Institute of Bioorganic Chemistry, Polish Academy of Sciences

Although serological and genetic studies of equine blood group systems have been conducted for many years, the molecular basis of erythrocyte antigens' variability has remained largely unexplored. In this study, we aimed to elucidate the genetic basis of serological variation within equine blood group K. Using mRNA extracted from peripheral blood samples (n = 100) collected from horses with known serological blood types (Ka or K-), we performed a transcriptome-wide association study (TWAS), which revealed a significantly associated region on equine chromosome 2 (ECA2). A detailed analysis of this region identified GYPA (glycophorin A) as the most promising candidate gene. Resequencing its entire coding sequence revealed the presence of a dinucleotide missense variant in exon 3 (ENSECAT00000026370.3:c.145_146delinsAT; p.Asp49Ile), which is predicted to potentially alter the function of the GYPA protein. Genotyping this variant in a large, breed-diverse cohort, which included family-based samples, confirmed perfect cosegregation between the identified GYPA missense substitution and serological K blood group typing results. Our findings demonstrate that blood transcriptome-based approaches, despite certain limitations, can effectively reveal the molecular basis of equine erythrocyte antigen variability.

The impact of specific chromatin modifications on meiotic crossover frequency has typically been inferred from correlative studies, leaving the question of causality unresolved. To directly test this, we used a catalytically inactive CRISPR-associated protein 9 (dCas9)-based system to recruit the histone demethylase JMJ14 to defined genomic loci. Recruitment of JMJ14 led to a reduction in local histone H3 lysine 4 trimethylation (H3K4me3) levels and a decrease in crossover frequency within the targeted interval. This was accompanied by reduced expression of a long noncoding RNA (lncRNA) at the hotspot and altered crossover topology. Suppressed recombination was also observed at neighboring, untargeted hotspots. In contrast, targeting the transcriptional activator VP64 to the same region increased lncRNA expression, elevated crossover frequency, and raised H3K4me3 levels. Together, these findings establish a causal link between H3K4me3, transcription, and local crossover activity, demonstrating that H3K4me3 levels are closely associated with both transcriptional output and recombination frequency at specific genomic loci.

One of the major applications of the CRISPR-Cas9 system is the visualization of DNA by using nuclease-deactivated Cas9 (dCas9), which, following complexation with a single-guide RNA (sgRNA), specifically binds to target genomic sequences without inducing DNA breaks. In this approach, either dCas9 or the sgRNA is labeled with fluorescent proteins or dyes, or they are engineered to recruit such molecules. A key advantage of CRISPR-based imaging is that genomic elements in living cells can be tracked, because of the ability to express all system components in vivo. Although CRISPR-based imaging has been successfully used to label repetitive sequences in living cells, the visualization of nonrepetitive loci remains a challenging issue. The primary obstacles are a low signal-to-noise ratio and the potential for nonspecific DNA binding by the dCas9-sgRNA complex, which can generate fluorescent puncta at off-target sites. Efficient intracellular delivery of system components and their sustained expression over time are also a major concern. Consequently, CRISPR-based imaging remains a highly time- and labor-intensive process that requires ongoing optimization. Here, we summarize recent advances in labeling nonrepetitive genomic loci, outline key challenges associated with CRISPR-based imaging, and present insights derived from our own experimental findings and research experience.

Abstract Artificial microRNAs (amiRNAs) offer a powerful strategy for targeted gene silencing, but their rational design is limited by complex sequence-structure-processing relationships and the lack of tools capable of optimizing efficacy and specificity. To address this need, we developed miRarchitect, a web-based platform that uses machine learning to support the customizable design of amiRNAs. miRarchitect integrates neural network-guided target-site selection, siRNA insert design, and scaffold choice, utilizing large-scale data from human primary microRNAs (pri-miRNAs) and next-generation sequencing. The platform generates molecules that closely resemble endogenous pri-miRNAs and includes comprehensive off-target analysis to enhance specificity. Experimental validation targeting TMPRSS2 and ACE-2 confirmed precise processing, robust knockdown, and high specificity of miRarchitect-designed amiRNAs. In comparative benchmarking, miRarchitect consistently produced functional amiRNAs, whereas only half of the top candidates generated by other tools showed measurable activity. miRarchitect is freely available at https://rnadrug.ichb.pl/mirarchitect and provides an intuitive interface with an automated workflow for generating, ranking, and selecting candidate amiRNAs for research and therapeutic applications.

