RNA Profiling Research Articles

HIF-1 regulates common bidirectional gene expression changes during hypoxia and iron deficiency in human induced pluripotent stem cell derived cardiomyocytes

Abstract Background Heart failure regardless of cause is characterised by extensive transcriptional reprogramming and a switch to foetal developmental programmes via unknown mechanisms (1-3). An additional factor that adversely impacts heart failure patients is iron deficiency (ID)(4). ID is also associated with worse prognosis in acute coronary syndromes (5). However, the mechanisms by which ID interferes with clinical outcomes in heart disease remain incompletely understood. Purpose The purpose of this study was to investigate gene expression changes in human cardiomyocytes during hypoxia and ID, two important stressors in the context of ischaemia and heart failure. Methods In this study, we use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) derived through a protocol that promotes left ventricle cardiomyocyte differentiation (6). Cells were harvested at different stages of differentiation (day 6 and day 32) and mature (day 32) hiPSC-CMs were treated with 30 μM deferoxamine to induce ID either under normoxic or hypoxic conditions with 1% O2. hiPSC contraction was assessed using musclemotion software and metabolism through use of a Seahorse extracellular flux analyser. RNA was isolated using standard isolation kits and RNA-seq (n=5) was performed. Data was analysed for gene expression by alignment with the human hg19 reference genome using the software STAR. A log2 fold change of 1 and P value of <0.0001 were considered significant. Results We show that both hypoxic and ID hiPSC-CMs demonstrate impaired contractility and reduced oxygen consumption, features of cardiomyocytes from failing hearts. Importantly, hiPSC-CMs exposed to hypoxia, ID or the combination of the two exhibit bidirectional gene expression changes with significant overlap (Figure 1). Focusing on the commonly affected gene set, it becomes evident that hypoxia & ID work via similar mechanisms involving activation of the HIF-1 pathway, that in turn orchestrates the metabolic switch to glycolysis and glutamine oxidation similar to foetal cells. Overall, hypoxia promotes a shift towards immature gene programmes, with 45% of dysregulated genes in hypoxia mimicking expression patterns of less mature cells. Interestingly changes in long non-coding RNA (lncRNA) profiles in hypoxia and ID mimic gene expression changes in these conditions (figure 2). Although restoring oxygen levels in hypoxic hiPSC-CMs rescues the hypoxic transcriptional signature, hiPSC-CMs previously exposed to hypoxia can use glycolytic pathways more efficiently upon further exposures to hypoxia. Conclusion These results shed light into the mechanisms ID impacts heart disease patients and are suggestive that HIF-1 is a key player in orchestrating the gene switch in ID and hypoxia.

A spatially-resolved multiomic cell atlas reveals gene regulatory networks underlying cell specification in the developing human heart

Abstract Introduction Cardiogenesis requires the differentiation and coordinated function of a large variety of cell types. Congenital heart disease (CHD), affecting 1% of live births[1], and several adult-onset heart diseases result when these processes go awry[2–4]. To understand the cellular and molecular mechanisms at play we must define the repertoire of cell types in the developing heart as well as the cellular niches within which they interact. Single-cell RNA sequencing is a powerful means of defining cell populations, however existing studies of the human foetal heart[5,6] have been hampered by low cell numbers which limits detection of rare but important cell types. To create an improved atlas we increased the number of cells, increased the age range of foetal hearts sampled, and generated spatial transcriptomic data to map the defined cells to multicellular niches of co-located and interacting cells. To better infer the transcription factors (TFs) governing cell identity using gene regulatory network (GRN) inference we also generated paired single-cell resolution RNA and chromatin accessibility profiles using Assay for Transposase-Accessible Chromatin (ATAC) sequencing. Methods We performed paired single-nuclei RNA and ATAC sequencing (10X Multiome) and single-cell RNAseq (10X 3’ RNAseq) on 23 human foetal hearts ranging from 4 to 20 post-conception weeks. In addition we generated spatial transcriptomic data (10X Visium) from these hearts. We validated cell types defined in single-cell data using protein-level immunofluorescence imaging. Results Our healthy reference atlas includes 300k cells and nuclei. Using their gene expression profile we resolved 46 fine-grained cell types (FIGURE 1). We used cell2location[7], a machine learning classifier trained on the atlas to map the defined cell types to the spatial data (FIGURE 2) and detected niches of co-locating cells, such as a macrophage population in the walls of the great vessels predicted to interact with mural cells. We were able to define location-specific transcriptional signatures such as cardiomyocytes of each chamber (as well as the compact and trabeculated myocardium) and smooth muscle cells specific to great vessels vs coronary arteries. Using a combination of RNA and ATAC data we inferred cell-specific GRNs, which implicated novel TFs such as MSX2 in sinoatrial node pacemaker cells and the enrichment of CHD genes amongst cardiomyocyte-specific GRNs. In the spatial data we showed that gene modules associated with different forms of CHD enrich in specific anatomical regions, such as the outflow tract. Discussion: Together these data constitute the most comprehensive cardiac developmental cell atlas to date. It sheds new light on the diversity of cell states, their niches, and their vulnerability to the genetic causes of CHD.Figure 1:UMAP showing atlasFigure 2:Spatial transcriptomic mapping

