High-throughput RNA Sequencing Analysis Research Articles

Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types.

GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase-driven expression of hemagglutinin A (HA)-tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government. Basic Protocol 1: Breeding mice to obtain RiboTag homozygosity Support Protocol 1: Detection of ectopic Cre recombinase expression Basic Protocol 2:The RiboTag assay Support Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA Support Protocol 3: Construction of cell-type-specific RNA-seq library Support Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset.

Inflammatory‐sensitive CHI3L1 protects nucleus pulposus via AKT3 signaling during intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is the main cause of low back pain and the mechanism of which is far from fully revealed. Although inflammation directed nucleus pulposus (NP) extracellular matrix metabolism dysregulation is known to be the main cause of the degeneration process, few is known about the protective factors. Using high-throughput label-free proteomics, we found that inflammation-related autocrine factor Chitinase-3-like protein 1 (CHI3L1, or YKL-40) is highly expressed in the NP cells during degeneration. Immunohistochemical analysis show that the expression of CHI3L1 is NP tissue specific, and increase significantly during degeneration. Overexpression of CHI3L1 significantly decrease the catabolism, and increase the anabolism of extracellular matrix. Knockdown of CHI3L1 using siRNAs show the opposite results, which imply that the protective role of CHI3L1 in IDD. Using high-throughput RNA sequencing and functional analyses, we find that AKT3 expression and its phosphorylation is mainly regulated by CHI3L1. And lastly, the mechanism of which is also validated using human and mouse degenerated NP tissues. In summary, our findings show that the inflammation-related autocrine factor CHI3L1 is NP specific, and it protects IDD by promoting the AKT3 signaling, which may serve as a potential therapeutic target in intervertebral disc degeneration.

P135 MAJOR GENE REGULATORS AFFECTED IN COLON AND BLOOD OF DEXTRAN SODIUM SULFATE ACUTE COLITIS MURINE MODEL

Abstract Background Dextran sulfate sodium (DSS) ulcerative colitis (UC) murine models have long been used for in vivo studies. DSS is a negatively charged polysaccharide with colitogenic properties. Although the mechanisms by which DSS induces intestinal inflammation are not fully understood, there are several good reasons why the DSS chemical colitis model for investigating the immunopathogenesis mechanism of UC is widely used. These include strong phenotypic clinical manifestations which emulate numerous clinical and histopathological features of human UC, ease of use, low mortality rate and high reproducibility. Here, by using high-throughput RNA sequencing analysis we set to investigate the major predicted gene regulators (GRs) affected by differentially expressed genes in the DSS treated UC model in order to obtain regulatory insights into the pathogenic mechanisms of UC development. Methods A DSS-induced mouse model of UC was established. Total RNA from colon tissue and blood of 3 healthy and 3 DSS-treated mice was extracted and sequenced by Illumina HiSeq 4000. Gene expression levels were obtained by mapping and quantification to the annotated mouse genome. Subsequently, differential gene expression analysis between DSS-treated and control mice both in colon and blood was performed. Ingenuity pathway analysis software (IPA®, Qiagen) was used to predict/identify major GRs affected by significantly differentially expressed genes (SDEGs, FC > |2|, p ≤0.05) in both colon and blood. Results Our analysis revealed how many and which major GRs are affected in DSS-treated mice and also the direction of change as compared to healthy (untreated) mice. In colon, 595 activated and 198 inhibited major GRs (p-value of overlap ≤0.05) in relation to ∼ 3180 SDEGs were identified, while in blood, we identified 205 activated and 62 inhibited GRs (in relation to ∼650 SDEGs). Colon and blood share 181 activated and 41 inhibited GRs. Identified GRs include transcription regulators, cytokines, transmembrane receptors and enzymes that mainly contribute to the development of inflammatory/immune responses. In colon and blood, the top 10 activated and inhibited regulators with the highest positive and negative activation z-score with target molecules as well as expression in the datasets are indicated in Figure 1a and 1b, respectively. Conclusion In this study, we analyzed linkage of GRs to SDEGs through coordinated expression and identified potential major regulators that have significant effect on UC pathogenic-related gene expression. These GRs seem to be the key regulators of transcriptomic changes induced by inflammation. These findings expand our molecular understanding of putative new targets that may be important in the pathophysiology of UC and provide biological insights into the observed expression changes between the UC and healthy controls.

