WEBINARS

WEBINAR: Controlling RNA-Seq Experiments Using Spike-In RNA Variants

Lukas Paul, PhD

Head of Services, Lexogen

Hardly anyone would run an RNA gel without a ladder, but transcriptomes are mostly sequenced without the use of external standards. The added layer of transcript isoform complexity in eukaryotes as well as incomplete or incorrect gene annotations further challenge RNA-Seq pipelines to correctly calculate and compare gene expression values. Lexogen, a specialized transcriptomics company, addresses this problem by providing Spike-In RNA Variant Control Mixes (SIRVs) to the RNA-Seq community. These controls are processed together with the RNA sample to allow for an evaluation of the RNA-Seq workflow and, in particular, of transcript isoform detection and gene expression quantification. The mixes contain 69 transcript variants that map to 7 human model genes and mirror the native transcriptome complexity by comprehensively representing splicing isoforms, transcription start-site and end-site variants, overlapping transcripts and antisense transcription. Lukas Paul, Head of Services at Lexogen, describes in this webinar how the SIRVs have been used to estimate absolute accuracy and consistency, as well as concordance in gene expression measurements at the level of workflows, experiments, and samples. A “SIRVs dashboard” is introduced that brings together spike-in derived NGS data, annotations and data evaluation in an easily navigable way, and the webinar will highlight how condensed SIRVs data can function as a “RNA-Seq fingerprint” that enables comparisons across experiments, samples and platforms.

WEBINAR: Mapping nuclear-exosome targeted poly(A) tails with 3´-RNA seq

Kevin Roy

Postdoctoral Scholar, Department of Genetics, Stanford University

A large fraction of the RNA transcribed in eukaryotic cells is rapidly degraded in the nucleus. A poly-adenylation complex distinct from the canonical poly(A) machinery is responsible for initiating 3´-5´ degradation of nuclear RNAs. This non-canonical poly(A) machinery, termed the Trf4/5-Air1/2-Mtr4 or TRAMP complex, catalyzes the addition of 3-4 adenosines on target RNA 3´-ends. This tags the transcript for 3´-5´ exonuclease digestion by the nuclear RNA exosome, which can either degrade or trim the RNA in a manner dependent on the presence of RNA structures or RNA-binding proteins. Inactivating the nuclear exosome stabilizes these otherwise short-lived RNAs, and subsequent cellular polyadenylation lengthens the oligo(A) tails to >30 adenosines.The majority of these poly(A)+ 3´-ends arise from non-coding and pervasive RNA polymerase II (Pol II) transcripts undergoing transcription termination by the Nrd1-Nab3-Sen1 (NNS) complex. 3´-sequencing of RNAs from exosome-inactivated cells enabled mapping the precise 3´-ends of these unstable RNAs, providing a high-resolution view of NNS termination genome-wide.Surprisingly, different NNS-dependent terminators display substantial heterogeneity in the width of the termination window, with some genes terminating the majority of transcripts in a window of 500 bp. Further analysis of NNS-terminators with a narrow termination window revealed that a particular set of DNA-binding proteins cooperate with NNS by roadblocking Pol II to promote efficient transcription termination genome-wide. Using the QuantSeq 3´ mRNA-Seq library prep kits, we were able to multiplex >40 samples per sequencing lane and obtain between 2 to 5 million reads per sample. This enabled us to analyze numerous different strains with various exosome and roadblocking factors inactivated, showing that inactivating roadblocks shifted the window of NNS termination downstream. Strikingly, disabling NNS enabled elongation of Pol II through the same roadblocks.These results explain how RNA processing signals control the outcome of collisions between Pol II and DNA binding proteins.

