Comparative RNA-seq Research Articles

Comparative RNA-Seq Analysis of High- and Low-Oil Yellow Horn During Embryonic Development

Yellow horn (Xanthoceras sorbifolium Bunge) is an endemic oil-rich shrub that has been widely cultivated in northern China for bioactive oil production. However, little is known regarding the molecular mechanisms that contribute to oil content in yellow horn. Herein, we measured the oil contents of high- and low-oil yellow horn embryo tissues at four developmental stages and investigated the global gene expression profiles through RNA-seq. The results found that at 40, 54, 68, and 81 days after anthesis, a total of 762, 664, 599, and 124 genes, respectively, were significantly differentially expressed between the high- and low-oil lines. Gene ontology (GO) enrichment analysis revealed some critical GO terms related to oil accumulation, including acyl-[acyl-carrier-protein] desaturase activity, pyruvate kinase activity, acetyl-CoA carboxylase activity, and seed oil body biogenesis. The identified differentially expressed genes also included several transcription factors, such as, AP2-EREBP family members, B3 domain proteins and C2C2-Dof proteins. Several genes involved in fatty acid (FA) biosynthesis, glycolysis/gluconeogenesis, and pyruvate metabolism were also up-regulated in the high-oil line at different developmental stages. Our findings indicate that the higher oil accumulation in high-oil yellow horn could be mostly driven by increased FA biosynthesis and carbon supply, i.e. a source effect.

Genome-wide identification of flowering time genes associated with vernalization and the regulatory flowering networks in Chinese cabbage

Flowering time (Ft) is the most important characteristic of Chinese cabbage with high leaf yields and late-flowering are favorable traits, while little knowledge on genes involved in Ft and the flowering mechanism in this crop. In this study, we conducted genome-wide RNA-seq analysis using an inbred Chinese cabbage ‘4004’ line in response to vernalization and compared the Ft gene expression with radish crop. A number of Ft genes which play roles in flowering pathways were performed quantitative RT-PCR analysis to verify the regulatory flowering gene network in Chinese cabbage. We found that a total of 223 Ft genes in Chinese cabbage, and 50 of these genes responded to vernalization. The majority of flowering enhancers were upregulated, whereas most flowering repressors were downregulated in response to vernalization as confirmed by RT-qPCR. Among the major Ft genes, the expression of BrCOL1-2, BrFT1/2, BrSOC1/2/3, BrFLC1/2/3/5, and BrMAF was strongly affected by vernalization. In reference to comparative RNA-seq profiling of Ft genes, Chinese cabbage and radish revealed substantially different vernalization response in particular GA flowering pathway. Thus, this study provides new insight into functional divergence in flowering pathways and the regulatory mechanisms in Brassicaceae crops. Further analysis of the major integrator genes between early and late-flowering inbred lines facilitates understanding flowering trait variation and molecular basis of flowering in Chinese cabbage.

A comparative analysis of small RNAs between two Upland cotton backcross inbred lines with different fiber length: Expression and distribution

The cotton fiber is the most important raw material for the textile industry and an ideal model system for studying cell elongation. However, the genetic variation of fiber elongation in relation to miRNA is poorly understood. A high-throughput comparative RNA-seq of two lines differing in fiber length (FL) from a backcross inbred line (BIL) population of G. hirsutum × G. barbadense revealed differentially expressed (DE) miRNAs and their targets in rapidly elongating fibers. A real-time quantitative PCR analysis was further performed to validate the results. A total of 463 (including 47 DE) miRNAs were identified, and seven DE miRNAs were co-localized with seven FL quantitative trait loci (QTL) identified in the G. hirsutum × G. barbadense population. Of 82 (including 21 DE) targets identified, nine (including one DE) were also co-localized with the seven FL QTL. The relationship between the allopolyploid and its diploid ancestral species with respect to miRNAs and their targets was also characterized. These results will facilitate the understanding of the molecular genetic mechanism of fiber elongation with regards to miRNAs in cotton.