Impaired cortical neuronal homeostasis and cognition after diffuse traumatic brain injury are dependent on microglia and type I interferon responses.

Neuropsychiatric complications including depression and cognitive decline develop in the years after traumatic brain injury (TBI), negatively affecting quality of life. Microglial and type 1 interferon (IFN-I) responses are associated with the transition from acute to chronic neuroinflammation after diffuse TBI in mice. Thus, the purpose of this study was to determine if impaired neuronal homeostasis and increased IFN-I responses intersected after TBI to cause cognitive impairment. Here, the RNA profile of neurons and microglia after TBI (single nucleus RNA-sequencing) with or without microglia depletion (CSF1R antagonist) was assessed 7 dpi. There was a TBI-dependent suppression of cortical neuronal homeostasis with reductions in CREB signaling, synaptogenesis, and synaptic migration and increases in RhoGDI and PTEN signaling (Ingenuity Pathway Analysis). Microglial depletion reversed 50% of TBI-induced gene changes in cortical neurons depending on subtype. Moreover, the microglial RNA signature 7 dpi was associated with increased stimulator of interferon genes (STING) activation and IFN-I responses. Therefore, we sought to reduce IFN-I signaling after TBI using STING knockout mice and a STING antagonist, chloroquine (CQ). TBI-associated cognitive deficits in novel object location and recognition (NOL/NOR) tasks at 7 and 30 dpi were STING dependent. In addition, TBI-induced STING expression, microglial morphological restructuring, inflammatory (Tnf, Cd68, Ccl2) and IFN-related (Irf3, Irf7, Ifi27) gene expression in the cortex were attenuated in STINGKO mice. CQ also reversed TBI-induced cognitive deficits and reduced TBI-induced inflammatory (Tnf, Cd68, Ccl2) and IFN (Irf7, Sting) cortical gene expression. Collectively, reducing IFN-I signaling after TBI with STING-dependent interventions attenuated the prolonged microglial activation and cognitive impairment.

Coordinated Regulation of Myonuclear DNA Methylation, mRNA, and miRNA Levels Associates With the Metabolic Response to Rapid Synergist Ablation-Induced Skeletal Muscle Hypertrophy in Female Mice.

The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature microRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our integrated multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth.

243 Translating Maternal Vitamin and Mineral Supplementation Effect on Early Developing Fetus Through Hepatic Long Non-Coding Rnas Profile in Beef Heifers