WEBINAR: Gene Expression Analysis Using 3’-RNA Sequencing

Behnam Abasht, PhD

Assistant Professor, University of Delaware

RNA sequencing (RNA-seq) has revolutionized the study of gene expression in animals, plants and microorganisms. However, because of its high cost, this technology has been mainly used in experiments with limited number of samples. To examine a cost-effective alternative, we used a method, which confines sequencing to the 3’-end of mRNA and produces just one fragment per transcript, resulting in a dramatic decrease in sequencing cost. Total RNA isolated from chicken adipose tissue samples was used for cDNA library preparation using QuantSeq 3’mRNA-seq library Prep Kit. Sixty-one uniquely indexed cDNA libraries were pooled and sequenced on one lane on the Illumina Hiseq 2500. On average, 2.24 million reads per sample were generated, 90.1% of which were mapped to the chicken reference genome (Ensembl Galgal4). For more than 70% of the genes with detectable expression, we redefined the 3’-end and identified alternative polyadenylation sites within the 3’-untranslated regions. To compare gene expression measures between 3’-RNA-seq and RNA-seq technologies, we used data from a subset of 20 samples that were previously used in a RNA-seq study of feed efficiency. The correlation of the log10(fold-change) for gene expression (high- vs. low-feed efficiency birds) between these two methods was 0.90. In conclusion, 3’-RNA-seq is a cost effective method amenable to global gene expression studies at population-level, e.g., expression QTL (eQTL) mapping.  Also, it allows for accurate detection of the 3’-end of transcripts, enabling verification of the current gene model annotations and global characterization of alternative polyadenylation.

SENSE

Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder

Dora C. Tărlungeanu, Elena Deliu, Christoph P. Dotter, Majdi Kara, Philipp Christoph Janiesch, Mariafrancesca Scalise, Michele Galluccio, Mateja Tesulov, Emanuela Morelli, Fatma Mujgan Sonmez, Kaya Bilguvar, Ryuichi Ohgaki, Yoshikatsu Kanai, Anide Johansen, Seham Esharif, Tawfeg Ben-Omran, Meral Topcu, Avner Schlessinger, Cesare Indiveri, Kent E. Duncan, Ahmet Okay Caglayan, Murat Gunel, Joseph G. Gleeson, Gaia Novarino

Cell. 2016 Dec 1, doi:10.1016/j.cell.2016.11.013

Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. We previously described abnormalities in the branched-chain amino acid (BCAA) catabolic pathway as a cause of ASD. Here, we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation, and severe neurological abnormalities. Furthermore, we identified several patients with autistic traits and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. Finally, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for the BCAA in human brain function.

Features SENSE mRNA‐Seq Library Prep Kit

InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data

Konstantin Okonechnikov, Aki Imai-Matsushima, Lukas Paul, Alexander Seitz, Thomas F. Meyer, Fernando Garcia-Alcalde

PLoS One. 2016 Dec 1, doi:10.1371/journal.pone.0167417

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit

Chemical Hybridization of Glucagon and Thyroid Hormone Optimizes Therapeutic Impact for Metabolic Disease.

Brian Finan, Christoffer Clemmensen, Zhimeng Zhu, Kerstin Stemmer, Karine Gauthier, Luisa Müller, Meri De Angelis, Kristin Moreth, Frauke Neff, Diego Perez-Tilve, Katrin Fischer, Dominik Lutter, Miguel A. Sánchez-Garrido, Peng Liu, Jan Tuckermann, Mohsen Malehmir, Marc E. Healy, Achim Weber, Mathias Heikenwalder, Martin Jastroch, Maximilian Kleinert, Sigrid Jall, Sara Brandt, Frédéric Flamant, Karl-Werner Schramm, Heike Biebermann, Yvonne Döring, Christian Weber, Kirk M. Habegger, Michaela Keuper, Vasily Gelfanov, Fa Liu, Josef Köhrle, Jan Rozman, Helmut Fuchs, Valerie Gailus-Durner, Martin Hrabě de Angelis, Susanna M. Hofmann, Bin Yang, Matthias H. Tschöp, Richard DiMarchi, Timo D. Müller

Cell, doi:10.1016/j.cell.2016.09.014

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

Features SENSE mRNA‐Seq Library Prep Kit

The complete nucleotide sequence and genomic characterization of grapevine asteroid mosaic associated virus