Comparative RNA-seq analysis of citrus fruit in response to infection with three major postharvest fungi

Green mold, blue mold and sour rot caused by Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii infections, respectively, are major fungal diseases of citrus fruit. The above-mentioned diseases present a great threat to fruit quality, it is very important to clarify the pathogenicity mechanisms of the pathogens and the resistance mechanism of the host, but the mechanisms remain largely unknown. In this study, RNA sequencing was employed to analyze the gene expression of citrus fruit to P. digitatum, P. italicum and G. citri-aurantii on a genome-wide scale. The results suggest that a total of 4,520, 4785 and 4683 genes of citrus fruit were differentially expressed (P-value < 0.05, ︱Log2 fold change︱≥1) in response to P. digitatum, P. italicum and G. citri-aurantii, respectively. These differentially expressed genes are involved in a variety of stress responses, such as host innate immunity, synthesis of secondary metabolites and hormone-related pathways, etc. Remarkably, the host reactions under different pathogen infections are similar, and the establishment of citrus fruit systemic resistance in response to P. digitatum, P. italicum and G. citri-aurantii is salicylic acid-reliant. Overall, our study presents a genome-wide analysis of the similarities and differences in citrus fruit responses to common fungal pathogens, which is not only beneficial for a better understanding of host-pathogen interactions but also for the development of novel measures for the prevention of common fungal diseases.

Comparative RNA-Seq analysis on the regulation of cucumber sex differentiation under different ratios of blue and red light

Cucumber (Cucumis sativus L.) is a typical monoecism vegetable with individual male and female flowers, which has been used as a plant model for sex determination. It is well known that light is one of the most important environmental stimuli, which control the timing of the transition from vegetative growth to reproductive development. However, whether light controls sex determination remains elusive. To unravel this problem, we performed high-throughput RNA-Seq analyses, which compared the transcriptomes of shoot apices between R2B1(Red light:Blue light = 2:1)-treated and R4B1(Red light:Blue light = 4:1)-treated cucumber seedlings. Results showed that the higher proportion of blue light in the R2B1 treatment significantly induced the formation of female flowers and accelerated female flowering time in this whole study. The genes related to flowering time, such as flowering locus T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), were up-regulated after R2B1 treatment. Furthermore, the transcriptome analysis showed that up-regulation and down-regulation of specific DEGs (the differentially expressed genes) were primarily the result of plant hormone signal transduction after treatments. The specific DEGs related with auxin formed the highest percentage of DEGs in the plant hormone signal transduction. In addition, the expression levels of transcription factors also changed after R2B1 treatment. Thus, sex differentiation affected by light quality might be induced by plant hormone signal transduction and transcription factors. These results provide a theoretical basis for further investigation of the regulatory mechanism of female flower formation under different light qualities in cucumber seedlings.

Comparative RNA-Seq analysis reveals a critical role for brassinosteroids in rose (Rosa hybrida) petal defense against Botrytis cinerea infection

BackgroundOne of the most popular ornamental plants worldwide, roses (Rosa sp.), are very susceptible to Botrytis gray mold disease. The necrotrophic infection of rose petals by B. cinerea causes the collapse and death of these tissues in both the growth and post-harvest stages, resulting in serious economic losses. To understand the molecular basis of rose resistance against B. cinerea, we profiled the petal transcriptome using RNA-Seq technology.ResultsWe identified differentially transcribed genes (DTGs) in petals during B. cinerea infection at 30 h post inoculation (hpi) and/or 48 hpi. Gene ontology term enrichment and pathway analyses revealed that metabolic, secondary metabolite biosynthesis, plant-pathogen interaction, and plant hormone signal transduction pathways were involved. The expression of 370 cell-surface immune receptors was upregulated during infection. In addition, 188 genes encoding transcription factors were upregulated, particularly in the ERF, WRKY, bHLH, MYB, and NAC families, implying their involvement in resistance against B. cinerea. We further identified 325 upregulated DTGs in the hormone signal transduction pathways. Among them, the brassinosteroid (BR)-related genes were the most significantly enriched. To confirm the role of BR in Botrytis resistance, exogenous BR was applied to rose flowers before the inoculation of B. cinerea, which enhanced the defense response in these petals.ConclusionsOur global transcriptome profiling provides insights into the complex gene regulatory networks mediating the rose petal response to B. cinerea. We further demonstrated the role of the phytohormone BR in the resistance of petals to necrotrophic fungal pathogens.