Abstract Maternal nutrition is critical during pregnancy as the fetus relies on the nutrients from the dam. Fetal organogenesis starts during the first trimester. Thus, an appropriate supply of nutrients, including vitamins and minerals, is indispensable. In the field of fetal developmental programming, there is still a lack of knowledge based on appropriate livestock models to explain the role of regulatory factors in translating the effect of maternal minerals and vitamins on fetal development and metabolic function. Therefore, this study was designed to investigate the effect of maternal vitamin and mineral supplementation (VTM -d 71 periconceptual to d 83 of pregnancy) on fetal liver expression of long non-coding RNAs (lncRNAs) and their potential regulatory metabolic pathways. Angus crossbreed beef heifers (n = 16) were supplemented (VTM, n = 8) or not (NoVTM, n = 8) with 113 g· heifer-1· day-1 of mineral premix (Purina Wind & Rain Storm All-Season 7.5 Complete) and all heifers were bred with female sexed semen from a single sire. Transcriptomic profiles were determined with RNA-Seq in fetal livers collected on d 83 ± 0.27 of gestation. After data quality control and read mapping, lncRNAs were predicted using the FEELnc pipeline. Using the DESeq2 software, we identified 50 differentially expressed novel lncRNAs (expression of 32 lncRNAs was upregulated and 18 were downregulated; FDR < 0.05) located either downstream, upstream, or in exonic, or intronic regions of 51 genes (including PFKP, SLC30A10, KCNH8, ZNF451, MINPP1, BCL9, PAH, ITPR2, AHCY, and ABCG8), potentially regulating their expression. These genes could affect pathways (FDR < 0.1) such as PI3K signaling, mineral storage and transport, and amino acids biosynthetic pathways (i.e., cysteine and methionine biosynthesis). Collectively, these results suggest that periconceptual maternal vitamin and mineral supplementation affects the expression of lncRNAs and potentially regulates the expression of genes involved in fetal liver development and function.

A Bayesian method to infer copy number clones from single-cell RNA and ATAC sequencing.

Single-cell RNA and ATAC sequencing technologies enable the examination of gene expression and chromatin accessibility in individual cells, providing insights into cellular phenotypes. In cancer research, it is important to consistently analyze these states within an evolutionary context on genetic clones. Here we present CONGAS+, a Bayesian model to map single-cell RNA and ATAC profiles onto the latent space of copy number clones. CONGAS+ clusters cells into tumour subclones with similar ploidy, rendering straightforward to compare their expression and chromatin profiles. The framework, implemented on GPU and tested on real and simulated data, scales to analyse seamlessly thousands of cells, demonstrating better performance than single-molecule models, and supporting new multi-omics assays. In prostate cancer, lymphoma and basal cell carcinoma, CONGAS+ successfully identifies complex subclonal architectures while providing a coherent mapping between ATAC and RNA, facilitating the study of genotype-phenotype maps and their connection to genomic instability.

Deep and Durable Cytogenetic and Molecular Responses with Pemigatinib in Myeloid/Lymphoid Neoplasms with Fibroblast Growth Factor Receptor 1 Rearrangement: The Fight-203 Study

