SENSE Total RNA-Seq Publications
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SENSE Total RNA-Seq Publications
Pavel Rossner, Jr., Kristyna Vrbova, Andrea Rossnerova, Tana Zavodna, Alena Milcova, Jiri Klema, Zbynek Vecera, Pavel Mikuska, Pavel Coufalik,Lukas Capka, Kamil Krumal, Bohumil Docekal, Vladimir Holan, Miroslav Machala and Jan Topinka
Abstract
We investigated the transcriptomic response and epigenetic changes in the lungs of mice exposed to inhalation of copper(II) oxide nanoparticles (CuO NPs) (8 × 105 NPs/m3) for periods of 3 days, 2 weeks, 6 weeks, and 3 months. A whole genome transcriptome and miRNA analysis was performed using next generation sequencing. Global DNA methylation was assessed by ELISA. The inhalation resulted in the deregulation of mRNA transcripts: we detected 170, 590, 534, and 1551 differentially expressed transcripts after 3 days, 2 weeks, 6 weeks, and 3 months of inhalation, respectively. Biological processes and pathways affected by inhalation, differed between 3 days exposure (collagen formation) and longer treatments (immune response). Periods of two weeks exposure further induced apoptotic processes, 6 weeks of inhalation affected the cell cycle, and 3 months of treatment impacted the processes related to cell adhesion. The expression of miRNA was not affected by 3 days of inhalation. Prolonged exposure periods modified miRNA levels, although the numbers were relatively low (17, 18, and 38 miRNAs, for periods of 2 weeks, 6 weeks, and 3 months, respectively). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis based on miRNA–mRNA interactions, revealed the deregulation of processes implicated in the immune response and carcinogenesis. Global DNA methylation was not significantly affected in any of the exposure periods. In summary, the inhalation of CuO NPs impacted on both mRNA and miRNA expression. A significant transcriptomic response was already observed after 3 days of exposure. The affected biological processes and pathways indicated the negative impacts on the immune system and potential role in carcinogenesis.
Features Poly(A) RNA Selection Kit and SENSE Total RNA-Seq Library Prep Kit
Stamatia Laidou, Gregorio Alanis-Lobato, Jan Pribyl, Tamás Raskó, Boris Tichy, Kamil Mikulasek, Maria Tsagiopoulou, Jan Oppelt, Georgia Kastrinaki, Maria Lefaki, Manvendra Singh, Annika Zink, Niki Chondrogianni,Fotis Psomopoulos, Alessandro Prigione, Zoltán Ivics, Sarka Pospisilova, Petr Skladal, Zsuzsanna Izsvák, Miguel A. Andrade-Navarro, Spyros Petrakis
Abstract
Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.
Arie Fridrich, Vengamanaidu Modepalli, Magda Lewandowska, Reuven Aharoni, Yehu Moran
Abstract
microRNAs (miRNAs), base-pair to messenger RNA targets and guide Argonaute proteins to mediate their silencing. This target regulation is considered crucial for animal physiology and development. However, this notion is based exclusively on studies in bilaterians, which comprise almost all lab model animals. To fill this glaring phylogenetic gap, we characterized the functions of two Argonaute paralogs in the sea anemone Nematostella vectensis of the phylum Cnidaria, which is separated from bilaterians by ∼600 million years. Using genetic manipulation, Argonaute-immunoprecipitations and high-throughput sequencing we provide experimental evidence for the developmental importance of miRNAs in a non-bilaterian animal. Additionally, we uncover unexpected differential distribution of distinct miRNAs between the two Argonautes and the ability of one of them to load additional types of small RNAs. This enables us to postulate a novel model for evolution of miRNA precursors in sea anemones and their relatives, revealing alternative trajectories for metazoan miRNA evolution.