Abstract B028: Comparative assessment of cellular functions for the ZBTB methyl-CpG binding proteins in prostate cancer

Abstract It is becoming increasingly evident that DNA methylation patterns are misappropriated in a number of cancers. This leads to downstream alterations in transcription that afford a survival advantage for the disease state cells. In many cases, DNA methylation directs gene expression through the recruitment of methyl-CpG binding proteins (MBPs). These specialized factors read the methylated DNA signal and translate this into recruitment of chromatin remodeling complexes. The ZBTB family of MBPs, consisting of ZBTB33, ZBTB4, and ZBTB38, are unique among MBPs in that they utilize a single protein domain to specifically target both methylated and sequence-specific nonmethylated DNA sites. This bimodality in DNA recognition affords these proteins the ability to effect either transcriptional repression or activation depending on the cellular context. Further, there is mounting evidence that overexpression of ZBTB MBPs and their subsequent transcriptional activities are directly associated with cancer, though the mechanism by which each protein functions to maintain and/or progress the disease state remains to be elucidated. Of particular note, ZBTB33 overexpression has been correlated with tumorigenicity in prostate cancer (PCa). In these studies we sought to begin more thoroughly characterizing the role of ZBTB33 in PCa, and simultaneously evaluating whether ZBTB38 and ZBTB4 may also participate in this disease. Utilizing both cell line models and patient PCa tumors, we demonstrate that protein levels for all three ZBTB MBPs increase in the cancerous state relative to normal cells/tissues. Notably, the expression levels of ZBTB4 appear to peak at earlier stage disease and decrease at later. To begin delineating the biologic pathways mediated by each ZBTB MBP, we evaluated global transcriptome (RNA-seq) and phenotypic alterations after systematic ZBTB MBP depletion in two phenotypically distinct PCa cell line models. Specifically, we evaluated the impact of each ZBTB MBP on cell proliferation, apoptosis, and migration. In short, we determined that cellular phenotypic response to depletion of the three ZBTB MBPs appears highly variable between proteins as well as cell line model. Further, comparative RNA-seq after depletion of each ZBTB MBP demonstrates that consistent with the phenotypic analyses, each ZBTB MBP appears to mediate protein-specific transcriptional responses. These findings suggest that these proteins may function in parallel within PCa. Combined, these studies represent the first comprehensive analysis of all three ZBTB MBPs in the same disease context and emphasize the potential for these proteins as novel therapeutic targets. Citation Format: Amir Pozner, Aneesa T. Al-Soodani, Sheryl Tripp, Justin Caron, Daniel J. Albertson, Bethany A. Buck-Koehntop. Comparative assessment of cellular functions for the ZBTB methyl-CpG binding proteins in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B028.

Identification of WRKY Gene Family from Dimocarpus longan and Its Expression Analysis during Flower Induction and Abiotic Stress Responses

Longan is an important fruit tree in the subtropical region of Southeast Asia and Australia. However, its blooming and its yield are susceptible to stresses such as droughts, high salinity, and high and low temperature. To date, the molecular mechanisms of abiotic stress tolerance and flower induction in longan have not been elucidated. WRKY transcription factors (TFs), which have been studied in various plant species, play important regulatory roles in plant growth, development, and responses to stresses. However, there is no report about WRKYs in longan. In this study, we identified 55 WRKY genes with the conserved WRKY domain and zinc finger motif in the longan genome. Based on the structural features of WRKY proteins and topology of the phylogenetic tree, the longan WRKY (DlWRKY) family was classified into three major groups (I–III) and five subgroups (IIa–IIe) in group II. Tissue expression analysis showed that 25 DlWRKYs were highly expressed in almost all organs, suggesting that these genes may be important for plant growth and organ development in longan. Comparative RNA-seq and qRT-PCR-based gene expression analysis revealed that 18 DlWRKY genes showed a specific expression during three stages of flower induction in “Sijimi” (“SJ”), which exhibited the “perpetual flowering” (PF) habit, indicating that these 18 DlWRKY genes may be involved in the flower induction and the genetic control of the perpetual flowering trait in longan. Furthermore, the RT-qPCR analysis illustrated the significant variation of 27, 18, 15, 17, 27, and 23 DlWRKY genes under SA (Salicylic acid), MeJA (Methyl Jasmonate), heat, cold, drought, or high salinity treatment, respectively, implicating that they might be stress- or hormone-responsive genes. In summary, we systematically and comprehensively analyzed the structure, evolution, and expression pattern of the DlWRKY genes. The results presented here increase our understanding of the WRKY family in fruit trees and provide a basis for the further elucidation of the biological function of DlWRKY genes in longan.