José Vargas-Asencio, Klaudia Wojciechowska, Maia Baskerville, Annika L. Gomez, Keith L. Perry, Jeremy R. Thompson

Virus Research, Volume 227, doi:10.1016/j.virusres.2016.10.001

In analyzing grapevine clones infected with grapevine red blotch associated virus, we identified a small number of isometric particles of approximately 30 nm in diameter from an enriched fraction of leaf extract. A dominant protein of 25 kDa was isolated from this fraction using SDS-PAGE and was identified by mass spectrometry as belonging to grapevine asteroid mosaic associated virus (GAMaV). Using a combination of three methods RNA-Seq, sRNA-Seq, and Sanger sequencing of RT- and RACE-PCR products, we obtained a full-length genome sequence consisting of 6719 nucleotides without the poly(A) tail. The virus possesses all of the typical conserved functional domains concordant with the genus Marafivirus and lies evolutionarily between citrus sudden death associated virus and oat blue dwarf virus. A large shift in RNA-Seq coverage coincided with the predicted location of the subgenomic RNA involved in coat protein (CP) expression. Genus wide sequence alignments confirmed the cleavage motif LxG(G/A) to be dominant between the helicase and RNA dependent RNA polymerase (RdRp), and the RdRp and CP domains. A putative overlapping protein (OP) ORF lacking a canonical translational start codon was identified with a reading frame context more consistent with the putative OPs of tymoviruses and fig fleck associated virus than with those of marafiviruses. BLAST analysis of the predicted GAMaV OP showed a unique relatedness to the OPs of members of the genus Tymovirus.

Features SENSE mRNA‐Seq Library Prep Kit

Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila

Vasiliy O. Sysoev, Bernd Fischer, Christian K. Frese, Ishaan Gupta, Jeroen Krijgsveld, Matthias W. Hentze, Alfredo Castello & Anne Ephrussi

Nature Communications 7, Article number: 12128, doi:10.1038/ncomms12128

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development.

Features SENSE mRNA‐Seq Library Prep Kit

Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution

Vitaly Sedlyarov, Jörg Fallmann, Florian Ebner, Jakob Huemer, Lucy Sneezum, Masa Ivin, Kristina Kreiner, Andrea Tanzer, Claus Vogl, Ivo Hofacker, Pavel Kovarik

Molecular Systems Biology (2016) 12, 868, doi: 10.15252/msb.20156628

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation‐associated mRNA decay remains to be established. Using time‐resolved high‐resolution RNA binding analysis of the mRNA‐destabilizing protein tristetraprolin (TTP), an inflammation‐limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non‐destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP‐controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA‐stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co‐regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome‐wide atlas (http://ttp-atlas.univie.ac.at) of cis‐acting elements controlling mRNA decay in inflammation.

Features SENSE mRNA‐Seq Library Prep Kit
Features Mix2 RNA-Seq Data Analysis Software

Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements

Ji-Eun Hwang, Jae Young Joung, Seung-Phil Shin, Moon-Kyung Choi, Jeong Eun Kim, Yon Hui Kim, Weon Seo Park, Sang-Jin Lee, Kang-Hyun Lee

Cancer Letters, 2015 doi: 10.1016/j.canlet.2015.12.018

Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1–100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker.

Features SENSE mRNA-Seq Library Prep Kit

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast.

Aronica L, Kasparek T, Ruchman D, Marquez Y, Cipak L, Cipakova I, Anrather D, Mikolaskova B, Radtke M, Sarkar S, Pai CC, Blaikley E, Walker C, Shen KF, Schroeder R, Barta A, Forsburg SL, Humphrey TC.

Nucleic Acids Res. 2015 Dec 17. pii: gkv1473.

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.

Features SENSE mRNA-Seq Library Prep Kit

A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain

Wenyuan Zhao, Tieqiang Zhao, Yuanjian Chen, Fengbo Zhao, Qingqing Gu, Robert W. Williams, Syamal K. Bhattacharya, Lu Lu , Yao Sun

PLoS One. 2015 Aug 4;10(8):e0133132. doi: 10.1371/journal.pone.0133132.

Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.

Features SENSE mRNA-Seq Library Prep Kit

Otx2 and Oct4 Drive Early Enhancer Activation during Embryonic Stem Cell Transition from Naive Pluripotency

Shen-Hsi Yang, Tüzer Kalkan, Claire Morissroe, Hendrik Marks, Hendrik Stunnenberg, Austin Smith, Andrew D. Sharrocks

Cell Rep. 2014 June 26; 7(6): 1968–1981. doi: 10.1016/j.celrep.2014.05.037

Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.

Features SENSE mRNA-Seq Library Prep Kit

Highly sensitive amplicon-based transcript quantification by semiconductor sequencing

Jitao David Zhang, Tobias Schindler, Erich Küng, Martin Ebeling, Ulrich Certa

BMC Genomics. 2014; 15(1): 565. doi: 10.1186/1471-2164-15-565

Background

In clinical and basic research custom panels for transcript profiling are gaining importance because only project specific informative genes are interrogated. This approach reduces costs and complexity of data analysis and allows multiplexing of samples. Polymerase-chain-reaction (PCR) based TaqMan assays have high sensitivity but suffer from a limited dynamic range and sample throughput. Hence, there is a gap for a technology able to measure expression of large gene sets in multiple samples.

Results

We have adapted a commercially available mRNA quantification assay (AmpliSeq-RNA) that measures mRNA abundance based on the frequency of PCR amplicons determined by high-throughput semiconductor sequencing. This approach allows for parallel, accurate quantification of about 1000 transcripts in multiple samples covering a dynamic range of five orders of magnitude. Using samples derived from a well-characterized stem cell differentiation model, we obtained a good correlation (r = 0.78) of transcript levels measured by AmpliSeq-RNA and DNA-microarrays. A significant portion of low abundant transcripts escapes detection by microarrays due to limited sensitivity. Standard quantitative RNA sequencing of the same samples confirms expression of low abundant genes with an overall correlation coefficient of r = 0.87. Based on digital AmpliSeq-RNA imaging we show switches of signaling cascades at four time points during differentiation of stem cells into cardiomyocytes.

Conclusions

The AmpliSeq-RNA technology adapted to high-throughput semiconductor sequencing allows robust transcript quantification based on amplicon frequency. Multiplexing of at least 900 parallel PCR reactions is feasible because sequencing-based quantification eliminates artefacts coming from off-target amplification. Using this approach, RNA quantification and detection of genetic variations can be performed in the same experiment.

Features SENSE mRNA-Seq Library Prep Kit

Mutations in CECR1 associated with a neutrophil signature in peripheral blood

Alexandre Belot, Evangeline Wassmer, Marinka Twilt, Jean-Christophe Lega, Leo AH Zeef, Anthony Oojageer, Paul R Kasher, Anne-Laure Mathieu, Christophe Malcus, Julie Demaret, Nicole Fabien, Sophie Collardeau-Frachon, Laura Mechtouff, Laurent Derex, Thierry Walzer, Gillian I Rice, Isabelle Durieu, Yanick J Crow

Pediatr Rheumatol Online J. 2014; 12: 44. doi: 10.1186/1546-0096-12-44

Background

A reduction of ADA2 activity due to autosomal recessive loss of function mutations in CECR1 results in a newly described vasculopathic phenotype reminiscent of polyarteritis nodosa, with manifestations ranging from fatal systemic vasculitis with multiple strokes in children to limited cutaneous disease in middle-aged individuals. Evidence indicates that ADA2 is essential for the endothelial integrity of small vessels. However, CECR1 is not expressed, nor is the ADA2 protein detectable, in cultured human endothelial cells, thus implicating additional cell types or circulating factors in disease pathogenesis.

Methods

Considering the phenotypic overlap of ADA2 deficiency with the type I interferonopathy Aicardi-Goutières syndrome due to mutations in SAMHD1, we looked for the presence of an interferon signature in the peripheral blood of two newly ascertained ADA2-deficient patients.