Johnny Yu, Bruce Culbertson, Hosseinali Asgharian, Albertas Navickas, Lisa Fish, John Paolo Olegario, Benjamin Hänisch, Ethan M Weinberg, Rodrigo Dienstmann, Robert S Warren, Hani Goodarzi
Abstract
Broad dysregulation of gene expression control is a hallmark of cancer progression. Identifying the underlying master regulators that drive pathological gene expression is a key challenge in precision oncology. Here, we have developed a network analytical framework, named PRADA, that identifies oncogenic RNA-binding proteins through the systematic detection of coordinated changes in their target regulons. Application of this approach to data collected from clinical samples, patient-derived xenografts, and cell line models of colon cancer metastasis revealed the RNA-binding protein RBMS1 as a suppressor of colon cancer progression. We observed that silencing RBMS1 results in increased metastatic capacity in xenograft mouse models, and that restoring its expression blunts metastatic liver colonization. We have found that RBMS1 functions as a post-transcriptional regulator of RNA stability by directly binding and stabilizing ~80 target mRNAs. Measurements in more than 180 clinical samples as well as survival analyses in publicly available datasets, have shown that RBMS1 silencing and the subsequent downregulation of its targets are strongly associated with disease progression and poor survival in colon cancer patients. Together, our findings establish a role for RBMS1 as a previously unknown regulator of RNA stability and as a suppressor of colon cancer metastasis with clinical utility for risk stratification of patients.
Significance By applying a new analytical approach to transcriptomic data from clinical samples and models of colon cancer progression, we have uncovered RBMS1 as a suppressor of metastasis and as a post-transcriptional regulator of RNA stability. Notably, RBMS1 silencing and downregulation of its targets are negatively associated with patient survival.
Abstract
The role of viruses in forming a stable holobiont has been a subject of extensive research in the recent years. However, many emerging model organisms still lack any data on the composition of the associated viral communities. Here, we re-analyzed seven publicly available transcriptome datasets of the starlet sea anemone Nematostella vectensis, the most commonly used anthozoan lab model, and searched for viral sequences. We applied a straightforward, yet powerful approach of de novo assembly followed by homology-based virus identification and a multi-step, thorough taxonomic validation. The comparison of different lab populations of N. vectensis revealed the existence of the core virome composed of 21 viral sequences, present in all adult datasets. Unexpectedly, we observed almost complete lack of viruses in the samples from the early developmental stages which together with the identification of the viruses shared with the major source of the food in the lab, the brine shrimp Artemia salina, shed new light on the course of viral species acquisition in N. vectensis. Our study provides an initial, yet comprehensive insight into N. vectensis virome and sets the first foundation for functional studies of viruses and antiviral systems in this lab model cnidarian.
Abstract
Background and aims
Intestinal epithelial cells (IECs) secrete cytokines that recruit immune cells to the mucosa and regulate immune responses that drive inflammation in inflammatory bowel disease (IBD). However, experiments in patient-derived IEC models are still scarce. Here, we aimed to investigate how innate immunity and IEC-specific pattern recognition receptor (PRR) signaling can be involved in an enhanced type I interferon (IFN) gene signature observed in colon epithelium from patients with active IBD, with a special focus on secreted ubiquitin-like protein ISG15.
Method
Gene and protein-expression in whole mucosa biopsies and in microdissected human colonic epithelial lining, in HT29 human intestinal epithelial cells and primary 3D colonoids treated with PRR-ligands and cytokines were detected by transcriptomics, in situ hybridisation, immunohistochemistry, western blots and ELISA. Effects of IECs secreted cytokines were examined in human peripheral blood mononuclear cells (PBMCs) by multiplex chemokine profiling and ELISA.
Results
The type I IFN gene signature in human mucosal biopsies was mimicked in TLR3 and to some extent TNF treated human IECs. In intestinal biopsies, ISG15 expression correlated with expression of the newly identified receptor for extracellular ISG15, LFA-1 integrin. ISG15 was expressed and secreted from HT29-cells and primary 3D colonoids through both JAK1-pSTAT-IRF9 dependent and independent pathways. In experiments using PBMCs we show that ISG15 releases IBD relevant proinflammatory cytokines such as CXCL1, CXCL5, CXCL8, CCL20, IL1, IL6, TNF and IFNγ.
Conclusion
ISG15 is secreted from primary IECs upon extracellular stimulation and mucosal ISG15 emerges as an intriguing candidate for immunotherapy in IBD.
Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit
Sakshi Talwar, Manitosh Pandey, Chandresh Sharma, Rintu Kutum, Josephine Lum, Daniel Carbajo, Renu Goel, Michael Poidinger, Debasis Dash, Amit Singhal, Amit Kumar Pandey
Abstract
A worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly non-replicating and metabolically inert subset of the Mycobacterium tuberculosis (Mtb) population, called ‘persisters’. The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a ribonuclease toxin (VapC12) inhibits translation by targeting proT tRNA in Mtb. This results in cholesterol-specific growth modulation that increases the frequency of the generation of persisters in a heterogeneous Mtb population. Also, a null mutant strain of this toxin (ΔvapC12) failed to persist and demonstrated an enhanced growth phenotype in a guinea pig model of Mtb infection. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin–antitoxin (TA) module in Mtb enhances persister formation during infection. In addition to identifying the mechanism, the study provides opportunity for targeting persisters, a new paradigm facilitating tuberculosis drug development.