Novel findings to the biosynthetic pathway of magnoflorine and taspine through transcriptomic and metabolomic analysis of Croton draco (Euphorbiaceae)

BackgroundCroton draco is an arboreal species and its latex as well as some other parts of the plant, are traditionally used in the treatment of a wide range of ailments and diseases. Alkaloids, such as magnoflorine, prevent early atherosclerosis progression while taspine, an abundant constituent of latex, has been described as a wound-healer and antitumor-agent. Despite the great interest for these and other secondary metabolites, no omics resources existed for the species and the biosynthetic pathways of these alkaloids remain largely unknown.ResultsTo gain insights into the pathways involved in magnoflorine and taspine biosynthesis by C. draco and identify the key enzymes in these processes, we performed an integrated analysis of the transcriptome and metabolome in the major organs (roots, stem, leaves, inflorescences, and flowers) of this species. Transcript profiles were generated through high-throughput RNA-sequencing analysis while targeted and high resolution untargeted metabolomic profiling was also performed. The biosynthesis of these compounds appears to occur in the plant organs examined, but intermediaries may be translocated from the cells in which they are produced to other cells in which they accumulate.ConclusionsOur results provide a framework to better understand magnoflorine and taspine biosynthesis in C. draco. In addition, we demonstrate the potential of multi-omics approaches to identify candidate genes involved in the biosynthetic pathways of interest.

High-Throughput RNA Sequencing Analysis Reveals Distinct Molecular Signature in NRAS and PTPN11 JMML Patients

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare pediatric hematological disease, characterized by aberrant proliferation of myeloid precursors and hypersensitivity to GM-CSF stimulation. Mutations in PTPN11, N/K-RAS, CBL, or NF1 genes are found in ~90% of the cases. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for those patients harboring somatic PTPN11 mutations, while in patients with somatic NRAS mutations, transplantation need a careful evaluation, as these patients may have spontaneous resolution of the disease. Here, we show data from a deep-sequencing analysis enlightening typical molecular features of CD33+/34+ and CD14+ primary cells from NRAS and PTPN11 mutated patients compared with healthy donors (HDs). Patients and Methods Four Cord Blood samples from healthy donors (HDs) and 16 Bone Marrow (BM) samples from PTPN11 (N=8) and NRAS (n=8) mutated JMML patients were collected after approvals from International Review Boards of each Institution. CD33+/34+ and CD14+ cells were magnetically sorted following MACS protocols (Miltenyi). TruSeq and NextSeq kits (Illumina) were used for libraries preparation in order to perform the global transcriptomic and smallRNAs sequencing analyses on Illumina platform, according to the company protocols. StringTie and DESeq2 were used for differential expression analysis and REDItools python scripts for RNA editing analysis. Results In our first analysis, we screened ~60000 gene transcripts, finding different expression signatures between HDs, PTPN11 and NRAS samples. We built Expression Heat-Maps reporting the top 100 most statistically significant (Fig. 1, 2; adjusted p < 0.001) deregulated sequences in all the possible pairwise comparisons. Among the top 100 transcripts, we identified differential expression of 5 different genes: ZNF185, MRLPL30 resulted upregulated in PTPN11 vs NRAS CD14+ cells while CD36, RAG2 and CAMK2A resulted downregulated. Interestingly, comparison of PTPN11 and NRAS cohorts in CD33+/34+ subset, these 5 transcripts showed the opposite expression trend. Moreover, among the upregulated genes, we identified a subset of PRAME paralog transcripts in both NRAS and PTPN11 patients when compared to HDs. Seven transcripts (PRAMEF4, 5, 6, 9, 11, 15, 26) were shared between CD33+/34+ and CD14+ populations, while PRAMEF7, 8, 20, 21 resulted upregulated in CD33+/34+ cells and PRAME, PRAMEF22, 23, 25 were upregulated in CD14+ cells, as compared to HDs. In addition, we found aberrant upregulation of Hemoglobin γ chains 1 and 2 (HBG1, 2, i.e. those needed for fetal hemoglobin) in all JMML vs HDs in CD14+ cells, while in CD33+/34+ a downregulation of Hemoglobin α and β chains (HBA1,2 and HBB) in PTPN11 samples vs NRAS was detected. We also report an enhanced LIN28B expression in JMML samples vs HDs but only in CD33+/34+ cells. A global ADAR-dependent RNA editing analysis revealed differences in CD33+/34+ cells showing an enhanced Adenine to Inosine conversion in NRAS patients vs PTPN11 and HDs. Finally, microRNAs sequencing reveals a significant upregulation of miR-144-3p in all JMML samples and cell lineages compared to HDs, while the miR-146a-5p was upregulated only in CD14+ cells. Also the miR-22 showed decreased expression in PTPN11 vs HDs in CD33+/34+ subset, while the miR-29a-3p was downregulated in NRAS vs HDs in CD14+ cells. Discussion We report different expression and editing signatures among samples cohorts and cellular lineages, showing novel insights in JMML molecular biology. Upregulation of several PRAME paralog genes depicts an interesting array of possible targets for immunotherapies (e.g., using genetically modified T cells with a PRAME-specific T-cell receptor), although their expression have to be further validated. Differences in the expression of microRNAs subsets, LIN28B and subunits of the HbA and HbF already described in JMML, but here related to specific mutations and cell lineages, could help to better understand the pathogenic processes and peculiar clinical behaviors. Finally, the novel evidence of differential RNA editing in JMML opens new perspectives for further studies regarding differential protein isoforms expression and regulation as well as microRNA targeting. Disclosures Niemeyer: Celgene: Consultancy. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy. Merli:Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy.

