Transcriptome-wide Changes Research Articles

Hippocampal adenosine-to-inosine RNA editing in sepsis: dynamic changes and influencing factors.

Sepsis-associated encephalopathy is a diffuse brain dysfunction secondary to infection. It has been established that factors such as age and sex can significantly contribute to the development of sepsis-associated encephalopathy. Our recent study implicated a possible link between adenosine-to-inosine RNA editing and sepsis-associated encephalopathy, yet the dynamics of adenosine-to-inosine RNA editing during sepsis-associated encephalopathy and how it could be influenced by factors such as age, sex and antidepressants remain uninvestigated. Our current study analysed and validated transcriptome-wide changes in adenosine-to-inosine RNA editing in the hippocampus of different septic mouse models. Seventy-four sites in 64 genes showed significant differential RNA editing over time in septic mice induced by caecal ligation and perforation. The differential RNA editing might contribute to the RNA expression regulation of the edited genes, with 42.2% differentially expressed. These differentially edited genes, especially those with missense editing, such as glutamate receptor, ionotropic, kainate 2 (Grik2, p.M620V), filamin A (Flna, p.S2331G) and capicua transcriptional repressor (Cic, p.E2270G), were mainly involved in abnormal social behaviour and neurodevelopmental and psychiatric disorders. Significant effects of age and sex were also observed on sepsis-associated RNA editing. Further comparison highlighted 40 common differential RNA editing sites that caecal ligation and perforation-induced and lipopolysaccharide-induced septic mouse models shared. Interestingly, these findings demonstrate temporal dynamics of adenosine-to-inosine RNA editing in the mouse hippocampus during sepsis, add to the understanding of age and sex differences in the disease and underscore the role of the epigenetic process in sepsis-associated encephalopathy.

IL-1β + TGF-β2 dual-licensed mesenchymal stem cells have reduced major histocompatibility class I expression and positively modulate tenocyte migration, metabolism, and gene expression.

The study objectives were to 1) determine the mesenchymal stem cell (MSC) surface expression of major histocompatibility complex (MHC) class I and transcriptome-wide gene expression changes following IL-1β + TGF-β2 dual licensing and 2) evaluate if IL-1β + TGF-β2 dual-licensed MSCs had a greater ability to positively modulate tenocyte function compared to naive MSCs. Equine bone marrow-derived MSCs from 6 donors and equine superficial digital flexor tenocytes from 3 donors. Experiments were performed in vitro. Flow cytometry and bulk RNA sequencing were utilized to determine naive and dual-licensed MSC phenotype and transcriptome-wide changes in gene expression. Conditioned media were generated from MSCs and utilized in tenocyte cell culture assays as a method to determine the effect of MSC paracrine factors on tenocyte function. Dual-licensed MSCs have a reduced expression of MHC class I and exhibit enrichment in functional pathways associated with the extracellular matrix, cell signaling, and tissue development. Additionally, dual-licensed MSC-conditioned media significantly improved in vitro tenocyte migration and metabolism to a greater degree than naive MSC-conditioned media. In tenocytes exposed to IL-1β, dual-licensed conditioned media also positively modulated tenocyte gene expression. Our data indicate that conditioned media containing paracrine factors secreted from dual-licensed MSCs significantly modulates in vitro tenocyte function, which may confer benefits in vivo to healing tendons following injury. Additionally, due to reduced MHC class I expression in dual-licensed MSCs, this technique may also provide an avenue to provide an effective "off-the-shelf" allogenic source of MSCs.

Transcriptome-wide characterization of alternative splicing regulation in Najran wheat (Triticum aestivum) under salt stress