Rachel E. Lackie, Abdul R. Razzaq, Sali M. K. Farhan, Lily R. Qiu, Gilli Moshitzky, Flavio H. Beraldo, Marilene H. Lopes, Andrzej Maciejewski, Robert Gros, Jue Fan, Wing‐Yiu Choy, David S. Greenberg, Vilma R. Martins, Martin L. Duennwald, Jason P. Lerch, Hermona Soreq, Vania F. Prado, Marco A. M. Prado
Abstract
Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress‐inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co‐chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in‐depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co‐chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co‐chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age‐dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age‐dependent hippocampal neurodegeneration in mice.
Phil Jun Kang, Daryeon Son, Tae Hee Ko, Wonjun Hong, Wonjin Yun, Jihoon Jang, Jong-Il Choi, Gwonhwa Song, Jangbo Lee, In Yong Kim, and Seungkwon You
Abstract
Human neural stem cells (NSCs) hold enormous promise for neurological disorders, typically requiring their expandable and differentiable properties for regeneration of damaged neural tissues. Despite the therapeutic potential of induced NSCs (iNSCs), a major challenge for clinical feasibility is the presence of integrated transgenes in the host genome, contributing to the risk for undesired genotoxicity and tumorigenesis. Here, we describe the advanced transgene-free generation of iNSCs from human urine-derived cells (HUCs) by combining a cocktail of defined small molecules with self-replicable mRNA delivery. The established iNSCs were completely transgene-free in their cytosol and genome and further resembled human embryonic stem cell-derived NSCs in the morphology, biological characteristics, global gene expression, and potential to differentiate into functional neurons, astrocytes, and oligodendrocytes. Moreover, iNSC colonies were observed within eight days under optimized conditions, and no teratomas formed in vivo, implying the absence of pluripotent cells. This study proposes an approach to generate transplantable iNSCs that can be broadly applied for neurological disorders in a safe, efficient, and patient-specific manner.
Yochai Wolf, Osnat Bartok, Sushant Patkar, Gitit Bar Eli, Sapir Cohen, Kevin Litchfield, Ronen Levy, Alejandro Jiménez-Sánchez, Sophie Trabish, Joo Sang Lee, Hiren Karathia, Eilon Barnea, Chi-Ping Day, Einat Cinnamon, Ilan Stein, Adam Solomon, Lital Bitton, Eva Pérez-Guijarro, Tania Dubovik, Shai S. Shen-Orr, Martin L. Miller, Glenn Merlino, Yishai Levin, Eli Pikarsky, Lea Eisenbach, Arie Admon, Charles Swanton, Eytan Ruppin, Yardena Samuels
Abstract
Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.
Roberta De Mori, Mariasavina Severino, Maria Margherita Mancardi, Danila Anello, Silvia Tardivo, Tommaso Biagini, Valeria Capra, Antonella Casella, Cristina Cereda, Brett R Copeland, Stella Gagliardi, Alessandra Gamucci, Monia Ginevrino, Barbara Illi, Elisa Lorefice, Damir Musaev, Valentina Stanley, Alessia Micalizzi, Joseph G Gleeson, Tommaso Mazza, Andrea Rossi, Enza Maria Valente
Abstract
Basal ganglia are subcortical grey nuclei that play essential roles in controlling voluntary movements, cognition and emotion. While basal ganglia dysfunction is observed in many neurodegenerative or metabolic disorders, congenital malformations are rare. In particular, dysplastic basal ganglia are part of the malformative spectrum of tubulinopathies and X-linked lissencephaly with abnormal genitalia, but neurodevelopmental syndromes characterized by basal ganglia agenesis are not known to date. We ascertained two unrelated children (both female) presenting with spastic tetraparesis, severe generalized dystonia and intellectual impairment, sharing a unique brain malformation characterized by agenesis of putamina and globi pallidi, dysgenesis of the caudate nuclei, olfactory bulbs hypoplasia, and anomaly of the diencephalic-mesencephalic junction with abnormal corticospinal tract course. Whole-exome sequencing identified two novel homozygous variants, c.26C>A; p.(S9*) and c.752A>G; p.(Q251R) in the GSX2 gene, a member of the family of homeobox transcription factors, which are key regulators of embryonic development. GSX2 is highly expressed in neural progenitors of the lateral and median ganglionic eminences, two protrusions of the ventral telencephalon from which the basal ganglia and olfactory tubercles originate, where it promotes neurogenesis while negatively regulating oligodendrogenesis. The truncating variant resulted in complete loss of protein expression, while the missense variant affected a highly conserved residue of the homeobox domain, was consistently predicted as pathogenic by bioinformatic tools, resulted in reduced protein expression and caused impaired structural stability of the homeobox domain and weaker interaction with DNA according to molecular dynamic simulations. Moreover, the nuclear localization of the mutant protein in transfected cells was significantly reduced compared to the wild-type protein. Expression studies on both patients’ fibroblasts demonstrated reduced expression of GSX2 itself, likely due to altered transcriptional self-regulation, as well as significant expression changes of related genes such as ASCL1 and PAX6. Whole transcriptome analysis revealed a global deregulation in genes implicated in apoptosis and immunity, two broad pathways known to be involved in brain development. This is the first report of the clinical phenotype and molecular basis associated to basal ganglia agenesis in humans.