Introduction:Myeloid/lymphoid neoplasmswith fibroblast growth factor receptor 1 rearrangement (MLN FGFR1) are aggressive hematologic neoplasms caused by various reciprocal translocations involving chromosome band 8p11, resulting in tyrosine kinase fusion genes (eg, ZMYM2::FGFR1 from t[8;13][p11;q12] or BCR::FGFR1 from t[8;22][p11;q11]) and constitutive FGFR1 activationand downstream signaling. Pemigatinib is a potent and selective oral inhibitor of FGFR1-3 approved for the treatment of adults with relapsed or refractory MLN FGFR1. Efficacy and safety data for 41 patients with MLN FGFR1 in the ongoing FIGHT-203 study of pemigatinib were previously reported. Median daily pemigatinib dose was 9.5 mg; treatment-emergent adverse events led to dose interruptions in 66% of patients. Rates of complete response (CR) among the 38 patients evaluable for clinical response and rates of complete cytogenetic response (CCyR) for the 40 patients evaluable for cytogenetic response were 74% and 70%, respectively. CR and CCyR occurred in >80% of patients with chronic phase disease and in >50% of patients with blast phase disease. Here we present an analysis of centralized fluorescence in situ hybridization (FISH) evaluation of bone marrow (BM) cells and serial characterization of levels of FGFR1 fusion transcripts in this same cohort. Methods: FIGHT-203 (NCT03011372) is an ongoing open-label, multicenter, phase 2 study evaluating pemigatinib in patients ≥18 years old with MLN FGFR1 and ≥1 prior therapy. The starting dose was pemigatinib 13.5 mg daily (2 weeks on, 1 week off). Following protocol amendments, patients without prior therapy were eligible to enroll, and the dosing regimen was changed to a continuous schedule. Patients were censored for cytogenetic and molecular follow-up at the time of transplantation. CCyR was defined as 0 metaphases positive for the respective reciprocal translocation in ≥20 evaluated BM metaphases on local and/or central karyotyping or 0 cells (or not exceeding the lower level of detection of the probe) with the FGFR1 rearrangement in ≥200 BM cells on local and/or central break-apart FISH assays (central FISH prioritized). As part of the translational research plan, fusion partner genes were identified with next-generation sequencing (NGS; FusionPlex, Archer, Boulder, CO, USA) and targeted PCR profiling of RNA extracted from whole blood; droplet digital PCR (ddPCR) assays were developed to monitor the frequencies of specific FGFR1 fusion transcripts in RNA extracted from whole blood samples (estimated limit of detection, 0.05 copies/μL). Results: As of June 30, 2021, 39 of 41 enrolled and treated patients were evaluable for translational analyses. Among the 39 patients with whole blood samples, FGFR1 fusion partner genes identified on translational testing were ZMYM2 (n=17), BCR (n=11), FGFR1OP (n=4), TPR (n=3), TRIM24 (n=2), and C14orf93, CCDC6, CNTRL and ETV6 (n=1 each). Two of 39 patients had 2 fusions detected ( ZMYM2 and BCR, n=1; ZMYM2 and TPR, n=1). C14orf93, CCDC6 and ETV6 have not been previously identified as FGFR1 fusion partner genes. Central FISH assessment of BM cells confirmed FGFR1-aberrantcells were reduced to undetectable levels in responding patients but rebounded in some patients during periods of treatment interruption (Figure A). Serial ddPCR of whole blood samples demonstrated marked reductions in FGFR1 fusion transcripts for the 34 patients with ≥3 measures while on treatment (Figure B). Among patients with CR, 81.0% had a >2-log reduction and 47.6% had a >3-log reduction in detectable FGFR1 fusion transcripts. Smaller reductions in FGFR1 fusion transcripts were observed in patients with stable disease. Changes in FGFR1 fusion transcripts correlated with FGFR1 FISH results (Pearson coefficient = 0.81) and reflected changes in treatment intensity. Conclusion:Several new FGFR1 fusion partner genes, including C14orf93, CCDC6, and ETV6 were identified with NGS and PCR profiling. Pemigatinib treatment resulted in marked decreases in the percentages of cells with the FGFR1 rearrangement on FISH, as well as 2-3 log reductions in FGFR1 fusion transcripts in patients with MLN FGFR1. Further investigations are ongoing to determine the relationship between pemigatinib dose intensity and the depth and durability of molecular response.

Spiny projection neurons exhibit transcriptional signatures within subregions of the dorsal striatum

The dorsal striatum is organized into functional territories defined by corticostriatal inputs onto both direct and indirect spiny projection neurons (SPNs), the major cell types within the striatum. In addition to circuit connectivity, striatal domains are likely defined by the spatially determined transcriptomes of SPNs themselves. To identify cell-type-specific spatiomolecular signatures of direct and indirect SPNs within dorsomedial, dorsolateral, and ventrolateral dorsal striatum, we used RNA profiling in situ hybridization with probes to >98% of protein coding genes. We demonstrate that the molecular identity of SPNs is mediated by hundreds of differentially expressed genes across territories of the striatum, revealing extraordinary heterogeneity in the expression of genes that mediate synaptic function in both direct and indirect SPNs. This deep insight into the complex spatiomolecular organization of the striatum provides a foundation for understanding both normal striatal function and for dissecting region-specific dysfunction in disorders of the striatum.

Coding and noncoding RNA profile of human heterotopic ossifications - Risk factors and biomarkers.