The process of alternative splicing (AS) has emerged as a crucial mechanism in plant responses to environmental stresses, contributing to the enhancement of the required transcriptome and proteome complexity. Despite the importance of AS, there remains a paucity of studies on the regulatory implications of AS in the responses of wheat to salt stress. In the current study, transcriptome-wide changes in AS profiles were established in roots and shoots of Najran wheat treated with 200 mM NaCl. Salt stress induced AS events increasing the complexity of the transcriptome; out of all expressed genes in all samples, 32,268 genes (22.5% of expressed genes) in the roots and 31,941 genes (23.1% of expressed genes) in the shoots were subjected to AS with 3’ Alternative splice site (A3) being the most frequent AS event and mutually exclusive exon (MX) being the least common event. Moreover, the results revealed that salt stress modulates AS patterns in a tissue-specific way where 82% of AS events were differentially expressed in either root or shoot tissues, participating in organ differentiation. In Total, 423 Differential AS events associated with cytoskeletal-related categories such as microtubule-based processes, actin filament-based movements, and cytoskeletal motor activity were identified in the roots. In contrast, 393 Differential AS events associated with biological categories related to metabolic and signalling processes such as catabolic processes, and response to gibberellin were identified in the shoots. The results presented in this study enhance our understanding of salt tolerance mechanisms in wheat and provide promising insights for future functional investigations and crop improvement efforts.

Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Pan-cancer ion transport signature reveals functional regulators of glioblastoma aggression

Ion channels, transporters, and other ion-flux controlling proteins, collectively comprising the “ion permeome”, are common drug targets, however, their roles in cancer remain understudied. Our integrative pan-cancer transcriptome analysis shows that genes encoding the ion permeome are significantly more often highly expressed in specific subsets of cancer samples, compared to pan-transcriptome expectations. To enable target selection, we identified 410 survival-associated IP genes in 33 cancer types using a machine-learning approach. Notably, GJB2 and SCN9A show prominent expression in neoplastic cells and are associated with poor prognosis in glioblastoma, the most common and aggressive brain cancer. GJB2 or SCN9A knockdown in patient-derived glioblastoma cells induces transcriptome-wide changes involving neuron projection and proliferation pathways, impairs cell viability and tumor sphere formation in vitro, perturbs tunneling nanotube dynamics, and extends the survival of glioblastoma-bearing mice. Thus, aberrant activation of genes encoding ion transport proteins appears as a pan-cancer feature defining tumor heterogeneity, which can be exploited for mechanistic insights and therapy development.

MeRIP-Seq for Identifying Stress-Responsive Transcriptome-Wide m6A Profiles in Plants.

Recent advancements in detection and mapping methods have enabled researchers to uncover the biological importance of RNA chemical modifications, which play a vital role in post-transcriptional gene regulation. Although numerous types of RNA modifications have been identified in higher eukaryotes, only a few have been extensively studied for their biological functions. Of these, N6-methyladenosine (m6A) is the most prevalent and important mRNA modification that influences various aspects of RNA metabolism, including mRNA stability, degradation, splicing, alternative polyadenylation, export, and localization, as well as translation. Thus, they have implications for a variety of biological processes, including growth, development, and stress responses. The m6A deposition or removal on transcripts is dynamic and is altered in response to internal and external cues. Because this mark can alter gene expression under stress conditions, it is essential to identify the transcripts that can acquire or lose this epitranscriptomic mark upon exposure to stress conditions. Here we describe a step-by-step protocol for identifying stress-responsive transcriptome-wide m6A changes using RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq).

Effects of miR-722 on gene expression and alternative splicing in the liver of half-smooth tongue sole after infection with Vibrio anguillarum

MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8, were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture.

Collective signalling drives rapid jumping between cell states.

Development can proceed in 'fits and starts', with rapid transitions between cell states involving concerted transcriptome-wide changes in gene expression. However, it is not clear how these transitions are regulated in complex cell populations, in which cells receive multiple inputs. We address this issue using Dictyostelium cells undergoing development in their physiological niche. A continuous single cell transcriptomics time series identifies a sharp 'jump' in global gene expression marking functionally different cell states. By simultaneously imaging the physiological dynamics of transcription and signalling, we show the jump coincides with the onset of collective oscillations of cAMP. Optogenetic control of cAMP pulses shows that different jump genes respond to distinct dynamic features of signalling. Late jump gene expression changes are almost completely dependent on cAMP, whereas transcript changes at the onset of the jump require additional input. The coupling of collective signalling with gene expression is a potentially powerful strategy to drive robust cell state transitions in heterogeneous signalling environments. Based on the context of the jump, we also conclude that sharp gene expression transitions may not be sufficient for commitment.