Aarif M. N. Batcha, Stefanos A. Bamopoulos, Paul Kerbs, Ashwini Kumar, Vindi Jurinovic, Maja Rothenberg-Thurley, Bianka Ksienzyk, Julia Philippou-Massier, Stefan Krebs, Helmut Blum, Stephanie Schneider, Nikola Konstandin, Stefan K. Bohlander, Caroline Heckman, Mika Kontro, Wolfgang Hiddemann, Karsten Spiekermann, Jan Braess, Klaus H. Metzeler, Philipp A. Greif, Ulrich Mansmann & Tobias Herold
Abstract
The patho-mechanism of somatic driver mutations in cancer usually involves transcription, but the proportion of mutations and wild-type alleles transcribed from DNA to RNA is largely unknown. We systematically compared the variant allele frequencies of recurrently mutated genes in DNA and RNA sequencing data of 246 acute myeloid leukaemia (AML) patients. We observed that 95% of all detected variants were transcribed while the rest were not detectable in RNA sequencing with a minimum read-depth cut-off (10x). Our analysis focusing on 11 genes harbouring recurring mutations demonstrated allelic imbalance (AI) in most patients. GATA2, RUNX1, TET2, SRSF2, IDH2, PTPN11, WT1, NPM1 and CEBPA showed significant AIs. While the effect size was small in general, GATA2 exhibited the largest allelic imbalance. By pooling heterogeneous data from three independent AML cohorts with paired DNA and RNA sequencing (N = 253), we could validate the preferential transcription of GATA2-mutated alleles. Differential expression analysis of the genes with significant AI showed no significant differential gene and isoform expression for the mutated genes, between mutated and wild-type patients. In conclusion, our analyses identified AI in nine out of eleven recurrently mutated genes. AI might be a common phenomenon in AML which potentially contributes to leukaemogenesis.
Penelope Kroustallaki, Lisa Lirussi,Sergio Carracedo, Panpan You, Q. Ying Esbensen, Alexandra Götz, Laure Jobert, Lene Alsøe, Pål Sætrom, Sarantis Gagos, Hilde Nilsen
Abstract
Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3′-polyadenylated and 3′-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice.
Features SIRVs (Spike-in RNA Variant Control Mixes) and SENSE Total RNA-Seq Library Prep Kit
Erez Eliyahu, Osnat Tirosh, Martina Dobesova, Aharon Nachshon, Michal Schwartz, Noam Stern-Ginossar
Abstract
Rho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here, we demonstrate that ROCK has antiviral properties, and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection, ROCK1 translocates to the nucleus and concentrates in the nucleolus, where it colocalizes with the stress-related chaperone heat shock cognate 71-kDa protein (Hsc70). Gene expression measurements show that inhibition of ROCK activity does not seem to affect the cellular stress response. We demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the antiviral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether, our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus.
IMPORTANCE ROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here we reveal a novel antiviral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus, where it mainly localizes to the nucleolus. Our findings suggest that ROCK’s antiviral activity may be related to activation of the actomyosin network and inhibition of capsid egress out of the nucleus.