Heterotopic ossification (HO) means the formation of bone in muscles and soft tissues, such as ligaments or tendons. HO could have a genetic history or develop after a traumatic event, as a result of muscle injury, fractures, burns, surgery, or neurological disorders. Many lines of evidence suggest that the formation of HO is related to the pathological differentiation of stem or progenitor cells present within soft tissues or mobilized from the bone marrow. The cells responsible for the initiation and progression of HO are generally called HO precursor cells. The exact mechanisms behind the development of HO are not fully understood. However, several factors have been identified as potential contributors. For example, local tissue injury and inflammation disturb soft tissue homeostasis. Inflammatory cells release growth factors and cytokines that promote osteogenic or chondrogenic differentiation of HO precursor cells. The bone morphogenetic protein (BMP) is one of the main factors involved in the development of HO. In this study, next-generation sequencing (NGS) and RT-qPCR were performed to analyze the differences in mRNA, miRNA, and lncRNA expression profiles between muscles, control bone samples, and HO samples coming from patients who underwent total hip replacement (THR). As a result, crucial changes in the level of gene expression between HO and healthy tissues were identified. The bioinformatic analysis allowed to describe the processes most severely impacted, as well as genes which level differed the most significantly between HO and control samples. Our analysis showed that the level of transcripts involved in leukocyte migration, differentiation, and activation, as well as markers of chronic inflammatory diseases, that is, miR-148, increased in HO, as compared to muscle. Furthermore, the levels of miR-195 and miR-143, which are involved in angiogenesis, were up-regulated in HO, as compared to bone. Thus, we suggested that inflammation and angiogenesis play an important role in HO formation. Importantly, we noticed that HO is characterized by a higher level of TLR3 expression, compared to muscle and bone. Thus, we suggest that infection may also be a risk factor in HO development. Furthermore, an increased level of transcripts coding proteins involved in osteogenesis and signaling pathways, such as ALPL, SP7, BGLAP, BMP8A, BMP8B, SMPD3 was noticed in HO, as compared to muscles. Interestingly, miR-99b, miR-146, miR-204, and LINC00320 were up-regulated in HO, comparing to muscles and bone. Therefore, we suggested that these molecules could be important biomarkers of HO formation and a potential target for therapies.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Analysis of Xylem Cells by Nucleus-Based Transcriptomics and Chromatin Profiling.

Nuclei contain essential information for cell states, including chromatin and RNA profiles - features which are nowadays accessible using high-throughput sequencing applications. Here, we describe analytical pipelines including nucleus isolation from differentiated xylem tissues by fluorescence-activated nucleus sorting (FANS), as well as subsequent SMART-seq2-based transcriptome profiling and assay for transposase-accessible chromatin (ATAC)-seq-based chromatin analysis. Combined with tissue-specific expression of nuclear fluorescent reporters, these pipelines allow obtaining tissue-specific data on gene expression and on chromatin structure and are applicable for a large spectrum of cell types, tissues, and organs. Considering, however, the extreme degree of differentiation found in xylem cells with programmed cell death happening during vessel element formation and their role as a long-term depository for atmospheric CO2 in the form of wood, xylem cells represent intriguing and relevant objects for large-scale profilings of their cellular signatures.

RNA Profiles of Tear Fluid Extracellular Vesicles in Patients with Dry Eye-Related Symptoms.

Currently, diagnosing and stratifying dry eye disease (DED) require multiple tests, motivating interest in a single definitive test. The purpose of this study was to investigate the potential for using tear fluid extracellular vesicle (EV)-RNA in DED diagnostics. With a role in intercellular communication, nanosized EVs facilitate the protected transport of diverse bioactive molecules in biofluids, including tears. Schirmer strips were used to collect tears from 10 patients presenting with dry eye-related symptoms at the Norwegian Dry Eye Clinic. The samples comprised two groups, five from patients with a tear film break-up time (TBUT) of 2 s and five from patients with a TBUT of 10 s. Tear fluid EV-RNA was isolated using a Qiagen exoRNeasy Midi Kit, and the RNA was characterized using Affymetrix ClariomTM D microarrays. The mean signal values of the two groups were compared using a one-way ANOVA. A total of 26,639 different RNA transcripts were identified, comprising both mRNA and ncRNA subtypes. Approximately 6% of transcripts showed statistically significant differential abundance between the two groups. The mRNA sodium channel modifier 1 (SCNM1) was detected at a level 3.8 times lower, and the immature microRNA-130b was detected at a level 1.5 times higher in the group with TBUT 2 s compared to the group with TBUT 10 s. This study demonstrates the potential for using tear fluid EV-RNA in DED diagnostics.

Leptospira transcriptome sequencing using long-read technology reveals unannotated transcripts and potential polyadenylation of RNA molecules.