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

Intraocular VEGF deprivation induces degeneration and fibrogenic response in retina.

VEGF is a critical driver of ocular neovascularization under disease conditions. Current therapeutic strategies rely on intraocular delivery of VEGF-antagonizing reagents, which results in sustained suppression of pathogenic vascularization. Although significant advancement has been achieved in VEGF antagonism, substantial adverse effects have been reported in retrospective clinical studies. To study mechanisms for VEGF antagonism-associated adverse effects in visual system, we intravitreally delivered recombinant adeno-associated virus-mediated expression of soluble Fms-related tyrosine kinase-1 (rAAV.sFLT-1), the extracellular domain of VEGF receptor, and analyzed the morphology and functions of retinal tissue. Here, we confirmed that intraocular VEGF antagonism induced retinal degeneration and gliosis. The functional deficit in retinal response to visual stimulation was also demonstrated in rAAV.sFLT-1-treated eyes by electroretinogram. Moreover, high-throughput RNA sequencing analysis suggests that VEGF antagonism activates retinal degeneration, inflammation, and other adverse effects. Taken together, our findings have shed light on pathogenic mechanisms for VEGF antagonism-associated adverse effects and potential therapeutic targets.-Xiao, M., Liu, Y., Wang, L., Liang, J., Wang, T., Zhai, Y., Wang, Y., Liu, S., Liu, W., Luo, X., Wang, F., Sun, X. Intraocular VEGF deprivation induces degeneration and fibrogenic response in retina.

Conjoint Analysis of Genome-Wide lncRNA and mRNA Expression of Heteromorphic Leavesin Response to Environmental Heterogeneityin Populus euphratica.

Heterophylly is the phenomenon of leaf forms varying along the longitudinal axis within a single plant. Populus euphratica, a heterophyllous woody plant, develops lanceolate leaves and dentate broad-ovate leaves on the bottom and top of the canopy, respectively, which are faced with different intensities of ambient solar radiation. However, the mechanism of the heteromorphic leaf response to the microenvironment in P. euphratica remains elusive. Here, we show that the dentate broad-ovate leaves have advantages in tolerating high light intensity, while lanceolate leaves are excellent at capturing light. Compared with lanceolate leaves, more trichomes, higher stomatal density, thicker lamina, and higher specific leaf weight were observed in dentate broad-ovate leaves. Furthermore, high-throughput RNA sequencing analysis revealed that the expression patterns of genes and long noncoding RNAs (lncRNAs) are different between the two heteromorphic leaves. A total of 36,492 genes and 1725 lncRNAs were detected, among which 586 genes and 54 lncRNAs were differentially expressed. Based on targets prediction, lncRNAs and target genes involved in light adaption, protein repair, stress response, and growth and development pathways were differentially expressed in heteromorphic leaves, 10 pairs of which were confirmed by quantitative real-time PCR. Additionally, the analysis of interactions indicated that lncRNA–mRNA interactions were involved in the response to the microenvironment of heteromorphic leaves. Taken together, these results suggest that the morphological features and joint regulation of lncRNA–mRNA in heteromorphic leaves may serve as survival strategies for P. euphratica, which could lead to optimal utilization of environmental factors.