Joel Hrit, Leeanne Goodrich, Cheng Li, Bang-An Wang, Ji Nie, Xiaolong Cui, Elizabeth Allene Martin, Eric Simental, Jenna Fernandez, Monica Yun Liu, Joseph R Nery, Rosa Castanon, Rahul M Kohli, Natalia Tretyakova, Chuan He, Joseph R Ecker, Mary Goll, Barbara Panning
Abstract
TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1’s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.
Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit
Jasmin Paris, Marcos Morgan, Joana Campos, Gary J. Spencer, Alena Shmakova, Ivayla Ivanova, Christopher Mapperley, Hannah Lawson, David A. Wotherspoon, Catarina Sepulveda, Milica Vukovic, Lewis Allen, Annika Sarapuu, Andrea Tavosanis, Amelie V. Guitart, Arnaud Villacreces, Christian Much, Junho Choe, Ali Azar, Louie N. van de Lagemaat, Douglas Vernimmen, Ali Nehme, Frederic Mazurier, Tim C.P. Somervaille, Richard I. Gregory, Dónal O’Carroll, Kamil R. Kranc
Abstract
Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.
Features SENSE Total RNA-Seq Library Prep Kit, QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina and SLAMseq Metabolic RNA Labeling Kit for RNA-Seq
Justin E. Ideozu, Vittobai Rangaraj, Hiam Abdala-Valencia, Xi Zhang, Manoj Kandpal, Marc A. Sala, Ramana V. Davuluri and Hara Levy
Abstract
Background
In cystic fibrosis (CF), impaired immune cell responses, driven by the dysfunctional CF transmembrane conductance regulator (CFTR) gene, may determine the disease severity but clinical heterogeneity remains a major therapeutic challenge. The characterization of molecular mechanisms underlying impaired immune responses in CF may reveal novel targets with therapeutic potential. Therefore, we utilized simultaneous RNA sequencing targeted at identifying differentially expressed genes, transcripts, and miRNAs that characterize impaired immune responses triggered by CF and its phenotypes.
Methods
Peripheral blood mononuclear cells (PBMCs) extracted from a healthy donor were stimulated with plasma from CF patients (n = 9) and healthy controls (n = 3). The PBMCs were cultured (1 × 105 cells/well) for 9 h at 37 ° C in 5% CO2. After culture, total RNA was extracted from each sample and used for simultaneous total RNA and miRNA sequencing.
Results
Analysis of expression signatures from peripheral blood mononuclear cells induced by plasma of CF patients and healthy controls identified 151 genes, 154 individual transcripts, and 41 miRNAs differentially expressed in CF compared to HC while the expression signatures of 285 genes, 241 individual transcripts, and seven miRNAs differed due to CF phenotypes. Top immune pathways influenced by CF included agranulocyte adhesion, diapedesis signaling, and IL17 signaling, while those influenced by CF phenotypes included natural killer cell signaling and PI3K signaling in B lymphocytes. Upstream regulator analysis indicated dysregulation of CCL5, NF-κB and IL1A due to CF while dysregulation of TREM1 and TP53 regulators were associated with CF phenotype. Five miRNAs showed inverse expression patterns with three target genes relevant in CF-associated impaired immune pathways while two miRNAs showed inverse expression patterns with two target genes relevant to a dysregulated immune pathway associated with CF phenotypes.
Conclusions
Our results indicate that miRNAs and individual transcript variants are relevant molecular targets contributing to impaired immune cell responses in CF.
Features SENSE Total RNA-Seq Library Prep Kit and RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)
Mingwei Min, Sabrina L. Spencer
Abstract
Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a noncycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous population and perform RNA sequencing to delineate the transcriptome underlying the slow-cycling state. We show that cellular stress responses—the p53 transcriptional response and the integrated stress response (ISR)—are the most salient causes of spontaneous entry into the slow-cycling state. Finally, we show that cells’ ability to enter the slow-cycling state enhances their survival in stressful conditions. Thus, the slow-cycling state is hardwired to stress responses to promote cellular survival in unpredictable environments.
