RiboCop

Natural Killer Cell transcriptome during primary EBV infection and EBV associated Hodgkin Lymphoma in children – a preliminary observation

Alka M., Shatakshee Chatterjee, Parchure A., Mahantesh S., Sravanthi Davuluri, Arun Kumar AR., Avinash T., Padma Me., Premalata CS., Mahua Sinha, Anup Chugani, Vishnu Priya R., Acharya KK, Jayshree R.S.

Immunobiology, doi:10.1016/j.imbio.2020.151907

Epstein Barr Viral infection is a common childhood infection in India and is also nearly 100 % etiologically associated with pediatric Hodgkin Lymphoma (HL). The main question in EBV immunobiology has been, why only a small subset of infected individuals develop EBV associated malignancies, while the vast majority carry this virus asymptomatically for life. Natural Killer (NK) cells, with a phenotype of CD56dim CD16+ exhibit potent cytotoxicity towards both virus infected cells and transformed cells and hence have been considered to be crucial in preventing the development of symptomatic EBV infection and lymphoma. In order to get an insight into the various possible molecular aspects of NK cells, in the pathogenesis of both these EBV mediated diseases in children we studied the whole transcriptome of MACS sorted CD56dim CD16 + NK cells from four patients from each of the three groups of children viz. Infectious Mononucleosis (IM), HL and age matched controls by using a massively parallel sequencing approach. NK cells from both IM and HL had down-regulated innate immunity and chemokine signaling genes. While down-regulation of genes responsible for polarization of the secretory apparatus, activated NK cell signaling and MAP kinase signaling were exclusive to NK cells in patients with IM, in NK cells of HL, specifically, genes involved in extracellular matrix (ECM) – receptor interaction, cytokine-cytokine receptor interaction, TNF signaling, Toll-like receptor signaling pathway and cytosolic DNA-sensing pathways were significantly down-regulated. Enrichment analysis showed STAT3 to be the most significant transcription factor (TF) for the down-regulated genes in IM, whereas, GATA1 was found to be the most significant TF for the genes down-regulated in HL. Analysis of protein interaction network identified functionally important protein clusters. Top clusters, comprised of down-regulated genes, involved in signaling and ubiquitin-related processes and pathways. These may perhaps be responsible for the hypo-responsiveness of NK cells in both diseases. These possibly point to different deficiencies in NK cell activation, loss of activating receptor signaling and degranulation in IM, versus loss of cytokine and chemokine signaling in HL, in the two EBV associated pathologies investigated. Various suppressed molecules and pathways were novel, which have not been reported earlier and could therefore be potential targets for immunotherapy of NK cell reactivation in both the diseases in future.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

The human malaria parasite Plasmodium falciparum uses mutually exclusive expression of the PfEMP1-encoding var gene family to evade the host immune system. Despite progress in the molecular understanding of the default silencing mechanism, the activation mechanism of the uniquely expressed var member remains elusive. A GC-rich noncoding RNA (ncRNA) gene family has coevolved with Plasmodium species that express var genes. Here, we show that this ncRNA family is transcribed in a clonally variant manner, with predominant transcription of a single member occurring when the ncRNA is located adjacent to and upstream of an active var gene. We developed a specific CRISPR interference (CRISPRi) strategy that allowed for the transcriptional repression of all GC-rich members. A lack of GC-rich ncRNA transcription led to the downregulation of the entire var gene family in ring-stage parasites. Strikingly, in mature blood-stage parasites, the GC-rich ncRNA CRISPRi affected the transcription patterns of other clonally variant gene families, including the downregulation of all Pfmc-2TM members. We provide evidence for the key role of GC-rich ncRNA transcription in var gene activation and discovered a molecular link between the transcriptional control of various clonally variant multigene families involved in parasite virulence. This work opens new avenues for elucidating the molecular processes that control immune evasion and pathogenesis in P. falciparum.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

Intestinal epithelial cells express immunomodulatory ISG15 during active ulcerative colitis and Crohn’s disease

Ann Elisabet Østvik, Tarjei Dahl Svendsen, Atle van Beelen Granlund, Berit Doseth, Helene Kolstad Skovdahl, Ingunn Bakke, Silje Thorsvik, Wahida Afroz, Gunnar Andreas Walaas, Tom Eirik Mollnes, Björn Inge Gustafsson, Arne Kristian Sandvik, Torunn Bruland

Journal of Crohn’s & colitis, doi:10.1093/ecco-jcc/jjaa022

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

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 & 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 RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit

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 RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit

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 RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit

Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs

Marek Vecera, Jiri Sana, Jan Oppelt, Boris Tichy, Kopkova Alena, Radim Lipina, Martin Smrcka, Radim Jancalek, Marketa Hermanova, Leos Kren, Ondrej Slaby

PLoS One, doi:10.1371/journal.pone.0211978

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit

Nociceptor Translational Profiling Reveals the Ragulator-Rag GTPase Complex as a Critical Generator of Neuropathic Pain