Oxygen-induced circRNA profiles and coregulatory networks in a retinopathy of prematurity mouse model.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. At present, the molecular mechanisms underlying ROP are still far from being clearly understood. Circular RNAs (circRNAs), a novel class of noncoding RNAs, have been reported to serve vital regulatory roles in several human diseases. However, it is still unclear how circRNAs are involved in ROP. In the present study, oxygen-induced retinopathy (OIR) murine retinal samples and paired normal tissues were chosen for high-throughput transcriptome RNA sequencing and bioinformatic analyses. As a result, a total of 236 differentially expressed circRNAs, 14 differentially expressed miRNAs, and 9,756 differentially expressed mRNAs were identified in the OIR samples. Gene ontology analysis showed that angiogenesis ranked in the top five upregulated biological processes associated with differential mRNA expression. Then, 66 co-expression pairs of circRNA-mRNA were predicted according to the mRNAs that were enriched in angiogenesis. Furthermore, coregulation prediction was separately performed to identify the differentially expressed miRNAs that targeted angiogenesis-associated circRNAs or mRNAs. Finally, nine differentially expressed circRNAs were predicted to be competing endogenous RNAs by constructing a circRNA-miRNA-mRNA network followed by reverse transcription-quantitative PCR validation. The results of the present study suggest that the identified set of circRNA transcripts and the potential regulatory mechanisms for the development of ROP are worthy of functional studies.

Coupling of COPII vesicle trafficking to nutrient availability by the IRE1α-XBP1s axis

The cytoplasmic coat protein complex-II (COPII) is evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of the nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII vesicle trafficking parallel the activation of transcription factor X-box binding protein 1 (XBP1s), a critical transcription factor in handling cellular endoplasmic reticulum (ER) stress in both live cells and mouse livers upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent trafficking, mediating the nutrient stimulatory effects. Chromatin immunoprecipitation (ChIP) coupled with high-throughput DNA sequencing (ChIP-seq) and RNA-sequencing analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII traffic-related genes, thereby driving the COPII-mediated trafficking process. Liver-specific disruption of the inositol-requiring enzyme 1α (IRE1α)-XBP1s signaling branch results in diminished COPII vesicle trafficking. Reactivation of XBP1s in mice lacking hepatic IRE1α restores COPII-mediated lipoprotein secretion and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid trafficking: The IRE1α-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII vesicle trafficking.

Transcriptional Analysis Shows a Robust Host Response toToxoplasma gondiiduring Early and Late Chronic Infection in Both Male and Female Mice

The long-term host effects caused by the protozoan parasite Toxoplasma gondii are poorly understood. High-throughput RNA sequencing analysis previously determined that the host response in the brain was greater and more complex at 28 days than at 10 days postinfection. Here, we analyzed the host transcriptional profile of age- and sex-matched mice during very early (21 days), early (28 days), mid (3 months), and late (6 months) chronic infection. We found that a majority of the host genes which increase in abundance at day 21 postinfection are still increased at 6 months postinfection for both male and female mice. While most of the differentially expressed genes were similar between sexes, females had far fewer genes that were significantly less abundant, which may have led to the slightly increased cyst burden in males. Transcripts for C-X-C motif chemokine ligand 13 and a C-C motif chemokine receptor 2 (CCR2) were significantly higher in females than in males during infection. As T. gondii chronic infection and profilin (PRF) confer resistance to Listeria monocytogenes infection in a CCR2-dependent manner, the differences in CCR2 expression led us to retest the protection of PRF in both sexes. Male mice were nearly as effective as female mice at reducing the bacterial burden either with a chronic infection or when treated with PRF. These data show that most of the host genes differentially expressed in response to T. gondii infection are similar between males and females. While differences in transcript abundance exist between the sexes, the infection phenotypes tested here did not show significant differences.

SUN-024 Uncovering a Novel Role for Nuclear Receptor Fxr and Shp in Regulating N-Linked Glycosylation

Nuclear receptor Farnesoid X Receptor (Fxr) and Small Heterodimer Partner (Shp) are key regulators of glucose, fat and bile acid homeostasis. As expected, liver-specific deletion of both Fxr and Shp (LDKO) sensitizes mice to bile acid excess. Surprisingly, high throughput RNA-sequencing analysis revealed increases in key glycosyltransferases involved in “decorating” various glycoproteins in LDKO livers. Remarkably, we were able to confirm increased glycan decoration and branching in LDKO livers using glycomics and glycoproteomics approaches. Further, electron microscopic analysis revealed disruption and dilation of Golgi network, which is consistent with aberrant glycosylation. LDKO mice were hypoglycemic, stored lower glycogen, indicating glucose being utilized to meet the increased glycosylation demand. Taken together, we uncover a novel role for hepatic Fxr-Shp axis in maintaining the glycosylation status of proteins in the liver.