Features SENSE Total RNA-Seq Library Prep Kit and RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)
Dagmara M. Wiatrek, Maria E. Candela, Jiří Sedmík, Jan Oppelt, Liam P. Keegan and Mary A. O’Connell
Abstract
Viral and cellular double-stranded RNA (dsRNA) is recognized by cytosolic innate immune sensors, including RIG-I-like receptors. Some cytoplasmic dsRNA is commonly present in cells, and one source is mitochondrial dsRNA, which results from bidirectional transcription of mitochondrial DNA (mtDNA). Here we demonstrate that Trp53 mutant mouse embryonic fibroblasts contain immune-stimulating endogenous dsRNA of mitochondrial origin. We show that the immune response induced by this dsRNA is mediated via RIG-I-like receptors and leads to the expression of type I interferon and proinflammatory cytokine genes. The mitochondrial dsRNA is cleaved by RNase L, which cleaves all cellular RNA including mitochondrial mRNAs, increasing activation of RIG-I-like receptors. When mitochondrial transcription is interrupted there is a subsequent decrease in this immune-stimulatory dsRNA. Our results reveal that the role of p53 in innate immunity is even more versatile and complex than previously anticipated. Our study, therefore, sheds new light on the role of endogenous RNA in diseases featuring aberrant immune responses.
Features SENSE Total RNA-Seq Library Prep Kit and RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)
M. Verheijen, M. Lienhard, Y. Schrooders, O. Clayton, R. Nudischer, S. Boerno, B. Timmermann, N. Selevsek, R. Schlapbach, H. Gmuender, S. Gotta, J. Geraedts, R. Herwig, J. Kleinjans & F. Caiment
Abstract
Though clinical trials for medical applications of dimethyl sulfoxide (DMSO) reported toxicity in the 1960s, later, the FDA classified DMSO in the safest solvent category. DMSO became widely used in many biomedical fields and biological effects were overlooked. Meanwhile, biomedical science has evolved towards sensitive high-throughput techniques and new research areas, including epigenomics and microRNAs. Considering its wide use, especially for cryopreservation and in vitro assays, we evaluated biological effect of DMSO using these technological innovations. We exposed 3D cardiac and hepatic microtissues to medium with or without 0.1% DMSO and analyzed the transcriptome, proteome and DNA methylation profiles. In both tissue types, transcriptome analysis detected >2000 differentially expressed genes affecting similar biological processes, thereby indicating consistent cross-organ actions of DMSO. Furthermore, microRNA analysis revealed large-scale deregulations of cardiac microRNAs and smaller, though still massive, effects in hepatic microtissues. Genome-wide methylation patterns also revealed tissue-specificity. While hepatic microtissues demonstrated non-significant changes, findings from cardiac microtissues suggested disruption of DNA methylation mechanisms leading to genome-wide changes. The extreme changes in microRNAs and alterations in the epigenetic landscape indicate that DMSO is not inert. Its use should be reconsidered, especially for cryopreservation of embryos and oocytes, since it may impact embryonic development.
Verheijen, M.C.T.
Abstract
Due to immense technological advancements over the last decade, biomedical science has evolved towards more sensitive high-throughput techniques and towards new areas of research, including regulatory mechanisms affecting transcription and translation. The research hypothesizes that the use of an experimental design better reflecting the human in vivo conditions combined with analysis of post-transcriptional mechanisms would aid the evolution of toxicogenomics research in order to improve drug safety assessments in the future. This dissertation describes possible options for realization of this evolution towards improved drug safety assessments.
Rachel Lackie, Marilene H Lopes, Sali M.K. Farhan, Abdul Razzaq, Gilli Moshitzky, Mariana B Prado, Flavio H Beraldo, Andrzej Maciejewski, Robert Gros, Jue Fan, Wing-Yiu Choy, David Greenberg, Vilma R Martins, Martin Duennwald, Hermona Soreq, Vania F Prado, Marco A. M. Prado
Abstract
The chaperone machinery is well conserved from yeast to mammals, however our knowledge of their impact on mammalian physiology is lagging. Stress-inducible phosphoprotein-1 (STI1; STIP1; Hop) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90 via three tetratricopeptide repeat (TPR) domains, of which TPR1 and TPR2B may be redundant in yeast. In-depth analysis of human datasets indicated that STI1 belongs to a set of co-chaperones that is essential in humans and that the TPR1 domain is evolutionarily conserved, suggesting that in mammals it may be required for optimal STI1 activity in vivo. We generated mice with a hypomorphic Stip1 allele lacking the TPR1 domain. While these mice are viable, they presented decreased levels of Hsp90 client proteins and co-chaperones, suggesting profound dysregulation of chaperone networks. We used this hypomorphic STI1 mutant mouse line to investigate the requirement of STI1-mediated regulation of chaperone networks in mouse physiology. Embryonic cell pluripotency was severely affected by decreased STI1 activity, contributing to the abnormal development in these mice. Moreover, adult TPR1-deprived STI1 mice presented age-related hippocampal neurodegeneration, resulting in compromised memory recall. Our findings reveal a requirement for optimal regulation of chaperone networks and their clients during development and strict dependence on full STI1 activity for healthy neuronal aging. These experiments demonstrate the unique experimental power of using hypomorphic alleles to reveal how chaperone networks regulate mammalian physiology.
