bacteria and fungi. To investigate the diversity and community structure of protists in sorghum leaves and roots, we employed amplicon sequencing of the eukaryotic 18S rRNA gene in 563 leaf and root samples collected from 57 locations across China. We found significant differences in the diversity and community structure of protists in sorghum leaves and roots. The leaf was taxonomically dominated by Evosea, Cercozoa and Ciliophora, while the root was dominated by Endomyxa, Cercozoa and Oomycota. The functional taxa of protists exhibited notable differences between leaves and roots, with the former being predominantly occupied by consumers and the latter by parasites. The community composition of protists in the leaf was predominantly influenced by mean annual precipitation, whereas soil pH played a more significant role in the root. The present study identified the most abundant and distributed protists in sorghum leaves and roots and elucidated the underlying factors that govern their community structure. The present study offers a novel perspective on the factors that shape plant-associated protist communities and their potential roles in enhancing the functionality of plant ecosystems.

Developing messenger RNA biomarkers: A workflow to characterise and identify transcript target sequences unaffected by alternative splicing for reproducible gene transcript quantification by reverse transcriptase quantitative polymerase chain reaction

AbstractMost eukaryotic genes generate multiple messenger RNA (mRNA) transcript variants by alternative splicing. The incomplete annotation of gene transcripts in genomic databases can result in improper primer design, adversely affecting the reliability of gene expression measurements by reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR). Hence, we present a workflow combining bioinformatics analyses, to select two to three evolutionarily conserved constitutive exons in rats, mice and humans as target sequences for PCR primer design, with experimental RT‐PCR amplification and amplicon sequencing to confirm the expression and identity of gene transcript targets. The application of this workflow to the characterization of neurodevelopmental biomarker genes identified an unannotated exon in the rat Map2 gene, illustrating the importance of target sequence validation for the development of translational mRNA biomarkers for toxicological and biomedical studies.

Evolution of the regulatory subunits for the heteromeric acetyl-CoA carboxylase.

The committed step for de novo fatty acid (FA) synthesis is the ATP-dependent carboxylation of acetyl-coenzyme A catalysed by acetyl-CoA carboxylase (ACCase). In most plants, ACCase is a multi-subunit complex orthologous to prokaryotes. However, unlike prokaryotes, the plant and algal orthologues are comprised both catalytic and additional dedicated regulatory subunits. Novel regulatory subunits, biotin lipoyl attachment domain-containing proteins (BADC) and carboxyltransferase interactors (CTI) (both three-gene families in Arabidopsis) represent new effectors specific to plants and certain algal species. The evolutionary history of these genes in autotrophic eukaryotes remains elusive, making it an ongoing area of research. Analyses of potential protein-protein and co-occurrence interactions, informed by gene network patterns using the STRING database, in Arabidopsis thaliana and Chlamydomonas reinhardtii unveil intricate gene associations with ACCase, suggesting a complex interplay between FA synthesis and other cellular processes. Among both species, a higher number of co-expressed genes was identified in Arabidopsis, indicating a wider potential regulatory network of ACCase in plants. This review investigates the extent to which these genes arose in autotrophic eukaryotes and provides insights into their evolutionary trajectory. This article is part of the theme issue 'The evolution of plant metabolism'.

The role of the U1 snRNP complex in the regulation of gene expression: recent reports

U1 snRNP (U1 small nuclear ribonucleoprotein) is a nuclear ribonucleoprotein complex involved mainly in pre-mRNA splicing, which is a key regulatory process in the eukaryotic gene expression pathway, but also in the process of preventing premature transcription termination (telescripting). U1 snRNP interacts directly with RNA polymerase II, thereby influencing the synthesis and maturation of transcripts in the cell nucleus, including the formation of the 3' end of mRNA and polyadenylation. At the level of cell physiology, it regulates the functioning of mitochondria and energy metabolism. The core of the U1 snRNP complex is U1 snRNA, encoded by many copies of genes that differ in sequence and expression level, and the expression of some of them leads to the formation of defective products. According to current reports, U1 snRNA can be used for therapeutic purposes to regulate gene expression and improve mRNA splicing defects, which are the cause of many diseases. Here we present selected recent discoveries and achievements related to U1 snRNP.

PPM1G and its diagnostic, prognostic and therapeutic potential in HCC (Review).

Global statistics indicate that hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer‑related death. Protein phosphatase Mg2+/Mn2+ dependent 1G (PPM1G, also termed PP2Cγ) is one of the 17 members of the PPM family. The enzymatic activity of PPM1G is highly reliant on Mg2+ or Mn2+ and serves as a dephosphorylation regulator for numerous key proteins. PPM1G, functioning as a phosphatase, is involved in a number of significant biological processes such as the regulation of eukaryotic gene expression, DNA damage response, cell cycle and apoptosis, cell migration ability, cell survival and embryonic nervous system development. Additionally, PPM1G serves a role in regulating various signaling pathways. In recent years, further research has increasingly highlighted PPM1G as an oncogene in HCC. A high expression level of PPM1G is closely associated with the occurrence, progression and poor prognosis of HCC, offering notable diagnostic and therapeutic value for this patient population. In the present review, the regulatory role of PPM1G in diverse biological processes and signaling pathway activation in eukaryotes is evaluated. Furthermore, its potential application as a biomarker in the diagnosis and prognosis evaluation of HCC is assessed, and future prospects for HCC treatment strategies centered on PPM1G are discussed.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.