Abstract 4751: APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer

Abstract Introduction: Apolipoprotein B mRNA Editing enzyme, Catalytic polypeptide 3A (A3A) is a C to U cytidine deaminase, preferentially deaminating the lagging strand of double-stranded DNA at specific sequence motifs. A3A is overexpressed across multiple tumor types, and in pancreatic ductal adenocarcinoma (PDA), high expression is associated with significantly decreased survival in early stage patients. Large scale sequencing studies of various human tumors (TCGA and ICGC), including PDA, have implicated A3A as a potential driver of tumor mutagenesis. However, the biological relevance of the deamination function of A3A has not yet been shown. Here, we utilize a novel genetically engineered mouse model to show that A3A supports the development of aggressive PDA independent of mutagenic capabilities. Methods: As opposed to humans, mice only contain one APOBEC3 isoform which has limited to no deaminating activity on genomic DNA. Thus, to dissect the function of A3A on PDA initiation and progression, we made mice with a germline knockin for the coding sequence for human A3A and bred to a well-established GEMM of PDA to yield LSL-KrasG12D; p53fl/+; Pdx1-Cre; RosaLSL-YFP; A3A+/- (KPCY;A3A) mice and compared to KPCY mice. We genotyped tumors from both cohorts using VarScan after exome capture sequencing. Tumor and immune cells were FACS sorted comparative RNAseq performed. The immune contexture of murine and 155 untreated and treated human PDAs was analyzed by performing 7-color multiplex staining and immune cell spatial interaction analysis. Results: All KPCY;A3A (n=11) mice developed tumors and expired significantly faster compared to KPCY (n=18) controls (3.9 v. 7.0 mo; p&amp;lt;0.01). One third of the KPCY; A3A mice developed macrometastatic disease compared to 3/12 in the KPCY control. Whole exome sequencing of tumors revealed no significant difference in the number of point mutations or neoantigen load between KPCY;A3A and KPCY tumors. However, histologically, KPCY;A3A tumors contained significantly more desmoplasia and altered immune cell infiltrates. Specifically, comparative RNAseq and IHC analyses revealed dramatic differences in the number and distribution of various T-cell and B-cell subsets in KPCY;A3A mice. Moreover, A3A expression led to abrogation of CD8+ T cell activation and proliferation. Concomitantly, a panel of immune checkpoint-related genes were upregulated in A3A high expressing tumors and cell lines compared to non-A3A expressing controls. This was confirmed in a panel of resected human PDA. Conclusions: Counter to our hypothesis, A3A supports the initiation and growth of PDA in vivo through effects independent of deamination or mutagenesis of cancer cell genomes but rather indirectly through suppression of the tumor immune response. Future studies will address the precise mechanisms underlying A3A-mediated tumor immune evasion. Citation Format: Sonja Woermann, Robert Cowan, Susan M. Ross, Andrew D. Rhim. APOBEC3A inhibits tumor immune response independent of deamination in a novel genetic model of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4751.

