Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3\' UTRs. Yet, the biological significance of 3\' UTR IRAlus remains largely unknown. Here, we find that IRAlus induce the silencing of genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3\' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3\' UTR IRAlus and its role in human pathophysiology. less

MicroRNAs (miRNAs) regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that differential expression of conserved miRNAs and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning. less

Hematopoietic stem and progenitor cells (HSPCs) are intended to deliver life-long, consistent output. However, with age, we observe changes in blood counts and clonal disorders. Better understanding of inter-individual variation in HSPC behavior is needed to understand the transition from health to age-related hematological disorders. Here we study 360K single circulating HSPCs (CD34+) from 99 healthy individuals together with clinical information and clonal hematopoiesis (CH) profiles to characterize population variability in hematopoiesis. Individuals with CH were linked with reduced frequencies of lymphocyte progenitors and higher RDW. We describe a Lamin-A transcriptional signature across the HSPC spectrum and show it is reduced in CH individuals. We define and estimate HSPC composition bias and an age-related increased S-phase gene signature and show how they form a heterogeneous and multifactorial aging trend in the blood. The new comprehensive model of normal HSPC variation will allow the study of stem cell-related disorders. As a proof of concept, we present methodologies for analyzing myeloid malignancies in comparison to our reference atlas. Together, our data and methodologies shed light on age-related changes in blood counts, CH and can be used to study stem cell-related disorders in the future. less

The precise mechanisms governing sequence-dependent positioning of nucleosomes on DNA remain unknown in detail. Existing algorithms, taking into account the sequence-dependent deformability of DNA and its interactions with the histone globular domains, predict rotational setting of only 65% of human nucleosomes mapped in vivo. To uncover novel factors responsible for the nucleosome positioning, we analyzed potential involvement of the histone N-tails in this process. To this aim, we reconstituted the H2A/H4 N-tailless nucleosomes on human BRCA1 DNA (~100 kb) and compared their positions and sequences with those of the wild-type nucleosomes. In the case of H2A tailless nucleosomes, the AT content of DNA sequences is changed locally at superhelical location (SHL) +/-4, while maintaining the same rotational setting as their wild-type counterparts. Conversely, the H4 tailless nucleosomes display widespread changes of the AT content near SHL +/-1 and SHL +/-2, where the H4 N-tails interact with DNA. Furthermore, a substantial number of H4 tailless nucleosomes exhibit rotational setting opposite to that of the wild-type nucleosomes. Thus, our findings strongly suggest that the histone N-tails are operative in selection of nucleosome positions, which may have wide ranging implications for epigenetic modulation of chromatin states. less

Despite the significant role plasmids play in microbial evolution, there is limited knowledge of their ecology, evolution, and transfer in microbial communities. Therefore, we developed and implemented a novel approach to identify rare plasmid hosts by combining Hi-C, a proximity ligation method, with enrichment for plasmid-specific DNA. We hereafter refer to this Hi-C enrichment approach as Hi-C+. Our experimental design mimicked scenarios in which the transfer of an antimicrobial resistance plasmid from a donor to a recipient in soil was increasingly rare. We established that Hi-C can link a plasmid to its host in soil when the relative abundance of that plasmid-host pair is as low as 0.001%. The Hi-C+ method further improved the detection limit of Hi-C 100-fold and allowed identification of plasmid hosts at the genus level. Therefore, Hi-C+ will facilitate the exploration of the ecological and evolutionary pathways that affect the spread of plasmids in natural environments. less

The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, which cohesin is thought to achieve by actively extruding DNA loops without topologically entrapping DNA. The \'loop extrusion\' hypothesis finds motivation from in vitro observations - whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, as well as acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion model and point to an alternative mechanism for cohesin-dependent chromatin organisation. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin\'s two roles in chromosome segregation and interphase genome organisation. less

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease, tick-borne encephalitis, and anaplasmosis. The study of gene function in I. scapularis and other ticks has been hampered by the lack of genetic tools, such as an inducible promoter to permit temporal control over transgenes encoding protein or double-stranded RNA expression. Studies of vector-pathogen relationships would also benefit from the capability to activate anti-pathogen genes at different times during pathogen infection and dissemination. We have characterized an intergenic sequence upstream of the heat shock protein 70 (HSP70) gene that can drive Renilla luciferase expression and mCherry fluorescence in the I. scapularis cell line ISE6. In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter in the synthetic 3xP3 promoter with a minimal portion of the I. scapularis HSP70 promoter and generated an I. scapularis specific 3xP3 (Is3xP3) promoter. Both promoter constructs, IsHSP70 and Is3xP3, allow for heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent positive cells upon exposure to a 2 h heat shock. These promoters described here will be valuable tools for gene function studies and temporal control of gene expression, including anti-pathogen genes. less