Non-redundant roles for the human mRNA decapping cofactor paralogs DCP1a and DCP1b.

Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions.

Functional Characterization of Accessible Chromatin in Common Wheat.

Eukaryotic gene transcription is fine-tuned by precise spatiotemporal interactions between cis-regulatory elements (CREs) and trans-acting factors. However, how CREs individually or coordinated with epigenetic marks function in regulating homoeolog bias expression is still largely unknown in wheat. In this study, through comprehensively characterizing open chromatin coupled with DNA methylation in the seedling and spikelet of common wheat, we observed that differential chromatin openness occurred between the seedling and spikelet, which plays important roles in tissue development through regulating the expression of related genes or through the transcription factor (TF)-centered regulatory network. Moreover, we found that CHH methylation may act as a key determinant affecting the differential binding of TFs, thereby resulting in differential expression of target genes. In addition, we found that sequence variations in MNase hypersensitive sites (MHSs) result in the differential expression of key genes responsible for important agronomic traits. Thus, our study provides new insights into the roles of CREs in regulating tissue or homoeolog bias expression, and controlling important agronomic traits in common wheat. It also provides potential CREs for genetic and epigenetic manipulation toward improving desirable traits for wheat molecule breeding.

Evolution of a Eukaryotic Transcription Factor's co-TF Dependence Involves Multiple Intrinsically Disordered Regions Affecting Activation and Autoinhibition.

Combinatorial control by multiple transcription factors (TFs) is a hallmark of eukaryotic gene regulation. Despite its prevalence and crucial roles in enhancing specificity and integrating information, the mechanisms behind why eukaryotic TFs depend on one another, and whether such interdependence evolves, are not well understood. We exploit natural variation in co-TF dependence in the yeast phosphate starvation (PHO) response to address this question. In the model yeast Saccharomyces cerevisiae, the main TF, Pho4, relies on the co-TF Pho2 to regulate ~28 genes. In a related yeast pathogen, Candida glabrata, its Pho4 exhibits significantly reduced Pho2 dependence and has an expanded target set of ~70 genes. Biochemical analyses showed C. glabrata Pho4 (CgPho4) binds to the same consensus motif with 3-4-fold higher affinity than ScPho4 does. A machine-learning-based prediction and yeast one-hybrid assay identified two Intrinsically Disordered Regions (IDRs) in CgPho4 that boost the activity of the main activation domain but showed little to no activity on their own. We also found evidence for autoinhibition behind the co-TF dependence in ScPho4. An IDR in ScPho4 next to its DNA binding domain was found to act as a double-edged sword: it both allows for enhanced activity with Pho2, and inhibits Pho4's activity without Pho2. This study provides a detailed molecular picture of how co-TF dependence is mediated and how its evolution, mainly driven by IDR divergence, can lead to significant rewiring of the regulatory network.

Conservation of antiviral systems across domains of life reveals immune genes in humans

Deciphering the immune organization of eukaryotes is important for human health and for understanding ecosystems. The recent discovery of antiphage systems revealed that various eukaryotic immune proteins originate from prokaryotic antiphage systems. However, whether bacterial antiphage proteins can illuminate immune organization in eukaryotes remains unexplored. Here, we use a phylogeny-driven approach to uncover eukaryotic immune proteins by searching for homologs of bacterial antiphage systems. We demonstrate that proteins displaying sequence similarity with recently discovered antiphage systems are widespread in eukaryotes and maintain a role in human immunity. Two eukaryotic proteins of the anti-transposon piRNA pathway are evolutionarily linked to the antiphage system Mokosh. Additionally, human GTPases of immunity-associated proteins (GIMAPs) as well as two genes encoded in microsynteny, FHAD1 and CTRC, are respectively related to the Eleos and Lamassu prokaryotic systems and exhibit antiviral activity. Our work illustrates how comparative genomics of immune mechanisms can uncover defense genes in eukaryotes.