Results

We identified biallelic CECR1 mutations in two patients consistent with ADA2 deficiency. Both patients demonstrated an upregulation of interferon stimulated gene transcripts in peripheral blood. More strikingly however, genome-wide analysis revealed a marked overexpression of neutrophil-derived genes, suggesting that the vasculitis seen in ADA2 deficiency may be an indirect effect resulting from chronic and marked activity of neutrophils.

Conclusions

We hypothesise that ADA2 may act as a regulator of neutrophil activation, and that a reduction of ADA2 activity results in significant endothelial damage via a neutrophil-driven process.

Features SENSE mRNA-Seq Library Prep Kit

QuantSeq

Targeting the PD-1/PD-L1 pathway potentiates immunoediting to counterbalance neutral evolution in a mouse model of colorectal cancer

Zlatko Trajanoski, Mirjana Efremova, Victoria Klepsch, Pornpimol Charoentong, Francesca Finotello, Dietmar Rieder, Hubert Hackl, Natasch Hermann-Kleiter, Gottfried Baier, Anne Krogsdam

BioRxiv, doi:10.1101/099747

Background: The cancer immunoediting hypothesis postulates a dual role of the immune system: protecting the host by eliminating tumor cells, and shaping the developing tumor by editing the cancer genome. However, to what extent immunoediting is shaping the cancer genome in common malignancies is still a matter of debate. Moreover, the impact of cancer immunotherapy with checkpoint blockers on modulating immunoediting remains largely unexplored. Results: Here we employed a mouse model of colorectal cancer (CRC), next-generation sequencing, and computational analyses to elucidate the impact of evolutionary and immune-related forces on editing the tumor. We first carried out genomic and transcriptomic analyses of a widely-used model, MC38 cell line and show that this is a valid model for hypermutated and microsatellite-unstable CRC. Analyses of the data from longitudinal samples of wild type and immunodeficient RAG1 knockout mice transplanted with MC38 cells revealed that upregulation of checkpoint molecules and infiltration of Tregs are the major tumor escape mechanisms. Strikingly, the impact of neutral evolution on sculpting the tumor outweighed immunoediting by T cell dependent and T cell independent mechanisms in the progressing tumors. We also show that targeting the PD-1/PD-L1 pathway potentiated immunoediting and rendered tumors more homogeneous. Conclusions: In summary, our study demonstrates that neutral evolution is the major force that sculpts the tumor during progression, and that checkpoint blockade effectively enforces T cell dependent immunoselective pressure in this model. The results have important implication for basic research studies on the mechanisms of resistance to checkpoint blockade and for clinical translation.

Features QuantSeq 3′ mRNA-Seq Library Prep Kit

Global analysis of regulatory divergence in the evolution of mouse alternative polyadenylation

Mei‐Sheng Xiao, Bin Zhang, Yi‐Sheng Li, Qingsong Gao, Wei Sun, Wei Chen

Molecular Systems Biology (2016), doi:10.15252/msb.20167375

Alternative polyadenylation (APA), which is regulated by both cis‐elements and trans‐factors, plays an important role in post‐transcriptional regulation of eukaryotic gene expression. However, comparing to the extensively studied transcription and alternative splicing, the extent of APA divergence during evolution and the relative cis‐ and trans‐contribution remain largely unexplored. To directly address these questions for the first time in mammals, by using deep sequencing‐based methods, we measured APA divergence between C57BL/6J and SPRET/EiJ mouse strains as well as allele‐specific APA pattern in their F1 hybrids. Among the 24,721 polyadenylation sites (pAs) from 7,271 genes expressing multiple pAs, we identified 3,747 pAs showing significant divergence between the two strains. After integrating the allele‐specific data from F1 hybrids, we demonstrated that these events could be predominately attributed to cis‐regulatory effects. Further systematic sequence analysis of the regions in proximity to cis‐divergent pAs revealed that the local RNA secondary structure and a poly(U) tract in the upstream region could negatively modulate the pAs usage.