Zinc Uptake and Storage During the Formation of the Cerebral Cortex in Mice.

The cerebral cortex (or neocortex) is a brain structure formed during embryogenesis. The present study seeks to provide a detailed characterization of the Zn homeostatic mechanisms during cerebral cortex formation and development. To reach that goal, we have combined high-throughput RNA-sequencing analysis of the whole murine genome, X-ray fluorescence nanoimaging (XRF), inductively coupled plasma-atomic emission spectrometry (ICP-AES), and live-cell imaging of dissociated cortical neurons loaded with the Zn fluorescent probe FluoZin-3. The transcriptomic analysis was conducted from mRNAs isolated from cortices collected at embryonic (E) days 11 (E11), E13, and E17 and on postnatal day 1 (PN1) pups. This permitted to characterize the temporal pattern of expression of the main genes participating in the cellular transport, storage, and release of Zn during corticogenesis. It appears that cells of the immature cortex express a wide diversity of actors involved in Zn homeostasis with Zip7, SOD1, and metallothioneins being the most abundant transcripts throughout corticogenesis. The quantification of total Zn with XRF and ICP-AES reveals a reduction of Zn levels. Moreover, this is accompanied by a diminution of the size of the internal pools of mobilizable Zn. This study illustrates the tight temporal and spatial regulation of Zn homeostasis during cerebral brain development.

Multiple microRNAs Regulate the Floral Development and Sex Differentiation in the Dioecious Cucurbit Coccinia grandis (L.) Voigt

Despite having a small genome and heteromorphic sex chromosomes, the molecular basis of sex differentiation in the dioecious cucurbit, Coccinia grandis, largely remains unclear. Increasing evidences indicate a vital regulatory role of microRNAs in the reproductive development of plants. In this study, we used a combination of high-throughput small RNA sequencing and computational analysis to identify sex-specific miRNAomes from male and female buds of C. grandis. Ninety-eight conserved miRNAs from 22 families and 44 novel miRNAs specific to C. grandis were detected. Comparative profiling together with Northern blot and qRT-PCR analysis revealed 41 significantly differentially expressed (DE) miRNAs, of which 16 could be fundamental to the regulation of sexual dimorphism. One hundred six target genes were predicted for 35 DE miRNAs that were significantly involved in flower organogenesis, phytohormone signaling, metabolism, transcription regulation, and DNA methylation. Temporal analysis of a set of 16 target genes at three stages of the bud development revealed a reciprocal alteration in their expression pattern with the complementary miRNAs. Further, the miR167a-1, miR393a, miR398b, and miRn9 mediated down-regulation of four predicted targets associated with reproductive organ development in C. grandis was confirmed through transient co-expression in Nicotiana benthaminana. Taken together, the present study represents the first report suggesting that multiple miRNA-mediated gene silencing cascade could be involved in regulating the molecular basis of sex differentiation in C. grandis.

Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.

Identification of a Novel Semi-Dominant Spotted-Leaf Mutant with Enhanced Resistance to Xanthomonas oryzae pv. oryzae in Rice.

Many spotted-leaf mutants show enhanced disease resistance to multiple pathogen attacks; however, the mechanisms are largely unknown. Here, we reported a novel semi-dominant spotted-leaf mutant 24 (spl24) obtained from an ethyl methane sulfonate (EMS)-induced IR64 mutant bank. spl24 developed tiny brown lesions on the leaf tip and spread down gradually to the leaf base as well as the sheath at the early heading stage. The performances of major agronomic traits such as the plant height, panicle length, number of panicles/plant, and 1000-grain weight were significantly altered in spl24 when compared to the wild-type IR64. Furthermore, spl24 exhibited a premature senescing phenotype with degeneration of nuclear acids, significantly reduced soluble protein content, increased level of malonaldehyde (MDA), and lowered activities of reactive oxygen species (ROS) scavenging enzymes. Disease evaluation indicated that spl24 showed enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial leaf blight in rice, with elevated expression of pathogenesis-related genes, salicylic acid (SA) signaling pathway-associated genes revealed by real-time quantitative PCR and high-throughput RNA sequencing analysis. Genetic analysis and gene mapping indicated that the lesion mimic phenotype was controlled by a novel semi-dominant nuclear gene. The mutation, tentatively termed as OsSPL24, was in a 110 kb region flanked by markers Indel-33 and Indel-12 in chromosome 11. Together, our data suggest that spl24 is a novel lesion mimic mutant with enhanced innate immunity and would facilitate the isolation and functional characterization of the target gene.