Salim Megat, Pradipta R. Ray, Jamie K. Moy, Tzu-Fang Lou, Paulino Barragán-Iglesias, Yan Li, Grishma Pradhan, Andi Wanghzou, Ayesha Ahmad, Michael D. Burton, Robert Y. North, Patrick M. Dougherty, Arkady Khoutorsky, Nahum Sonenberg, Kevin R. Webster, Gregory Dussor, Zachary T. Campbell and Theodore J. Price

The Journal of neuroscience, doi:10.1523/JNEUROSCI.2661-18.2018

Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase (MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

Compared with conventional serologic, culture-based, and molecular-based diagnostic tests, next-generation sequencing (NGS) provides sequence-evidenced detection of various microbes, without prior knowledge, and thus is becoming a novel diagnostic approach. Herein we describe an RNA-based metatranscriptomic NGS (mtNGS) protocol for culture-independent detection of potential infectious pathogens, using clinical bronchoalveolar lavage specimens as an example. We present both an optimized workflow for experimental sequence data collection and a simplified pipeline for bioinformatics sequence data processing. As shown, the whole protocol takes approximately 24 to 36 hours to detect a wide range of Gram-positive and -negative bacteria and possibly other viral and/or fungal pathogens. In particular, we introduce a spike-in RNA mix as an internal control, which plays a critical role in mitigating false-positive and false-negative results of clinical diagnostic tests. Moreover, our mtNGS method can detect antibiotic resistance genes and virulence factors; although it may not be comprehensive, such information is imperative and helpful for the clinician to make better treatment decisions. Results from our preliminary testing suggest that the mtNGS approach is a useful alterative in diagnostic detection of emerging infectious pathogens in clinical laboratories. However, further improvements are needed to achieve better sensitivity and accuracy.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

Unbiased dynamic characterization of RNA-protein interactions by OOPS

Rayner M. L. Queiroz, Tom Smith, Eneko Villanueva, Mie Monti, Mariavittoria Pizzinga, Maria Marti-Solano, Dan-Mircea Mirea, Manasa Ramakrishna, Robert F. Harvey, Veronica Dezi, Sven Degroeve, Lennart Martens, Gavin H. Thomas, Anne E. Willis, Kathryn S. Lilley

bioRxiv, doi:10.1101/333336

Current methods for the identification of RNA–protein interactions require a quantity and quality of sample that hinders their application, especially for dynamic biological systems or when sample material is limiting. Here, we present a new approach to enrich RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is compatible with downstream proteomics and RNA sequencing. OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. By applying OOPS to human cell lines, we extract the majority of known RBPs, and importantly identify additional novel RBPs, including those from previously under-represented cellular compartments. The high yield and unbiased nature of OOPS facilitates its application in both dynamic and inaccessible systems. Thus, we have identified changes in RNA-protein interactions in mammalian cells following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. Overall, OOPS provides an easy-to-use and flexible technique that opens new opportunities to characterize RNA-protein interactions and explore their dynamic behaviour.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit

Alternative polyadenylation drives genome-to-phenome information detours in the AMPKα1 and AMPKα2 knockout mice

Shuwen Zhang, Yangzi Zhang, Xiang Zhou, Xing Fu, Jennifer J. Michal, Guoli Ji, Min Du, Jon F. Davis & Zhihua Jiang

Scientific Reports, doi:10.1038/s41598-018-24683-7

Currently available mouse knockout (KO) lines remain largely uncharacterized for genome-to-phenome (G2P) information flows. Here we test our hypothesis that altered myogenesis seen in AMPKα1- and AMPKα2-KO mice is caused by use of alternative polyadenylation sites (APSs). AMPKα1 and AMPKα2 are two α subunits of adenosine monophosphate-activated protein kinase (AMPK), which serves as a cellular sensor in regulation of many biological events. A total of 56,483 APSs were derived from gastrocnemius muscles. The differentially expressed APSs (DE-APSs) that were down-regulated tended to be distal. The DE-APSs that were related to reduced and increased muscle mass were down-regulated in AMPKα1-KO mice, but up-regulated in AMPKα2-KO mice, respectively. Five genes: Car3 (carbonic anhydrase 3), Mylk4 (myosin light chain kinase family, member 4), Neb (nebulin), Obscn (obscurin) and Pfkm (phosphofructokinase, muscle) utilized different APSs with potentially antagonistic effects on muscle function. Overall, gene knockout triggers genome plasticity via use of APSs, completing the G2P processes. However, gene-based analysis failed to reach such a resolution. Therefore, we propose that alternative transcripts are minimal functional units in genomes and the traditional central dogma concept should be now examined under a systems biology approach.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

Differences between dorsal root and trigeminal ganglion nociceptors in mice revealed by translational profiling

Salim Megat, Pradipta R. Ray, Diana Tavares-Ferreira, Jamie K. Moy, Ishwarya Sankaranarayanan, Andi Wanghzou, Tzu Fang Lou, Paulino Barragan-Iglesias, Zachary T. Campbell, Gregory Dussor, Theodore J. Price