Abstract 52: A genomic editing approach for purification of viable quiescent cancer cells

Abstract Recurrent and metastatic cancer lesions often undergo a period of dormancy, which is closely associated with cancer cell quiescence, a state whereby cancer cells exit the cell cycle and are reversibly arrested in G0 phase. Quiescent cancer cells are inherently resistant to cell death and refractory to therapeutic drugs. However, the mechanisms responsible for the resistance remain largely undefined. This is closely associated with the lack of understanding of their biological properties as a consequence of technical hurdles in the isolation and analysis of viable quiescent cells. Nevertheless, it is known that quiescent cells are characteristically negative for the proliferation marker Ki67 and express high levels of the cyclin-dependent kinase (CDK) inhibitor p27. In this study, we developed a CRISPR/Cas9-based system to fuse a green fluorescent protein (EGFP) gene with endogenous CDKN1B, the gene encoding p27, and a red fluorescent protein (mCherry) gene with endogenous MKI67, the gene encoding Ki67 in the genome of human melanoma cells. By using this system, we have successfully isolated viable p27high/Ki67- melanoma cells using fluorescence-activated cell sorting (FACS). The quiescent state of these cells was confirmed by dual nucleic acid staining (DNA with Hoechst-33342, and RNA with Pyronin Y). This approach provides a unique research tool for further characterization of the biological properties of quiescent cells. Comparative RNA-seq and proteomics analyses are being currently carried out to reveal molecular networks involved in regulating cell quiescence, and to identify molecular targets for overcoming resistance of quiescent melanoma cells to treatment. Citation Format: Ting La, Margaret Farrelly, Xu Guang Yan, Hamed Yari, Yuanyuan Zhang, Yuchen Feng, Seyed Hessamedin Tabatabaeehatambakhsh, Tao Liu, Lei Jin, Xu Dong Zhang. A genomic editing approach for purification of viable quiescent cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 52.

Abstract 2972: Co-clinical trial of olaparib and temozolomide in SCLC PDX models uncovers new biomarkers of sensitivity

Abstract Introduction: Small cell lung cancer (SCLC) is a common and rapidly fatal malignancy for which no biomarker-targeted therapies have been developed. Despite a critical need, progress suffers from (1) scarcity of cutting-edge laboratory models and (2) absence of promising targets. Patient-derived xenografts (PDX) may faithfully model the clinical disease, but because SCLC is rarely biopsied or resected, specimens for PDX generation are scarce. PARP inhibition has recently emerged as a compelling strategy for SCLC, and an ongoing phase 1/2 trial of combination olaparib tablets and temozolomide (O/T) has shown promising activity in patients. However, biomarkers for patient selection remain elusive. Methods: We generated SCLC PDX models from circulating tumor cells (CTCs), biopsies and malignant effusions. CTCs were enriched with an automated microfluidic device, the CTC-iChip. To assess the genomic fidelity of the models, we performed comparative whole exome sequencing (WES) and RNA-seq on 6 sets of corresponding patient biopsies, founder (P0) PDX tumors, and early-passage PDXs. We then assessed the activity of combination O/T in a panel of PDX models, and compared PDX responses with molecular profiles to identify candidate biomarkers. Results: 44 PDXs were generated from 32 patients, including 6 sets of serial models and 4 synchronous CTC- and biopsy-derived models. PDXs were derived with high efficiency from both CTCs (35% per blood draw) and biopsies/effusions (82% per implant). WES demonstrated that somatic alterations in tumor biopsies were stably maintained in both CTC and biopsy-derived models, without significant accumulation of new mutations, and transcriptional profiles remained consistent through early passages. Six models were derived from O/T trial patients, including two sets of serial models before and after durable responses. The serial models faithfully recapitulated patient responses to O/T: pre-trial models were highly sensitive and post-relapse were highly resistant. The co-clinical trial was expanded to 30 models, using the models derived from trial patients to delineate the margins of clinical sensitivity (6 models), intermediate sensitivity (6 models) and resistance (18 models). Among the molecular features evaluated, basal protein PARylation best distinguished the O/T-sensitive category from both intermediate (p &amp;lt; 0.001) and resistant models (p &amp;lt; 0.0001). In addition, PARylation decreased after relapse in serial models from O/T trial patients. Conclusions: Both biopsy- and CTC-derived SCLC PDX models faithfully recapitulate the genomic and functional features of the donor patient tumor. O/T sensitivity in this panel correlated with basal PARylation. The value of the co-clinical trial is the potential to refine the clinical application of O/T in real time, to optimize follow-on clinical trials and to develop biomarker-directed therapy for SCLC. Citation Format: Benjamin J. Drapkin, Julie George, Marcello Stanzione, Beow Y. Yeap, Mari Mino-Kenudson, Camilla L. Christensen, Ruben Dries, Sarah Phat, Jun Zhong, David T. Myers, Joseph A. Licausi, Tilak Sundaresan, Marina Kem, Nima Abedpour, Leica V. Sequist, Alice T. Shaw, Aaron N. Hata, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber, Martin Peifer, Roman K. Thomas, Anna F. Farago, Nicholas J. Dyson. Co-clinical trial of olaparib and temozolomide in SCLC PDX models uncovers new biomarkers of sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2972.