Functional inactivation of OsGCNT induces enhanced disease resistance to Xanthomonas oryzae pv. oryzae in rice

BackgroundSpotted-leaf mutants are important to reveal programmed cell death and defense-related pathways in rice. We previously characterized the phenotype performance of a rice spotted-leaf mutant spl21 and narrowed down the causal gene locus spl21(t) to an 87-kb region in chromosome 12 by map-based cloning.ResultWe showed that a single base substitution from A to G at position 836 in the coding sequence of Oryza sativa beta-1,6-N-acetylglucosaminyl transferase (OsGCNT), effectively mutating Tyr to Cys at position 279 in the translated protein sequence, was responsible for the spotted-leaf phenotype as it could be rescued by functional complementation. Compared to the wild type IR64, the spotted-leaf mutant spl21 exhibited loss of chlorophyll, breakdown of chloroplasts, down-regulation of photosynthesis-related genes, and up-regulation of senescence associated genes, which indicated that OsGCNT regulates premature leaf senescence. Moreover, the enhanced resistance to the bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae, up-regulation of pathogenesis-related genes and increased level of jasmonate which suggested that OsGCNT is a negative regulator of defense response in rice. OsGCNT was expressed constitutively in the leaves, sheaths, stems, roots, and panicles, and OsGCNT-GFP was localized to the Golgi apparatus. High throughput RNA sequencing analysis provided further evidence for the biological effects of loss of OsGCNT function on cell death, premature leaf senescence and enhanced disease resistance in rice. Thus, we demonstrated that the novel OsGCNT regulated rice innate immunity and immunity-associated leaf senescence probably by changing the jasmonate metabolic pathway.ConclusionsThese results reveal that a novel gene Oryza sativa beta-1,6-N-acetylglucosaminyl transferase (OsGCNT) is responsible for the spotted-leaf mutant spl21, and OsGCNT acts as a negative-regulator mediating defense response and immunity-associated premature leaf senescence probably by activating jasmonate signaling pathway.

Identification of miRNAs and their targets in regulating tuberous root development in radish using small RNA and degradome analyses.

High-throughput small RNA sequencing and degradome analysis were used in this study to thoroughly investigate the role of miRNA-mediated regulatory network in tuberous root development of radish. Samples from the early seedling stage (RE) and the cortex splitting stage (RL) were used for the construction of six small RNA libraries and one degradome library. A total of 518 known and 976 novel miRNAs were identified, of which, 338 known and 18 novel miRNAs were expressed in all six libraries, respectively. A total of 52 known and 57 novel miRNAs were identified to be significantly differentially expressed between RE and RL, and 195 mRNAs were verified to be the targets of 194 miRNAs by degradome sequencing. According to the degradome analysis, 11 differentially expressed miRNAs had miRNA-mRNA targets, and 13 targets were identified for these 11 miRNAs. Of the 13 miRNA-mRNA targets, 4 genes (RSG11079.t1, RSG11844.t1, RSG16775.t1, and RSG42419.t1) were involved in hormone-mediated signaling pathway, 2 gens (RSG11079.t1 and RSG16775.t1) were related to post-embryonic root development, and 1 gene (RSG23799.t1) was involved in anatomical structure morphogenesis, according to the GO function analysis for biological process. Target Genes participated in these processes are important candidates for further studies. This study provides valuable information for a better understanding of the molecular mechanisms involved in radish tuberous root formation and development.