Leptospirosis, caused by the spirochete bacteria Leptospira, is an emerging zoonosis that causes life-threatening disease in humans and animals. However, a comprehensive understanding of leptospiral RNA profiles is limited. In this study, we sequenced and analyzed the transcriptome of multiple Leptospira strains using Oxford Nanopore Technologies' direct cDNA and direct RNA sequencing methods. We identified new operons, RNA molecules, and evidence of potential posttranscriptional polyadenylation in Leptospira transcriptomes. Some RNA molecules that had not been previously annotated could be potential sRNA or noncoding RNA molecules that play a role in gene expression regulation in Leptospira. Interestingly, the majority of the new RNA molecules identified in this study were not detected in the nonpathogenic Leptospira, suggesting potential virulence-related functions for these molecules. Overall, our study highlights the utility of Oxford Nanopore Technologies's sequencing in studying prokaryotic transcriptome profiles and offers a tool to improve our understanding of prokaryotic RNA landscapes and polyadenylation. Nonetheless, the findings from our study also warrant that the presence of homopolymers of adenine bases in the transcripts may interfere with the interpretation of bacterial transcriptome profiles. Carefully designed experiments are needed to explore the role of the features described in this study in Leptospira virulence and pathogenesis. IMPORTANCE Leptospirosis, caused by the spirochete bacteria Leptospira, is a zoonotic disease of humans and animals, accounting for over 1 million annual human cases and over 60,000 deaths. We have characterized operon transcriptional units, identified novel RNA coding regions, and reported evidence of potential posttranscriptional polyadenylation in the Leptospira transcriptomes for the first time using Oxford Nanopore Technology RNA sequencing protocols. The newly identified RNA coding regions and operon transcriptional units were detected only in the pathogenic Leptospira transcriptomes, suggesting their significance in virulence-related functions. This article integrates bioinformatics, infectious diseases, microbiology, molecular biology, veterinary sciences, and public health. Given the current knowledge gap in the regulation of leptospiral pathogenicity, our findings offer valuable insights to researchers studying leptospiral pathogenicity and provide both a basis and a tool for researchers focusing on prokaryotic molecular studies for the understanding of RNA compositions and prokaryotic polyadenylation for their organisms of interest.

Transcriptome analysis reveals involvement of thiopurine S-methyltransferase in oxidation-reduction processes

Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in the deactivation of thiopurines and represents a major determinant of thiopurine-related toxicities. Despite its well-known importance in thiopurine metabolism, the understanding of its endogenous role is lacking. In the present study, we aimed to gain insight into the molecular processes involving TPMT by applying a data fusion approach to analyze whole-genome expression data. The RNA profiling was done on whole blood samples from 1017 adult male and female donors to the Estonian biobank using Illumina HTv3 arrays. Our results suggest that TPMT is closely related to genes involved in oxidoreductive processes. The in vitro experiments on different cell models confirmed that TPMT influences redox capacity of the cell by altering S-adenosylmethionine (SAM) consumption and consequently glutathione (GSH) synthesis. Furthermore, by comparing gene networks of subgroups of individuals, we identified genes, which could have a role in regulating TPMT activity. The biological relevance of identified genes and pathways will have to be further evaluated in molecular studies.

Identification of transcriptome characteristics of granulosa cells and the possible role of UBE2C in the pathogenesis of premature ovarian insufficiency

BackgroundPremature ovarian insufficiency (POI) is an important cause of infertility characterized by the functional decline of the ovary. Granulosa cells (GCs) around oocytes are critical for folliculogenesis, and GC dysfunction is one of the important etiologies of POI. The aim of this study was to explore the potential biomarkers of POI by identifying hub genes and analyze the correlation of biomarkers with immune infiltration in POI using RNA profiling and bioinformatics analysis.MethodsRNA sequencing was performed on GCs from biochemical POI (bPOI) patients and controls. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were used to explore the candidate genes. qRT‒PCR was performed to verify the expression of hub genes. Western blot, Cell Counting Kit-8, 5‐ethynyl‐2’‐deoxyuridine (EdU) assays, TUNEL (TdT-mediated dUTP Nick-End Labeling) and flow cytometry analysis were used to validate the possible role of ubiquitin-conjugating enzyme 2C (UBE2C) in POI. CIBERSORT was adopted to explore immune cell infiltration and the correlation between UBE2C and immune cells in bPOI.ResultsThrough analysis of differentially expressed genes (DEGs) and WGCNA, we obtained 143 candidate genes. After construction of the protein‒protein interaction (PPI) network and analysis with Cytoscape, 10 hub genes, including UBE2C, PBK, BUB1, CDC20, NUSAP1, CENPA, CCNB2, TOP2A, AURKB, and FOXM1, were identified and verified by qRT‒PCR. Subsequently, UBE2C was chosen as a possible biomarker of POI because knockdown of UBE2C could inhibit the proliferation and promote the apoptosis of GCs. Immune infiltration analysis indicated that monocytes and M1 macrophages may be associated with the pathogenesis of POI. In addition, UBE2C was negatively correlated with monocytes and M1 macrophages in POI.ConclusionsThis study identified a hub gene in GCs that might be important in the pathogenesis of POI and revealed the key role of UBE2C in driving POI. Immune infiltration may be highly related with the onset and etiology of POI.