Histone acetylation by the plant NuA4 complex promotes high expression of growth-related genes while preventing gene body H2A.Z depletion and spurious activation of stress-responsive loci. How this NuA4-dependent chromatin state is modulated by histone deacetylases (HDACs) in plants remains unclear. Here, we investigate the interplay between NuA4 and HDACs in Arabidopsis by generating a collection of HDAC loss-of-function mutants in NuA4-proficient and NuA4-deficient backgrounds. Loss of individual HDACs did not rescue the severe growth defects of the NuA4(-) mutant; instead, most combinations further aggravated the phenotype, indicating that HDACs predominantly support rather than counteract NuA4 function. Focusing on the hda19-5Atepl1b allele, we show that loss of HDA19 causes strong developmental defects and constitutive activation of biotic stress-responsive genes, but not canonical heat-response genes. 3′ RNA-seq and ChIP-seq reveal that upregulated genes in hda19-5Atepl1b display increased H3K9ac and H2A.Zac with largely unchanged gene-body H2A.Z, and substantially overlap with genes induced in the NuA4-null Atepl1-2 mutant and in wild-type plants exposed to elevated temperature. Integration of our datasets with published HDA19 ChIP-seq maps shows that H2A.Zac is selectively increased at HDA19-bound H2A.Z peaks in hda19-5Atepl1b, consistent with a direct role for HDA19 in H2A.Z deacetylation. However, elevated H2A.Zac is neither necessary nor sufficient for transcriptional activation, whereas changes in H3K9ac correlate strongly with gene induction. We propose that NuA4 and HDA19 cooperate to tune chromatin at stress-related loci, with H3K9 acetylation as the primary driver of transcription and H2A.Z acetylation acting as a modulatory mark that shapes stress gene responsiveness.

Secondary metabolites, which are natural products of fungi, perform key biological functions and have applications in medicine. The production of these compounds is subject to the control of clusters of biosynthetic genes located within the genome. It has been observed that these genes can become dormant under standard culture conditions. The advent of modern genetic and epigenetic methodologies has rendered the activation of these elements possible, thus facilitating the identification of novel bioactive compounds. The present article delineates classes of metabolites, the mechanisms of their biosynthesis, and current research strategies, combining knowledge from biology, genetics, and biotechnology.

Numerous studies have already demonstrated that cytokinin (CK) distribution plays a pivotal role in shaping plant morphology in response to environmental changes. However, the contribution of short-distance CK translocation to root mineral nutrition remains poorly understood, and the specific roles of CK transporters in root morphology are still unclear. Identifying the molecular identity of CK transporters is, therefore, essential for advancing our understanding of root plasticity, under varying soil fertility conditions and common nutritional deficiencies. In this study, we identified and characterised MtABCG40, a full-size ATP-binding cassette (ABC) transporter of the G subfamily in Medicago truncatula, as a CK transporter. MtABCG40 expression is root-specific, and it is induced both by nitrogen deficiency and CK treatment. We propose that MtABCG40 mediates the lateral translocation of CKs from the xylem to surrounding cells, giving rise to lateral organs, and thereby influencing root morphology, by suppressing the lateral root and nodule formation under nitrogen-limited conditions. Furthermore, MtABCG40 reduces apoplastic CK concentrations in the root meristematic zone, lowering the responsiveness of the root apical meristem to CKs. In summary, the activity of MtABCG40 indirectly influences CK perception and, consequently, modulates auxin-mediated cellular responses.