Abstract 2015: Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration

Abstract Lung cancer is a highly metastatic disease. Contrary to the common belief that metastasis is a late event, recent findings have suggested that micrometastasis may also occur as an early phenomenon. An increasing body of evidence supports a role for epithelial-mesenchymal transition (EMT) in inducing field cancerization. Reports suggest that cancer-derived exosomes can act via intra-cellular communication to induce EMT and migration. However, the role of premalignant cell-derived exosomes in EMT induction, cellular migration and establishment of field cancerization in unknown. Herein, we describe the role of premalignant cell-derived exosomes on early metastatic behavior. Our laboratory has recently discovered a unique highly motile (HM) subpopulation of human bronchial epithelial cells (HBECs), using a novel “constricted migration” selection strategy, with enhanced metastatic potential in vivo. The HBECs used for selection were modified with changes reciprocating premalignancy (p53null, activated Kras-G12D). Comparative RNA-seq datasets illustrate increased expression of key EMT genes in HM-HBECs. This unique subpopulation of HM-HBECs offer a unique model to investigate premalignant cell migration. We used exosomes derived from HM, SM (slow motile) and unselected (UN) HBECs to evaluate EMT induction, migration, and invasion in vitro and in vivo. Exosomes were isolated by ultracentrifugation and characterized for size distribution using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy, and characterized by western blotting with antibodies specific for Alix, Flotillin, CD9 and CD63. Exosome labeling for cell uptake studies was performed using Dil dye entrapment and by transient transfection with CD63-GFP, releasing GFP+ exosomes. 6X108 HBEC cells produced approximately 6 x 108 exosomes with size ranging from 50 to 130 nm. The exosome signature of HM-, SM-, and UN-HBECs were characterized by mass spectroscopy and micro RNA sequencing. Exosomes from HM-HBECs induce UN-HBECs to obtain a HM phenotype with enhanced EMT, faster migration and increased invasive capacity. Western blot and qPCR data suggest a time-dependent activation of an EMT signature. Further in vivo experiments are in progress to evaluate the role of HM-HBEC derived exosomes on metastatic homing, tumor formation and spread of premalignant HBEC cells and tumor cells. Our data suggest that premalignant cell-derived exosomes possess distinct molecular signatures and could potentially mediate early migration, resulting in the creation of a premalignant field. As such, exosomes afford novel targets in premalignancy for early intervention and cancer interception. Citation Format: Manash K. Paul, Suman Dutta, Bharti Bisht, Salehi-Rad Ramin, Paul Pagano, Gal Bitan, John D. Minna, Steven M. Dubinett. Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2015.