Features QuantSeq 3′ mRNA-Seq Library Prep Kit

Genome-wide genetic screening with chemically mutagenized haploid embryonic stem cells

Josep V Forment, Mareike Herzog, Julia Coates, Tomasz Konopka, Bianca V Gapp, Sebastian M Nijman, David J Adams, Thomas M Keane & Stephen P Jackson

Nature Chemical Biology (2016), doi:10.1038/nchembio.2226

In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.

Features QuantSeq 3′ mRNA-Seq Library Prep Kit

Transgenic Expression of Mitochondrial Chaperone TRAP1 Accelerates Prostate Cancer Development

Sofia Lisanti, David S. Garlick, Kelly G. Bryant, Michele Tavecchio, Gordon B. Mills, Yiling Lu, Andrew V. Kossenkov, Louise C. Showe, Lucia R. Languino and Dario C. Altieri

JBC, doi: 10.1074/jbc.M116.745950

Protein homeostasis, or proteostasis is required for mitochondrial function, but its role in cancer is controversial. Here, we show that transgenic mice expressing the mitochondrial chaperone, TRAP1 in the prostate develop epithelial hyperplasia and cellular atypia. When examined on a Pten+/- background, a common alteration in human prostate cancer, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ. Conversely, homozygous deletion of TRAP1 delays prostatic tumorigenesis in Pten+/- mice, without affecting hyperplasia or prostatic intraepithelial neoplasia. Global profiling of Pten+/–TRAP1 transgenic mice by RNA sequencing and reverse phase protein array reveals modulation of oncogenic networks of cell proliferation, apoptosis, cell motility, and DNA damage. Mechanistically, reconstitution of Pten+/- prostatic epithelial cells with TRAP1 increases cell proliferation, reduces apoptosis, and promotes cell invasion, without changes in mitochondrial bioenergetics. Therefore, TRAP1 is a driver of prostate cancer, in vivo, and “actionable” therapeutic target.

Features QuantSeq 3′ mRNA-Seq Library Prep Kit

Dissecting cis-regulatory effect on alternative polyadenylation using hybrid mice * DISSERTATION

Meisheng Xiao

Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin

The 3’ ends of most eukaryotic mRNAs are cleaved and polyadenylated at the last step of transcription. Recent studies revealed that more than 70% mammalian genes have multiple polyadenylation sites (pAs) leading to the generation of multiple mRNA isoforms with different coding region or 3’ untranslated region (3’ UTR) from the same gene locus and contributes to the complexity of transcriptome and proteome by regulating their stability, localization, translation, and function. Boosted by the large-scale analysis technologies, extensive and dynamic regulation of 3’ UTR by alternative polyadenylation (APA) has been observed in different tissues; different cellular conditions (proliferation, differentiation, and development); and response to stimuli. Although the exact underlying mechanisms of APA remains under investigation, it should be in general regulated via the interaction between cisregulatory elements residing at the DNA/RNA and trans-factors including polyadenylation cleavage core protein complex as well as other accessory RNA binding proteins (RBP). Change of APA pattern during evolution remains underexplored. Such changes could arise from the divergence in cis-regulatory elements and/or transacting RBPs. The divergences of the two factors with different extent of pleiotropic consequences undergo distinct evolutionary trajectories. Therefore, to better understand evolution in APA, it is important to distinguish the relative contribution of cis– and trans-effects. In this project, to comprehensively investigate the contribution of cis-elements and trans-factors in the process of APA in a mammalian system, we identified and quantified pAs usage difference between two parental strains (C57BL/6J and SPRET/EiJ) and between the two alleles in the F1 hybrids with 3’ read capturing and sequencing (3’ READS) and 3’ mRNA-Seq methods, respectively. In total, we identified 3747 parental divergent pAs across five types of APA, between the two parental mouse strains. By comparing the parental divergent pAs with those in F1 hybrids, we observed a predominant contribution of cis-regulatory effect on pAs usage, which is mediated by genetic variants between two species around the pAs. Further sequence feature analysis demonstrated that the unstable secondary structure and a novel hexamer UUUUUU in the upstream region of pAs could enhance and inhibit the pAs usage, respectively.