MotivationMost widely used methods for evaluating RNA 3D structure models require experimental reference structures, which restricts their use for novel RNAs. They also often overlook recurrent structural features shared across multiple predictions of the same sequence. Although consensus approaches have proven effective in RNA sequence analysis and evolutionary studies, no existing tool applies these principles to evaluate ensembles of 3D models. This gap hampers the identification of native-like folds in computational predictions, particularly as AI-driven methods become increasingly prevalent.ResultsThis paper presents RNAtive, the first computational tool to apply consensus-derived secondary structures for reference-free evaluation of RNA 3D models. RNAtive aggregates recurrent base-pairing and stacking interactions across ensembles of predicted 3D structures to construct a consensus secondary structure. It introduces a novel conditionally weighted consensus mode that treats interaction networks as fuzzy sets and uniquely allows integration of user-defined 2D structural constraints, enabling evaluation guided by experimental data. Input RNA models are ranked using two adapted binary-classification-based scores. Benchmarking against CASP15 competition data shows that models consistent with the consensus exhibit native-like structural features. The RNAtive web server offers an intuitive platform for comparing and prioritizing RNA 3D predictions, providing a scalable solution to address the variability inherent in deep learning and fragment-assembly methods. By bridging consensus principles with 3D structural analysis, RNAtive advances the exploration of RNA conformational landscapes and has potential applications in fields like therapeutic RNA design.AvailabilityRNAtive is a freely accessible web server with a modern, user-friendly interface, available for scientific, educational, and commercial use at https://rnative.cs.put.poznan.pl/.

Abstract RNA-binding proteins (RBPs) play a central role in post-transcriptional regulation during brain development, yet their specific functions in coordinating human neural lineage decisions remain poorly understood. Here, we investigate for the first time the role of the double-stranded RBP Staufen2 (STAU2) in human neurogenesis. Characterization of STAU2 knockout iPSC derived cells using scRNA-seq shows that loss of STAU2 disrupts neuroepithelial cell identity and accelerates neural differentiation by altering the activity of key transcription factors and driving early metabolic transitions. Additionally, STAU2 regulates the expression of miRNA host genes and alters miRNA-mediated post- transcriptional control in progenitor cells, which exerts additional effects on STAU2 regulated gene regulatory networks. These changes result in neural progenitor exhaustion, unstructured neural rosettes, and reduced organoid size. Together, our work uncovers a previously unrecognized role for STAU2 as a central regulator of early human neurogenesis, acting through both miRNA-mediated and transcriptional pathways to coordinate progenitor maintenance and neuronal fate specification.

We presents the discovery and molecular characterization of a novel lytic enzyme from the extremophilic Thermus thermophilus MAT72 phage vB_Tt72. The protein of 346-aa (MW=39,705) functions as phage vB_Tt72 endolysin and shows low sequence identity (<37%) to members of M23 family of peptidoglycan hydrolases, except for two uncharacterized endopeptidases of T. thermophilus phages: φYS40 (87%) and φTMA (88%). The enzyme exhibits lytic activity mainly against bacteria of the genus Thermus and, to a lesser extent, against other Gram-negative and Gram-positive bacteria. The protein is monomeric in solution and is highly thermostable (Tm=98.3°C). It retains∼50% of its lytic activity after 90min of incubation at 99°C. Crystallographic analysis, at 2.2Å resolution, revealed a fold characteristic of M23 metallopeptidases, accounting for 40% of the structure. The remaining parts of the molecule are folded in a manner that was previously undescribed. The M23 fold contains a Zn2+ ion coordinated by a conserved His-Asp-His triad, and two conserved His residues essential for catalysis. The active site is occupied by a phosphate or a sulfate anion, while the substrate-binding groove contains a ligand, which is a fragment of E. coli peptidoglycan. The common sequence-based criteria failed to identify the protein as (hyper)thermophilic. It is likely that the protein's thermal stability is owed to peculiar features of its three-dimensional structure. Instead of trimmed surface loops, observed in many thermostable proteins, the catalytic domain contains two long loops that interlace and form an α-helical bundle with its own hydrophobic core.