Abstract 2451: Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27

Abstract Evading apoptosis and limitless replicative potential are two hallmarks of malignant cells. Here we report that melanoma cells surviving the induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) or the Mcl-1 inhibitor UMI-77 have even higher proliferative potential, and that this is associated with the increased expression of the long noncoding RNA (lncRNA) OVAAL which competes with p27 mRNA for binding to polypyrimidine tract binding protein 1 (PTBP1) and thus represses p27 mRNA translation. Prolonged exposure to TRAIL or Mcl-1 resulted in stable melanoma cell populations with increased proliferation activity. Comparative RNA-seq analysis identified OVAAL as the most significantly upregulated lncRNA in melanoma cells resistance to TRAIL and Mcl-1. ShRNA knockdown of OVVAL reversed the increased proliferative potential of resistant cells, and also inhibited proliferation in a subset of parental melanoma cell lines that constitutively expressed relatively high levels of OVAAL. RNA pulldown followed by Mass Spectrometry analysis revealed that among proteins that were physically associated with OVAAL was a protein called PTBP1 that bound to and enhanced the activity of the internal ribosomal entry site (IRES) in the 5' untranslated region (5'-UTR) of p27 mRNA. Indeed, OVAAL knockdown resulted in an increase in the association between PTBP1 and p27 IRES leading to upregulation of the p27 protein, whereas overexpression of OVAAL caused reduction in binding of PTBP1 to the IRES of p27. Furthermore, co-knockdown of PTBP1 abolished upregulation of p27 triggered by OVAAL knockdown. These results suggest that OVAAL competes with p27 mRNA for binding to PTBP1 and thus inhibits p27 expression and promotes cell proliferation. This was substantiated by binding assays using in vitro-synthesized biotin-labelled OVAAL, p27 IRES and purified PTBP1 in a cell-free system. Our results showed that the increased c-Myc activity was responsible for upregulation of OVAAL in melanoma cells selected for resistance to apoptosis induced by TRAIL or UMI-77. Collectively, these results identify the expression of OVAAL as an important mechanism promoting melanoma cell proliferation, with potential implications for targeting OVAAL in combination with apoptosis-inducing therapeutics in the treatment of melanoma. Citation Format: YUNA YUAN ZHANG, Xu Guang Yan, Margaret Farrelly, Hamed Yari, Yuchen Feng, Ting La, Hessam Tabatabaee, Lei Jin, Xu Dong Zhang. Long noncoding RNA OVAAL promotes melanoma cell proliferation through translational suppression of p27 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2451.

Low-phosphate-selected Auxenochlorella protothecoides redirects phosphate to essential pathways while producing more biomass.

Despite the capacity to accumulate ~70% w/w of lipids, commercially produced unicellular green alga A. protothecoides may become compromised due to the high cost of phosphate fertilizers. To address this limitation A. protothecoides was selected for adaptation to conditions of 100× and 5× lower phosphate and peptone, respectively, compared to ‘wild-type media’. The A. protothecoides showed initial signs of adaptation by 45–50 days, and steady state growth at ~100 days. The low phosphate (P)-adapted strain produced up to ~30% greater biomass, while total lipids (~10% w/w) remained about the same, compared to the wild-type strain. Metabolomic analyses indicated that the low P-adapted produced 3.3-fold more saturated palmitic acid (16:0) and 2.2-fold less linolenic acid (18:3), compared to the wild-type strain, resulting in an ~11% increase in caloric value, from 19.5kJ/g for the wild-type strain to 21.6kJ/g for the low P-adapted strain, due to the amounts and composition of certain saturated fatty acids, compared to the wild type strain. Biochemical changes in A. protothecoides adapted to lower phosphate conditions were assessed by comparative RNA-Seq analysis, which yielded 27,279 transcripts. Among them, 2,667 and 15 genes were significantly down- and up-regulated, at >999-fold and >3-fold (adjusted p-value <0.1), respectively. The expression of genes encoding proteins involved in cellular processes such as division, growth, and membrane biosynthesis, showed a trend toward down-regulation. At the genomic level, synonymous SNPs and Indels were observed primarily in coding regions, with the 40S ribosomal subunit gene harboring substantial SNPs. Overall, the adapted strain out-performed the wild-type strain by prioritizing the use of its limited phosphate supply for essential biological processes. The low P-adapted A. protothecoides is expected to be more economical to grow over the wild-type strain, based on overall greater productivity and caloric content, while importantly, also requiring 100-fold less phosphate.

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in Pinctada fucata.

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control.

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function.