Features QuantSeq 3′ mRNA-Seq Library Prep Kit

The role of the microbiota in prey capture behavior * DISSERTATION

Levi W. Simonson

Department of Biology and the Graduate School of the University of Oregon

There is a growing body of evidence that normal nervous system activity requires signals from resident microbes. We have yet to discover the mechanisms by which the microbiota influence brain function. However, we know that the enteric nervous system (ENS) serves as an important interface between the developing host and its microbiota. In this dissertation I will introduce a novel computer-assisted method for ENS characterization and a novel, incredibly specific mechanism of host-microbe interactions. With new ENS characterization method I developed, it will be possible to better understand the role of the ENS during development, by more rapidly and algorithmically assessing ENS phenotypes. Furthermore, my discovery of a single microbially-sourced protein that influences vertebrate host prey capture behavior and visual system development, will provide a new appreciation for the role resident microbes, both in model organisms and in ourselves. By both establishing a new, less biased, approach to image analysis and describing a surprising new regulatory host-microbe interaction, the work I describe in this dissertation should provide the foundation for an explosion of exciting discoveries in the near future.

Features QuantSeq 3′ mRNA-Seq Library Prep Kit

Pan-cancer analysis of the Mediator complex transcriptome identifies CDK19 and CDK8 as therapeutic targets in advanced prostate cancer.

Brägelmann J, Klümper N, Offermann A, von Mässenhausen A, Böhm D, Deng M, Queisser A, Sanders C, Syring I, Merseburger AS, Vogel W, Sievers E, Vlasic I, Carlsson J, Andrén O, Brossart P, Duensing S, Svensson MA, Shaikhibrahim Z, Kirfel J, Perner S.

Clinical Cancer Research, doi: 10.1158/1078-0432.CCR-16-0094

PURPOSE:
The Mediator complex is a multi-protein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Since gene expression is frequently altered in cancer a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches.

EXPERIMENTAL DESIGN:
We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities (n=8568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer (PCa) specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced PCa (n=622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA mediated gene knock-down and inhibitor treatment in PCa cell lines with functional assays and gene expression analysis by RNAseq.

RESULTS:
Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type specific Mediator complex compositions. Only PCa was marked by high expression of CDK19. In primary PCa CDK19 was associated with increased aggressiveness and shorter disease free survival. During cancer progression highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro, inhibition of CDK19 and CDK8 by knock-down or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion.

CONCLUSIONS:
Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover it identified CDK19 and CDK8 to be specifically overexpressed during PCa progression, highlighting their potential as novel therapeutic targets in advanced PCa.

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Molecular basis for cytoplasmic RNA surveillance by uridylation‐triggered decay in Drosophila

Madalena M Reimão‐Pinto, Raphael A Manzenreither, Thomas R Burkard, Pawel Sledz, Martin Jinek, Karl Mechtler, Stefan L Ameres

The EMBO Journal (2016) e201695164; doi: 10.15252/embj.201695164

The posttranscriptional addition of nucleotides to the 3′ end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. Here, we show that in flies, 3′ terminal RNA uridylation triggers the processive, 3′‐to‐5′ exoribonucleolytic decay via the RNase II/R enzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation‐mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA. RNA immunoprecipitation and high‐throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non‐coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP. Based on genetic and biochemical evidence, we propose a key function of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high‐throughput biochemical characterization of dmDis3l2 and bacterial RNase R, our results imply a conserved molecular function of RNase II/R enzymes as “readers” of destabilizing posttranscriptional marks—uridylation in eukaryotes and adenylation in prokaryotes—that play important roles in RNA surveillance.