Circular code identified by the codon usage

Since 1996, circular codes in genes have been identified thanks to the development of 6 statistical approaches: trinucleotide frequencies per frame (Arquès and Michel, 1996), correlation functions per frame (Arquès and Michel, 1997), frame permuted trinucleotide frequencies (Frey and Michel, 2003, 2006), advanced statistical functions at the gene population level (Michel, 2015) and at the gene level (Michel, 2017). All these 3-frame statistical methods analyse the trinucleotide information in the 3 frames of genes: the reading frame and the 2 shifted frames. Notably, codon usage does not allow for the identification of circular codes (Michel, 2020). This has been a long-standing problem since 1996, hindering biologists’ access to circular code theory.By considering circular code conditions resulting from code theory, particularly the concept of permutation class, and building upon previous statistical work, a new statistical approach based solely on the codon usage, i.e. a 1-frame statistical method, surprisingly reveals the maximal C3 self-complementary trinucleotide circular code X in bacterial genes and in average (bacterial, archaeal, eukaryotic) genes, and almost in archaeal genes. Additionally, a new parameter definition indicates that bacterial and archaeal genes exhibit codon usage dispersion of the same order of magnitude, but significantly higher than that observed in eukaryotic genes. This statistical finding may explain the greater variability of codes in eukaryotic genes compared to bacterial and archaeal genes, an issue that has been open for many years. Finally, biologists can now search for new (variant) circular codes at both the genome level (across all genes in a given genome) and the gene level using only codon usage, without the need for analysing the shifted frames.

Protein Arginine Methylation of the Translation Initiation Factor eIF1A Increases Usage of a Near-cognate Start Codon.

Protein arginine methylation has emerged as a key post-translational modification responsible for many facets of eukaryotic gene expression. To better understand the extent of this modification in cellular pathways, we carried out bioorthogonal methylation profiling in Saccharomyces cerevisiae to comprehensively identify the in vivo substrates of the major yeast protein arginine methyltransferase Hmt1. Gene ontology analysis of candidate substrates revealed an enrichment of proteins involved in the process of translation. We verified one such factor, eIF1A, by in vitro methylation. Three sites on eIF1A were found to be responsible for its methylation: R13, R14, and R62, with varied capacity by which each site contributed to the overall methylation capacity in vitro. To determine the role of methylation in eIF1A function, we used a battery of arginine-to-alanine substitution mutants to evaluate translation fidelity in these mutants. Our data show that substitution mutants at R13 and R14 in the N-terminal tail improved the fidelity of start codon recognition in an initiation fidelity assay. Overall, our data suggest that Hmt1-mediated methylation of eIF1A fine-tunes the fidelity of start codon recognition for proper translation initiation.

Light regulates widespread plant alternative polyadenylation through the chloroplast

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

Spray-induced gene silencing (SIGS) as a tool for the management of Pine Pitch Canker forest disease.

Global change is exacerbating the prevalence of plant diseases caused by pathogenic fungi in forests worldwide. The conventional use of chemical fungicides, which is commonplace in agricultural settings, is not sanctioned for application in forest ecosystems, so novel control strategies are imperative. SIGS (Spray-Induced Gene Silencing) is a promising approach that can modulate the expression of target genes in eukaryotes in response to double-stranded RNA (dsRNA) present in the environment that triggers the RNA interference (RNAi) mechanism. SIGS exhibited notable success in reducing virulence when deployed against some crop fungal pathogens, such as Fusarium graminearum, Botrytis cinerea and Sclerotinia sclerotiorum, among others. However, there is a conspicuous dearth of studies evaluating the applicability of SIGS for managing forest pathogens. This research aimed to determine whether SIGS could be used to control Fusarium circinatum, a widely impactful forest pathogen that causes Pine Pitch Canker disease. Through a bacterial synthesis, we produced dsRNA molecules to target fungal essential genes involved to vesicle trafficking (Vps51, DCTN1, and SAC1), signal transduction (Pp2a, Sit4, Ppg1, and Tap42), and cell wall biogenesis (Chs1, Chs2, Chs3b, Gls1) metabolic pathways. We confirmed that F. circinatum is able to uptake externally applied dsRNA, triggering an inhibition of the pathogen's virulence. Furthermore, this study pioneers the demonstration that recurrent applications of dsRNAs in SIGS are more effective in protecting plants than single applications. Therefore, SIGS emerges as an effective and sustainable approach for managing plant pathogens, showcasing its efficacy in controlling a globally significant forest pathogen subject to quarantine measures.

Variation in a Poaceae-conserved fatty acid metabolic gene cluster controls rice yield by regulating male fertility

A wide variety of metabolic gene clusters exist in eukaryotic genomes, but fatty acid metabolic gene clusters have not been discovered. Here, combining with metabolic and phenotypic genome-wide association studies, we identify a major locus containing a six-gene fatty acid metabolic gene cluster on chromosome 3 (FGC3) that controls the cutin monomer hydroxymonoacylglycerols (HMGs) contents and rice yield, possibly through variation in the transcription of FGC3 members. We show that HMGs are sequentially synthesized in the endoplasmic reticulum by OsFAR2, OsKCS11, OsGPAT6, OsCYP704B2 and subsequently transported to the apoplast by OsABCG22 and OsLTPL82. Mutation of FGC3 members reduces HMGs, leading to defective male reproductive development and a significant decrease in yield. OsMADS6 and OsMADS17 directly regulate FGC3 and thus influence male reproduction and yield. FGC3 is conserved in Poaceae and likely formed prior to the divergence of Pharus latifolius. The eukaryotic fatty acid and plant primary metabolic gene cluster we identified show a significant impact on the origin and evolution of Poaceae and has potential for application in hybrid crop breeding.