Mapping the spatial transcriptomic signature of the hippocampus during memory consolidation

Memory consolidation involves discrete patterns of transcriptional events in the hippocampus. Despite the emergence of single-cell transcriptomic profiling techniques, mapping the transcriptomic signature across subregions of the hippocampus has remained challenging. Here, we utilized unbiased spatial sequencing to delineate transcriptome-wide gene expression changes across subregions of the dorsal hippocampus of male mice following learning. We find that each subregion of the hippocampus exhibits distinct yet overlapping transcriptomic signatures. The CA1 region exhibited increased expression of genes related to transcriptional regulation, while the DG showed upregulation of genes associated with protein folding. Importantly, our approach enabled us to define the transcriptomic signature of learning within two less-defined hippocampal subregions, CA1 stratum radiatum, and oriens. We demonstrated that CA1 subregion-specific expression of a transcription factor subfamily has a critical functional role in the consolidation of long-term memory. This work demonstrates the power of spatial molecular approaches to reveal simultaneous transcriptional events across the hippocampus during memory consolidation.

Divergent molecular events underlying initial T-cell commitment in human prenatal and postnatal thymus.

Intrathymic T-cell development is a coordinated process accompanied by dynamic changes in gene expression. Although the transcriptome characteristics of developing T cells in both human fetal and postnatal thymus at single-cell resolution have been revealed recently, the differences between human prenatal and postnatal thymocytes regarding the ontogeny and early events of T-cell development still remain obscure. Moreover, the transcriptional heterogeneity and posttranscriptional gene expression regulation such as alternative polyadenylation at different stages are also unknown. In this study, we performed integrative single-cell analyses of thymocytes at distinct developmental stages. The subsets of prenatal CD4-CD8- double-negative (DN) cells, the most immature thymocytes responsible for T-cell lineage commitment, were characterized. By comprehensively comparing prenatal and postnatal DN cells, we revealed significant differences in some key gene expressions. Specifically, prenatal DN subpopulations exhibited distinct biological processes and markedly activated several metabolic programs that may be coordinated to meet the required bioenergetic demands. Although showing similar gene expression patterns along the developmental path, prenatal and postnatal thymocytes were remarkably varied regarding the expression dynamics of some pivotal genes for cell cycle, metabolism, signaling pathway, thymus homing, and T-cell commitment. Finally, we quantified the transcriptome-wide changes in alternative polyadenylation across T-cell development and found diverse preferences of polyadenylation site usage in divergent populations along the T-cell commitment trajectory. In summary, our results revealed transcriptional heterogeneity and a dynamic landscape of alternative polyadenylation during T-cell development in both human prenatal and postnatal thymus, providing a comprehensive resource for understanding T lymphopoiesis in human thymus.

IDESC: identifying differential expression in single-cell RNA sequencing data with multiple subjects

BackgroundSingle-cell RNA sequencing (scRNA-seq) technology has enabled assessment of transcriptome-wide changes at single-cell resolution. Due to the heterogeneity in environmental exposure and genetic background across subjects, subject effect contributes to the major source of variation in scRNA-seq data with multiple subjects, which severely confounds cell type specific differential expression (DE) analysis. Moreover, dropout events are prevalent in scRNA-seq data, leading to excessive number of zeroes in the data, which further aggravates the challenge in DE analysis.ResultsWe developed iDESC to detect cell type specific DE genes between two groups of subjects in scRNA-seq data. iDESC uses a zero-inflated negative binomial mixed model to consider both subject effect and dropouts. The prevalence of dropout events (dropout rate) was demonstrated to be dependent on gene expression level, which is modeled by pooling information across genes. Subject effect is modeled as a random effect in the log-mean of the negative binomial component. We evaluated and compared the performance of iDESC with eleven existing DE analysis methods. Using simulated data, we demonstrated that iDESC had well-controlled type I error and higher power compared to the existing methods. Applications of those methods with well-controlled type I error to three real scRNA-seq datasets from the same tissue and disease showed that the results of iDESC achieved the best consistency between datasets and the best disease relevance.ConclusionsiDESC was able to achieve more accurate and robust DE analysis results by separating subject effect from disease effect with consideration of dropouts to identify DE genes, suggesting the importance of considering subject effect and dropouts in the DE analysis of scRNA-seq data with multiple subjects.

