SENSE Total RNA-Seq Publications

Targeting the RNA m6A Reader YTHDF2 Selectively Compromises Cancer Stem Cells in Acute Myeloid Leukemia

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

Cell Stem Cell, doi:10.1016/j.stem.2019.03.021

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

Transcriptional consequences of impaired immune cell responses induced by cystic fibrosis plasma characterized via dual RNA sequencing

Justin E. Ideozu, Vittobai Rangaraj, Hiam Abdala-Valencia, Xi Zhang, Manoj Kandpal, Marc A. Sala, Ramana V. Davuluri and Hara Levy

BMC Medical Genomics, doi:10.1186/s12920-019-0529-0

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)

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)

Activation of innate immunity by mitochondrial dsRNA in mouse cells lacking p53 protein

Dagmara M. Wiatrek, Maria E. Candela, Jiří Sedmík, Jan Oppelt, Liam P. Keegan and Mary A. O’Connell

RNA, doi:10.1261/rna.069625.118

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)

DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro

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

Scientific Reports, doi:10.1038/s41598-019-40660-0

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.

Features SENSE Total RNA-Seq Library Prep Kit

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.

Features SENSE Total RNA-Seq Library Prep Kit

A hypomorphic Stip1 allele reveals the requirement for chaperone networks in mouse development and aging

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

biorXiv, doi:10.1101/258673

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.

Features SENSE Total RNA-Seq Library Prep Kit

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.

Features SENSE Total RNA-Seq Library Prep Kit

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.

Features SENSE Total RNA-Seq Library Prep Kit

Epistatic and allelic interactions control expression of ribosomal RNA gene clusters in Arabidopsis thaliana

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

Genome Biology 2017, doi:10.1186/s13059-017-1209-z

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.

Features SENSE Total RNA-Seq Library Prep Kit