Extrachromosomal circular DNA (eccDNA) enhances genomic plasticity, augmenting its coding and regulatory potential. Advances in high-throughput sequencing have enabled the investigation of these structural variants. Although eccDNAs have been investigated in numerous taxa, they remained understudied in euglenids. Therefore, we examined eccDNAs predicted from Illumina sequencing data of Euglena gracilis Z SAG 1224-5/25, grown under optimal photoperiod and exposed to UV irradiation. We identified approximately 1000 unique eccDNA candidates, about 20% of which were shared across conditions. We also observed a significant enrichment of mitochondrially encoded eccDNA in the UV-irradiated sample. Furthermore, we found that the heterogeneity of eccDNA was reduced in UV-exposed samples compared to cells that were grown in optimal conditions. Hence, eccDNA appears to play a role in the response to oxidative stress in Euglena, as it does in other studied organisms. In addition to contributing to the understanding of Euglena genomes, our results contribute to the validation of bioinformatics pipelines on a large, non-model genome. less

Lyme borreliosis is a disease caused by Borrelia burgdorferi sensu lato bacteria. Borrelia burgdorferi is known to induce prolonged extrafollicular immune responses and abnormal germinal center formation. However, the mechanism behind this is poorly understood. The extrafollicular response is characterized by strong plasmablast induction and by an IgM, IgG3, and IgG2b dominant antibody production. These antibodies do not generate a neutralizing type of immunity, and the bacteria eventually establish a persistent infection. Here, we performed single-cell RNA sequencing to characterize the immune landscape of lymph node lymphocytes in the early Borrelia burgdorferi infection in a murine model. Our results indicate that four days after Borrelia burgdorferi infection, a notable B cell proliferation, immunoglobulin class switching to IgG3 and IgG2b isotypes, and plasma cell differentiation are induced, all of which are hallmarks of the extrafollicular immune response. In addition, we found infection-derived upregulation of suppressor of cytokine signalling genes Socs1 and Socs3, and downregulation of genes involved in MHC II antigen presentation in B cells. Our results support the central role of B cells in the immune response of a Borrelia burgdorferi infection, and provide cues of mechanisms behind the determination between extrafollicular and germinal center responses during Borrelia burgdorferi infection. less

Mutational patterns consistent with the activity of the APOBEC3 cytidine deaminases are evident in more than half of human cancer genomes. APOBEC3-mediated mutagenesis is genotoxic when uncontrolled due to accumulation of base mutations, replication stress, and DNA breaks. In particular, the APOBEC3A family member is a potent enzyme with nuclear localization that causes substantial DNA damage in experimental systems and human tumors. However, the spectrum of genome-protective mechanisms that ensure genome stability in cells with active APOBEC3A is unknown. Through a genome-wide functional screen, we identify the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is expressed. Cells depleted of SMC5/6 incurred substantial DNA damage when APOBEC3A was active, as reflected by increased DNA breaks, DNA damage signaling, and defective proliferation. We observed an absence of APOBEC3A mutagenesis in human tumors with dysfunction of SMC5/6, consistent with synthetic lethality. APOBEC3A is known to act on ssDNA at replication forks. We observed increased DNA damage in replicating cells in the absence of SMC5/6, suggestive of replication forks as a source of DNA breaks. We interrogated replication fork dynamics by DNA fiber spreading and found a consistent increase in the length of replication tracks upon APOBEC3A activity across multiple cell lines. Increased replication fork length was dependent on Primpol, consistent with a repriming mechanism downstream of APOBEC3A-induced lesions. Loss of SMC5/6 resulted in abrogation of fork elongation in cells with active APOBEC3A, along with increased DNA breaks. Our findings indicate that increased length of replication forks in response to APOBEC3A is a genome-protective response and is dependent on intact SMC5/6. Therefore, SMC5/6 may be a therapeutic vulnerability in tumors in which APOBEC3A is active. less