PEPseq quantifies transcriptome-wide changes in protein occupancy and reveals selective translational repression after translational stress.

Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal an increase of protein interactions in the coding region of a distinct set of mRNAs, including mRNAs coding for the majority of cytosolic ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.

Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

462 RNA-Based Therapeutics Targeting the Oncogenic Splicing Factor TRA2β Rewire Glioblastoma Cell Transcriptomes and Inhibit Their Proliferation

INTRODUCTION: Glioblastomas are an aggressive brain tumor that, despite current treatment paradigms, carry a poor prognosis. Novel therapies aimed at specific glioblastoma alterations are needed. Alternative RNA splicing is a critical step in gene expression that allows a single gene to encode multiple RNA and subsequently protein isoforms. Alternative splicing becomes dysregulated in human tumors, including glioblastoma, through alterations in upstream regulatory splicing factor proteins such as TRA2β, an oncogene that enhances glioma cell proliferation. Despite these known alterations, targeting alternative splicing in glioblastomas has not been leveraged therapeutically. METHODS: U87MG glioblastoma cells were treated with splice-switching antisense oligonucleotides (ASOs) targeting the TRA2β transcript (ASO-1570) or non-targeting control (ASO-CTL). Cancer-relevant phenotypes, including proliferation and apoptosis, were measured using high-content imaging. Transcriptomic splicing signatures were defined using high-depth RNA-sequencing with computational tools that quantify percent splicing event inclusion (PSI) and validated using RT-PCR and western blotting. RESULTS: ASO-1570 treatment increased inclusion of a poison-exon in TRA2β leading to decreased coding transcripts and subsequent reductions in TRA2β protein expression compared to ASO-CTL. ASO-1570 significantly decreased cell proliferation and increased apoptosis. Mechanistically, ASO-1570 treated cells exhibited significant differences in alternatively spliced events, impacting genes involved in cancer-relevant pathways such as p53 and mTOR signaling. For example, ASO-1570 led to increased exon skipping in MDM2 leading to decreased MDM2 protein expression and increased p53 wild-type expression. ASO-1570 treatment also impacted growth of U87MG 3D-neurospheres, suggesting efficacy in advanced models that better capture in vivo conditions. CONCLUSIONS: Glioblastoma cells are sensitive to ASO-mediated TRA2β knockdown, which mechanistically induced transcriptome wide changes that impact cancer-related pathways. Further testing of this ASO in vivo may open novel avenues for its use as a targeted glioblastoma therapeutic.

Characterization of the sublethal toxicity and transcriptome-wide biological changes induced by λ-cyhalothrin in Bombyx mori.

λ-Cyhalothrin (λ-cyh) is widely used in agricultural production and has been reported to cause damages to numerous nontarget insects. As an important economic and model insect of Lepidoptera, Bombyx mori was extremely sensitive to λ-cyh, and pesticide drift often leads to silkworm poisoning. However, little is known about the persistence of sublethal effects or the potential recovery from short-term exposure to sublethal doses of pesticides. In this study, we estimated the sublethal effects caused by short-term exposure (24 h) of λ-cyh LC1 , LC10 , LC25 , and LC50 , respectively, and investigated the persistent negative effects on the growth, survival, and pupal metamorphosis of silkworm larvae. Silkworm growth was mostly retarded after λ-cyh exposure, with dose-dependent recovery observed at delayed time points. Relative to the control, the treatment groups showed significantly higher larval mortalities and abnormal pupa rates. Additionally, transcriptome sequencing was conducted to investigate the effects of λ-cyh LC10 on the normal physiological functions in the midgut of B. mori. A total of 2697 differentially expressed genes were identified, and 57.1% of DEGs were down-regulated. Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis further revealed that energy and nutrient metabolisms were negatively affected. Moreover, we demonstrated that sublethal λ-cyh inhibited the oxidative phosphorylation pathway by reducing the expression of mitochondrial electron transport chain complex genes and consequently the synthesis of ATP. This study has provided useful transcriptome-wide expression resources to facilitate the overall knowledge of the molecular basis of sublethal toxicity caused by λ-cyh in the midgut of B. mori.