Transcriptome Analysis Provides Insight into the Molecular Mechanisms Underlying gametophyte factor 2-Mediated Cross-Incompatibility in Maize

In maize (Zea mays L.), unilateral cross-incompatibility (UCI) is controlled by Gametophyte factors (Ga), including Ga1, Ga2, and Tcb1; however, the molecular mechanisms underpinning this process remain unexplored. Here, we report the pollination phenotype of an inbred line, 511L, which carries a near-dominant Ga2-S allele. We performed a high-throughput RNA sequencing (RNA-Seq) analysis of the compatible and incompatible crosses between 511L and B73, to identify the transcriptomic differences associated with Ga2-mediated UCI. An in vivo kinetics analysis revealed that the growth of non-self pollen tubes was blocked at the early stages after pollination in 511L, maintaining the UCI barrier in Ga2. In total, 25,759 genes were expressed, of which, 2063 differentially expressed genes (DEGs) were induced by pollination (G_GG, G_GB, B_BB, B_BG). A gene ontology (GO) enrichment analysis revealed that these genes were specifically enriched in functions involved in cell wall strength and pectic product modification. Moreover, 1839, 4382, and 5041 genes were detected to differentially express under same pollination treatments, including B_G, BG_GG, and BB_GB, respectively. A total of 1467 DEGs were constitutively expressed between the two inbred lines following pollination treatments, which were enriched in metabolic processes, flavonoid biosynthesis, cysteine biosynthesis, and vacuole functions. Furthermore, we confirmed 14 DEGs related to cell wall modification and stress by qRT-PCR, which might be involved in Ga2-S-mediated UCI. Our results provide a comprehensive foundation for the molecular mechanisms involved in silks of UCI mediated by Ga2-S.

Abstract PR06: Contribution of mutant microenvironment to hereditary cancer: Single-cell gene expression profiling of a genetically engineered mouse model of human hereditary BRCA1-related breast cancer

Abstract Carriers of germline mutations in tumor suppressor genes such as BRCA1 suffer from highly increased cancer risk with few effective measures for prevention and treatment. Development of better medical management options requires a more comprehensive understanding of the underlying mechanism of these hereditary cancers. Recent studies suggest that this cancer predisposition, most specifically in the breast and ovary, cannot be sufficiently explained by the DNA-damage repair defect alone in BRCA1-mutant cells, suggesting that additional factors are at play. Further bolstering this argument is the recent finding that BRCA1-heterozygous mutant cells show striking haplo-insufficient phenotypes. Based on these observations as well as preliminary studies in our laboratory, we propose that the heterozygous BRCA1-mutant mammary tissue microenvironment in germline BRCA1-mutation carriers may significantly contribute to breast cancer development by creating a pro-tumorigenic niche. To functionally interrogate this question, we have recently developed a genetically engineered mouse (GEM) model to mimic breast tumor predisposition in human carriers of germline BRCA1 mutation. This model combines Cre/Lox and CRISPR/Cas9 technologies to allow temporal-spatial conditional inactivation of Brca1 as well as other frequently co-mutated genes (e.g., Trp53 and Pten) to initiate breast tumors. Importantly, the surrounding cells in the tissue microenvironment uniformly harbor constitutive heterozygous Brca1 mutation, thereby recapitulating the tissue microenvironment in the human hereditary disease. Use of this mouse model allows us to systematically investigate the aberrations that occur in the heterozygous Brca1-mutant mammary tissue microenvironment, while avoiding confounding variations in age, parity, menstrual cycle, and obesity, which are nearly impossible to eliminate in human samples. Using this GEM model, we have performed high-throughput single-cell RNA-sequencing analysis to identify protumorigenic signaling molecules that are aberrantly upregulated in Brca1-heterozygous cells in the mammary microenvironment. Furthermore, functional in vitro experiments using human cells have demonstrated that Brca1-heterozygous cells create a proliferation-favoring environment compared to wild type, suggesting a role in promoting tumor development. Ongoing in vivo studies using our GEM model as well as in vitro experiments using its derivative organoids will further shed light on the detailed mechanisms that mediate this microenvironmental effect caused by heterozygous Brca1 mutation. Overall, this work may provide significant insights into our fundamental understanding of Brca1-hereditary breast cancer and thereby inform more effective strategies for its prevention, prognosis, and treatment. Furthermore, insights from this work may also be relevant to sporadic cancers with microenvironments altered due to physiologic stress. Thus, our findings may have important implications for both hereditary and sporadic cancers. This abstract is also being presented as Poster B16. Citation Format: Carman M. Li, Yaara Oren, Aviv Regev, Joan S. Brugge. Contribution of mutant microenvironment to hereditary cancer: Single-cell gene expression profiling of a genetically engineered mouse model of human hereditary BRCA1-related breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR06.