Noncoding RNA Profile in Reovirus Treated KRAS-Mutated Colorectal Cancer Patients.

To investigate the alterations in the expression of noncoding, micro, and small RNA expression during treatment with oncolytic reovirus in KRAS-mutated colorectal cancer. Oncolytic reovirus treatment was administered in phase 1 clinical trial (NCT01274624) for 5 days every 28 days, and blood samples were collected before the administration of the reovirus and 48 h, 8 days, and 15 days after its administration on day 1. Data from the blood samples were sorted using Transcriptome Analysis Software (TAC) 4.0, where a two-tailed t-test and a fold change filter were used to ascertain which sample signals had a statistically significant relative fold change of greater than 2 at multiple timepoints before or after oncolytic reovirus administration. The long noncoding RNA's RP11-332M2.1 (-6.1 x), LINC01506 (-16.18 x), and LINC00534 (-1.94 x) were downregulated at 48 h after reovirus administration [p < 0.05]. ncRNA's EPB41L4A-AS1 (-6.34 x, 48 h; 11.99 x, day 8), JAK2 (2.2 x, 48 h; -2.23 x, day 8), ANXA4 (20.47 x, day 8; -7.54 x, day 15), and PCDH9 (-2.09, day 8; 1.82 x, day 15) were affected by the reovirus treatment and reflected the progress of the treatment [p < 0.05]. The small RNA SNORA26 (-1.59 x, day 8) was downregulated 48 h after the reovirus administration [p < 0.05]. The microRNA MIR-4461 (6.18 x, day 8; -3.76 x, day 15) was also affected by the reovirus administration [p < 0.05]. The administration of oncolytic reovirus to treat KRAS-mutated colorectal cancer is reflected in a noncoding RNA profile, and expression levels of the ncRNAs in that profile may thus be able to be used as a potential predictive marker for reovirus-treated colorectal cancer.

Small RNA profiling reveals that an ovule-specific microRNA, cja-miR5179, targets a B-class MADS-box gene in Camellia japonica.

The functional specialization of microRNA and its target genes is often an important factor in the establishment of spatiotemporal patterns of gene expression that are essential to plant development and growth. In different plant lineages, understanding the functional conservation and divergence of microRNAs remains to be explored. To identify small regulatory RNAs underlying floral patterning, we performed a tissue-specific profiling of small RNAs in various floral organs from single and double flower varieties (flowers characterized by multiple layers of petals) in Camellia japonica. We identified cja-miR5179, which belongs to a deeply conserved microRNA family that is conserved between angiosperms and basal plants but frequently lost in eudicots. We characterized the molecular function of cja-miR5179 and its target - a B-function MADS-box gene - through gene expression analysis and transient expression assays. We showed that cja-miR5179 is exclusively expressed in ovule tissues at the early stage of floral development. We found that cja-miR5179 targets the coding sequences of a DEFICIENS-like B-class gene (CjDEF) mRNA, which is located in the K motif of the MADS-box domain; and the target sites of miR5179/MADS-box were consistent in Camellia and orchids. Furthermore, through a petal transient-expression assay, we showed that the BASIC PENTACYSTEINE proteins bind to the GA-rich motifs in the cja-miR5179 promoter region and suppresses its expression. We propose that the regulation between miR5179 and a B-class MADS-box gene in C. japonica has a deep evolutionary origin before the separation of monocots and dicots. During floral development of C. japonica, cja-miR5179 is specifically expressed in the ovule, which may be required for the inhibition of CjDEF function. This work highlights the evolutionary conservation as well as functional divergence of small RNAs in floral development.