SpliceTools, a suite of downstream RNA splicing analysis tools to investigate mechanisms and impact of alternative splicing.

As a fundamental aspect of normal cell signaling and disease states, there is great interest in determining alternative splicing (AS) changes in physiologic, pathologic, and pharmacologic settings. High throughput RNA sequencing and specialized software to detect AS has greatly enhanced our ability to determine transcriptome-wide splicing changes. Despite the richness of this data, deriving meaning from sometimes thousands of AS events is a substantial bottleneck for most investigators. We present SpliceTools, a suite of data processing modules that arms investigators with the ability to quickly produce summary statistics, mechanistic insights, and functional significance of AS changes through command line or through an online user interface. Utilizing RNA-seq datasets for 186 RNA binding protein knockdowns, nonsense mediated RNA decay inhibition, and pharmacologic splicing inhibition, we illustrate the utility of SpliceTools to distinguish splicing disruption from regulated transcript isoform changes, we show the broad transcriptome footprint of the pharmacologic splicing inhibitor, indisulam, we illustrate the utility in uncovering mechanistic underpinnings of splicing inhibition, we identify predicted neo-epitopes in pharmacologic splicing inhibition, and we show the impact of splicing alterations induced by indisulam on cell cycle progression. Together, SpliceTools puts rapid and easy downstream analysis at the fingertips of any investigator studying AS.

Transcriptome profiling of bovine endometrial epithelial cells induced by lipopolysaccharides in vitro

Endometritis is an inflammation of the surface of the endometrium that does not penetrate the submucosa and can cause infertility and increase the elimination rate in cows. Endometrial epithelial cells are the first barrier of the endometrium against foreign stimuli and bacterial infection. Understanding the genetic changes in stimulated endometrial epithelial cells will help in the efforts to prevent and treat endometritis. This study investigated changes in bovine endometrial epithelial (BEEC) gene expression induced by lipopolysaccharide (LPS)-induced inflammation and compared transcriptome-wide gene changes between LPS- and phosphate-buffered saline (PBS)- treated BEECs by RNA sequencing. Compared with the PBS group, the LPS group showed 60 differentially expressed genes (DEGs) (36 upregulated, 24 downregulated). Gene Ontology enrichment analysis revealed that most enrichment occurred during CXCR chemokine receptor binding, inflammatory response, and neutrophil migration. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed DEGs mainly concentrated in cytokine–cytokine receptor interactions; IL-17, tumor necrosis factor, NOD-like receptor, chemokine, Toll-like receptor, and nuclear factor-κB signaling pathways; and the cytoplasmic DNA sensing pathway. Moreover, results revealed that cytokines SAA3 and HP increased significantly after LPS treatment. These effects of LPS on BEECs transcriptome and the molecular mechanism of endometritis provide a basis for improved clinical treatment and novel drug development.

A conditional Smg6 mutant mouse model reveals circadian clock regulation through the nonsense-mediated mRNA decay pathway.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in physiological control of mRNA stability have emerged, possible functions in mammalian physiology in vivo remain unclear. Here, we created a conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We find that NMD down-regulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. Specifically, we uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. Transcriptome-wide changes in daily mRNA accumulation patterns in the entrained liver, as well as an altered response to food entrainment, expand the known scope of NMD regulation in mammalian gene expression and physiology.

LCM-Seq for Retinal Cell Layer-Specific Responses During Optic Nerve Regeneration.

LCM-seq is a powerful tool for gene expression analysis from individual or groups of cells that can be spatially isolated. Within the visual system, retinal ganglion cells (RGCs), the cells that connect the eye to the brain through the optic nerve, reside in the retinal ganglion cell layer of the retina. This well-defined location provides a unique opportunity to harvest RNA by laser capture microdissection (LCM) from a highly enriched cell population. Using this method, it is possible to explore transcriptome-wide changes in gene expression following optic nerve injury. In the zebrafish model, this method can be used to identify molecular events driving successful optic nerve regeneration in contrast to mammals that fail to regenerate axons in the central nervous system. Here we provide a method for LCM from the different retinal layers of zebrafish following optic nerve injury and during the process of optic nerve regeneration. Purified RNA from this protocol is sufficient for RNA-seq or other downstream analysis.

G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.