Krisztina Koczka, Philipp Peters, Wolfgang Ernst, Heinz Himmelbauer, Lisa Nika, Reingard Grabherr
Abstract
The baculovirus insect cell expression system has become a firmly established production platform in biotechnology. Various complex proteins, multi-subunit particles including veterinary and human vaccines are manufactured with this system on a commercial scale. Apart from baculovirus infected Spodoptera frugiperda (Sf9) cells, the Trichoplusia ni(HighFive) cell line is alternatively used as host organism. In this study, we explored the protein production capabilities of Tnms42 insect cells, a new derivative of HighFive, which is free of latent nodavirus infection. As a model system, a cytosolic (mCherry) and a secreted (hemagglutinin) protein were overexpressed in Tnms42 cells. The response of the host cells was followed in a time course experiment over the infection cycle by comparative transcriptome analysis (RNA-seq). As expected, the baculovirus infection per se had a massive impact on the host cell transcriptome, which was observed by the huge total number of differentially expressed transcripts (>14,000). Despite this severe overall cellular reaction, a specific response could be clearly attributed to the overexpression of secreted hemagglutinin, revealing limits in the secretory capacity of the host cell. About 400 significantly regulated transcripts were identified and assigned to biochemical pathways and gene ontology (GO) categories, all related to protein processing, folding and response to unfolded protein. The identification of relevant target genes will serve to design specific virus engineering concepts for improving the yield of proteins that are dependent on the secretory pathway.
Rebecca A. Zabinsky, Brett M. Weum, Mingxue Cui and Min Han
Abstract
Extensive studies have suggested that most miRNA functions are executed through complex miRNA-target interaction networks, and such networks function semiredundantly with other regulatory systems to shape gene expression dynamics for proper physiological functions. We found that knocking down vgln-1, which encodes a conserved RNA-binding protein associated with diverse functions, causes severe larval arrest at the early L1 stage in animals with compromised miRISC functions (an ain-2/GW182 mutant). Through an enhancer screen, we identified five specific miRNAs, and miRNA families, that act semiredundantly with VGLN-1 to regulate larval development. By RIP-Seq analysis, we identified mRNAs that are directly bound by VGLN-1, and highly enriched for miRNA binding sites, leading to a hypothesis that VGLN-1 may share common targets with miRNAs to regulate gene expression dynamics for development.
Fernando A. Rabanal, Terezie Mandáková, Luz M. Soto-Jiménez, Robert Greenhalgh, David L. Parrott, Stefan Lutzmayer, Joshua G. Steffen, Viktoria Nizhynska, Richard Mott, Martin A. Lysak, Richard M. Clark and Magnus Nordborg
Abstract
Background
Ribosomal RNA (rRNA) accounts for the majority of the RNA in eukaryotic cells, and is encoded by hundreds to thousands of nearly identical gene copies, only a subset of which are active at any given time. In Arabidopsis thaliana, 45S rRNA genes are found in two large ribosomal DNA (rDNA) clusters and little is known about the contribution of each to the overall transcription pattern in the species.
Results
By taking advantage of genome sequencing data from the 1001 Genomes Consortium, we characterize rRNA gene sequence variation within and among accessions. Notably, variation is not restricted to the pre-rRNA sequences removed during processing, but it is also present within the highly conserved ribosomal subunits. Through linkage mapping we assign these variants to a particular rDNA cluster unambiguously and use them as reporters of rDNA cluster-specific expression. We demonstrate that rDNA cluster-usage varies greatly among accessions and that rDNA cluster-specific expression and silencing is controlled via genetic interactions between entire rDNA cluster haplotypes (alleles).
Conclusions
We show that rRNA gene cluster expression is controlled via complex epistatic and allelic interactions between rDNA haplotypes that apparently regulate the entire rRNA gene cluster. Furthermore, the sequence polymorphism we discovered implies that the pool of rRNA in a cell may be heterogeneous, which could have functional consequences.