Dynamic roles of small RNAs and DNA methylation associated with heterosis in allotetraploid cotton (Gossypium hirsutum L.)

BackgroundHeterosis is a complex phenomenon wherein the hybrids outperform their parents. Understanding the underlying molecular mechanism by which hybridization leads to higher yields in allopolyploid cotton is critical for effective breeding programs. Here, we integrated DNA methylation, transcriptomes, and small RNA profiles to comprehend the genetic and molecular basis of heterosis in allopolyploid cotton at three developmental stages.ResultsTranscriptome analysis revealed that numerous DEGs responsive to phytohormones (auxin and salicylic acid) were drastically altered in F1 hybrid compared to the parental lines. DEGs involved in energy metabolism and plant growth were upregulated, whereas DEGs related to basal defense were downregulated. Differences in homoeologous gene expression in F1 hybrid were greatly reduced after hybridization, suggesting that higher levels of parental expression have a vital role in heterosis. Small RNAome and methylome studies showed that the degree of DNA methylation in hybrid is higher when compared to the parents. A substantial number of allele-specific expression genes were found to be strongly regulated by CG allele-specific methylation levels. The hybrid exhibited higher 24-nt-small RNA (siRNA) expression levels than the parents. The regions in the genome with increased levels of 24-nt-siRNA were chiefly related to genes and their flanking regulatory regions, demonstrating a possible effect of these molecules on gene expression. The transposable elements correlated with siRNA clusters in the F1 hybrid had higher methylation levels but lower expression levels, which suggest that these non-additively expressed siRNA clusters, reduced the activity of transposable elements through DNA methylation in the hybrid.ConclusionsThese multi-omics data provide insights into how changes in epigenetic mechanisms and gene expression patterns can lead to heterosis in allopolyploid cotton. This makes heterosis a viable tool in cotton breeding.

A novel sorting signal for RNA packaging into small extracellular vesicles

Extracellular vesicles (EVs) play a critical role in the transport of functional RNAs to target recipient cells in numerous physiological processes. The RNA profiles present in EVs differed significantly from those in the originating cells, suggesting selective and active loading of specific RNAs into EVs. Small EVs (sEVs) obtained by stepwise ultracentrifugation have been reported to contain non-sEV components. Analysis of sEVs separated from non-sEVs components revealed that microRNAs may not be released by sEVs. This has raised interest in other RNA types, such as mRNA, which may be functional molecules released by sEVs. However, the molecular mechanisms underlying selective loading of mRNA into sEVs remain unclear. Here, we show that the part of 3′ untranslated region (UTR) sequence of RAB13 selectively enriches RNA in sEVs and serves as an RNA signal for loading into sEVs. Our results demonstrate that RAB13 is the most enriched RNA in sEVs, and this enrichment is primarily driven by its 3′UTR sequence. These findings highlight the potential of the RAB13 3′UTR sequence as an RNA signal that enables the loading of target RNA into sEVs. This technology has the potential to improve EV-based drug delivery and other applications.

Alzheimer's disease-induced phagocytic microglia express a specific profile of coding and non-coding RNAs.

Alzheimer's disease (AD) is a neurodegenerative disease and the main cause of dementia in the elderly. AD pathology is characterized by accumulation of microglia around the beta-amyloid (Aβ) plaques which assumes disease-specific transcriptional signatures, as for the disease-associated microglia (DAM). However, the regulators of microglial phagocytosis are still unknown. We isolated Aβ-laden microglia from the brain of 5xFAD mice for RNA sequencing to characterize the transcriptional signature in phagocytic microglia and to identify the key non-coding RNAs capable of regulating microglial phagocytosis. Through spatial sequencing, we show the transcriptional changes of microglia in the AD mouse brain in relation to Aβ proximity. Finally, we show that phagocytic messenger RNAs are regulated by miR-7a-5p, miR-29a-3p and miR-146a-5p microRNAs and segregate the DAM population into phagocytic and non-phagocytic states. Our study pinpoints key regulators of microglial Aβ clearing capacity suggesting new targets for future therapeutic approaches.