Comparative transcriptome RNA-Seq analysis of Listeria monocytogenes with sodium lactate adaptation

Listeria monocytogenes is an important foodborne pathogen that can cause listeriosis with high mortality rates. Sodium lactate has been used as an antimicrobial in meat products to inhibit the growth of foodborne pathogens. However, the adaptation mechanism of L. monocytogenes to sodium lactate remains unclear. In this paper, the growth/survival of L. monocytogenes was determined in ready-to-eat (RTE) meats with 4% sodium lactate treatment. RNA-Seq was conducted to compare the transcriptional profiles between cells cultured in Brain Heart Infusion broth with and without 4% sodium lactate to identify differentially expressed genes (DEGs). DEGs (766) including 329 up-regulated and 437 down-regulated genes were identified from RNA-Seq analysis in response to sodium lactate treatment. RNA-Seq data were validated using quantitative reverse transcription PCR (qRT-PCR) assays. The DEGs include genes involved in pathways including signal transduction, ABC transporter and PTS systems. Interestingly, the DEGs involved in bacterial chemotaxis and flagellar assembly were exclusively down-regulated, indicating that flagellar synthesis was inhibited by sodium lactate treatment. Most importantly, virulence-related genes were induced, suggesting that sodium lactate may increase the virulence potential of L. monocytogenes. Our study provides insight on the adaptation mechanism of L. monocytogenes with treatment of sodium lactate, and may aid in searching for strategies that may have a synergistic effect in combination with sodium lactate to inhibit L. monocytogenes in RTE meat.

A new allele of the Brachytic2 gene in maize can efficiently modify plant architecture.

The applications of semi-dwarf genes such as sd1 and Rht1 in rice and wheat resulted in the first "green revolution" in the 1960s. However, such semi-dwarf genes that can efficiently reduce plant stature and have few negative yield traits have not yet been identified in maize. In this study, a new allele of Brachytic2 gene (qpa1) encoding P-glycoprotein was rapidly fine-mapped using a modified method. The qpa1, containing a 241-bp deletion in the last exon, had no negative effect on yield, but greatly modified the plant architecture including significantly reduced plant height and ear height, increased stalk diameter and erected leaf. A common variant similar to maize qpa1 was also present in the sorghum orthologous dw3 locus. Comparative RNA-seq analysis next showed 99 differentially co-expressed genes affected by Br2 in maize and dw3 in sorghum, including four plant height genes D3, BAK1, Actin7 and Csld1, which are involved in gibberellin and brassinosteroid biosynthesis, auxin transport and cellulose synthesis. The qpa1 can be applied to efficiently modify plant stature in maize and in combination with D3, BAK1, Actin7, Csld1 and the other 95 differentially co-expressed genes, can be edited using new genomic editing tools for further applications and studies.

The vesicle trafficking regulator PN_SCD1 is demethylated and overexpressed in florets of apomictic Paspalum notatum genotypes

Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the development of clonal progenies genetically identical to the mother plant. Here we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize cytosine methylation patterns occurring in florets of sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, later renamed PN_SCD1) was selected due to its relevant annotation and differential representation in apomictic and sexual floral transcriptome libraries. PN_SCD1 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and acts as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are expressed in P. notatum florets and upregulated in apomictic plants. Our results disclosed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis.

Regulation of sporangium formation by the orphan response regulator TcrA in the rare actinomycete Actinoplanes missouriensis.

The rare actinomycete Actinoplanes missouriensis forms terminal sporangia containing a few hundred flagellated spores, which can swim in aquatic environments after release from sporangium. However, gene regulation for its characteristic morphological development is largely unknown. Here, we report the functional analysis of an orphan response regulator, TcrA, which is encoded next to the chemotaxis-flagellar gene cluster. The tcrA null (ΔtcrA) mutant formed sporangium, in which sporulation proceeded. However, many distorted spores were produced and some spores ectopically germinated in the mutant sporangia. In addition, spores were hardly released from the mutant sporangia. A comparative RNA-Seq analysis between the wild-type and ΔtcrA strains showed that TcrA upregulated the transcription of more than 263 genes, which were integrated into 185 transcriptional units. In silico searches identified a 21-bp direct repeat sequence, 5'-nnGCA(A/C)CCG-n4 -GCA(A/C)CCGn-3', as the TcrA box, which was confirmed by electrophoretic mobility shift assays. Finally, we identified 34 transcriptional units as the TcrA regulon. TcrA seems to regulate a few hundred genes through the transcriptional activation of three FliA-family sigma factor genes besides its own regulon. We concluded that TcrA is a global transcriptional activator that controls many aspects of sporangium formation, including flagellar biogenesis, spore dormancy and sporangium dehiscence.