The Journal of Neuroscience, doi:10.1523/JNEUROSCI.2663-18.2019

Nociceptors located in the TG and DRG are the primary sensors of damaging or potentially damaging stimuli for the head and body, respectively, and are key drivers of chronic pain states. While nociceptors in these two tissues show a high degree of functional similarity, there are important differences in their development lineages, their functional connections to the central nervous system, and recent genome-wide analyses of gene expression suggest that they possess some unique genomic signatures. Here, we used translating ribosome affinity purification (TRAP) to comprehensively characterize and compare mRNA translation in Scn10a-positive nociceptors in the TG and DRG of male and female mice. This unbiased method independently confirms several findings of differences between TG and DRG nociceptors described in the literature but also suggests preferential utilization of key signaling pathways. Most prominently, we provide evidence that translational efficiency in mechanistic target of rapamycin (mTOR)-related genes is higher in the TG compared to DRG while several genes associated with the negative regulator of mTOR, AMPK activated protein kinase (AMPK), have higher translational efficiency in DRG nociceptors. Using capsaicin as a sensitizing stimulus we show that behavioral responses are greater in the TG region and this effect is completely reversible with mTOR inhibition. These findings have implications for the relative capacity of these nociceptors to be sensitized upon injury. Altogether, our data provide a comprehensive, comparative view of transcriptome and translatome activity in TG and DRG nociceptors that enhances our understanding of nociceptor biology.

SIGNIFICANCE STATEMENT

The DRG and TG provide sensory information from the body and head, respectively. Nociceptors in these tissues are critical first neurons in the pain pathway. Injury to peripheral neurons in these tissues can cause chronic pain. Interestingly, clinical and preclinical findings support the conclusion that injury to TG neurons is more likely to cause chronic pain and chronic pain in the TG area is more intense and more difficult to treat. We used TRAP technology to gain new insight into potential differences in the translatomes of DRG and TG neurons. Our findings demonstrate previously unrecognized differences between TG and DRG nociceptors that provide new insight into how injury may differentially drive plasticity states in nociceptors in these two tissues.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

OGT binds a conserved C-terminal domain of TET1 to regulate TET1 activity and function in development

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

eLife, doi:10.7554/eLife.34870

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

IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner

Simon Müller, Markus Glaß, Anurag K Singh, Jacob Haase, Nadine Bley, Tommy Fuchs, Marcell Lederer, Andreas Dahl, Huilin Huang, Jianjun Chen, Guido Posern, Stefan Hüttelmaier

Nucleic Acids Research, doi:10.1093/nar/gky1012

The oncofetal mRNA-binding protein IGF2BP1 and the transcriptional regulator SRF modulate gene expression in cancer. In cancer cells, we demonstrate that IGF2BP1 promotes the expression of SRF in a conserved and N6-methyladenosine (m6A)-dependent manner by impairing the miRNA-directed decay of the SRF mRNA. This results in enhanced SRF-dependent transcriptional activity and promotes tumor cell growth and invasion. At the post-transcriptional level, IGF2BP1 sustains the expression of various SRF-target genes. The majority of these SRF/IGF2BP1-enhanced genes, including PDLIM7 and FOXK1, show conserved upregulation with SRF and IGF2BP1 synthesis in cancer. PDLIM7 and FOXK1 promote tumor cell growth and were reported to enhance cell invasion. Consistently, 35 SRF/IGF2BP1-dependent genes showing conserved association with SRF and IGF2BP1 expression indicate a poor overall survival probability in ovarian, liver and lung cancer. In conclusion, these findings identify the SRF/IGF2BP1-, miRNome- and m6A-dependent control of gene expression as a conserved oncogenic driver network in cancer.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation* REVIEW

Ethan Edward Ford,Matthew R. Grimmer, Sabine Stolzenburg, Ozren Bogdanovic, Alex de Mendoza, Peggy J. Farnham, Pilar Blancafort, Ryan Lister

Biorxiv, doi:10.1101/170506

It is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression. The effect of forced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we conducted artificial methylation of thousands of promoters in human cells using an artificial zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription and histone modifications, and the durability of DNA methylation after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

eIF5A is an essential protein involved in protein synthesis, cell proliferation and animal development. High eIF5A expression is observed in many tumor types and has been linked to cancer metastasis. Recent studies have shown that eIF5A facilitates the translation elongation of stretches of consecutive prolines. Activated eIF5A binds to the empty E-site of stalled ribosomes, where it is thought to interact with the peptidyl-tRNA situated at the P-site. Here, we report a genome-wide analysis of ribosome stalling in Saccharomyces cerevisiae eIF5A depleted cells using 5Pseq. We confirm that, in the absence of eIF5A, ribosomes stall at proline stretches, and extend previous studies by identifying eIF5A-dependent ribosome pauses at termination and at >200 tripeptide motifs. We show that presence of proline, glycine and charged amino acids at the peptidyl transferase center and at the beginning of the peptide exit tunnel arrest ribosomes in eIF5A-depleted cells. Lack of eIF5A also renders ribosome accumulation at the stop codons. Our data indicate specific protein functional groups under the control of eIF5A, including ER-coupled translation and GTPases in yeast and cytoskeleton organization, collagen metabolism and cell differentiation in humans. Our results support a broad mRNA-specific role of eIF5A in translation and identify the conserved motifs that affect translation elongation from yeast to humans.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)