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BET Bromodomain Inhibition Promotes Anti-tumor Immunity by Suppressing PD-L1 Expression

Hengrui Zhu, Fee Bengsch, Nikolaos Svoronos, Melanie R. Rutkowski, Benjamin G. Bitler, Michael J. Allegrezza, Yuhki Yokoyama, Andrew V. Kossenkov, James E. Bradner, Jose R. Conejo-Garcia, Rugang Zhang

Cell Reports 16, 2829–2837, September 13, 2016; doi: 10.1016/j.celrep.2016.08.032

Restoration of anti-tumor immunity by blocking PD-L1 signaling through the use of antibodies has proven to be beneficial in cancer therapy. Here, we show that BET bromodomain inhibition suppresses PD-L1 expression and limits tumor progression in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of anti-tumor cytotoxic T cells. The BET inhibitor limited tumor progression in a cytotoxic T-cell-dependent manner. Together, these data demonstrate a small-molecule approach to block PD-L1 signaling. Given the fact that BET inhibitors have been proven to be safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a treatment strategy for targeting PD-L1 expression.

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An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication

Mario Mauthe, Martijn Langereis, Jennifer Jung, Xingdong Zhou, Alex Jones, Wienand Omta, Sharon A. Tooze, Björn Stork, Søren Riis Paludan, Tero Ahola, Dave Egan, Christian Behrends, Michal Mokry, Cornelis de Haan, Frank van Kuppevel, Fulvio Reggiori

The Rockefeller University Press / JCB vol. 214 no. 5 619-635 / doi: 10.1083/jcb.201602046

Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins. We determined the effects of the depletion of each ATG proteome component on the replication of six different viruses. Our screen reveals that up to 36% of the ATG proteins significantly alter the replication of at least one virus in an unconventional fashion. Detailed analysis of two candidates revealed an undocumented role for ATG13 and FIP200 in picornavirus replication that is independent of their function in autophagy as part of the ULK complex. The high numbers of unveiled ATG gene-specific and pathogen-specific functions of the ATG proteins calls for caution in the interpretation of data, which rely solely on the depletion of a single ATG protein to specifically ablate autophagy.

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Parallel reverse genetic screening in mutant human cells using transcriptomics

Bianca V Gapp, Tomasz Konopka, Thomas Penz, Vineet Dalal, Tilmann Bürckstümmer, Christoph Bock, Sebastian MB Nijman

Molecular Systems Biology (2016) 12, 879; doi: 10.15252/msb.20166890

Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease.

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Nuclear Innate Immune DNA Sensor IFI16 is Degraded During Lytic Reactivation of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency

Arunava Roy, Dipanjan Dutta, Jawed Iqbal, Gina Pisano, Olsi Gjyshi, Mairaj Ahmed Ansari, Binod Kumar and Bala Chandran

JVI.01003-16; doi: 10.1128/JVI.01003-16

IFI16 (Interferon-γ-inducible protein 16) recognizes the nuclear episomal herpes virus (KSHV, EBV and HSV-1) genomes and induces the inflammasome and interferon-β responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (HCMV and HPV) and transcription (HSV-1, HCMV and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in KSHV latently infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation, increase in KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch RTA gene. Overexpression of IFI16 reduced lytic gene induction by chemical agent TPA. IFI16 protein levels were significantly reduced or absent in TPA or doxycycline induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. IFI16’s degradation was absent in phosphonoacetic acid treated cells that blocks KSHV DNA replication and consequently late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation.

Importance Like all herpesviruses, latency is an integral part of Kaposi’s sarcoma-associated herpesvirus (KSHV) life cycle, an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to an active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoters and acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving the transcriptional repression. This is the first report to demonstrate IFI16’s role in latency maintenance of a herpes virus and further understanding will lead to the development of strategies to eliminate latent infection.

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Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line

Mireia Borràs-Fresneda, Joan-Francesc Barquinero, Maria Gomolka, Sabine Hornhardt, Ute Rössler, Gemma Armengol & Leonardo Barrios

Sci. Rep. 6, 27043; doi:10.1038/srep27043 (2016)

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

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The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway

Crina Popa, Liang Li, Sergio Gil, Laura Tatjer, Keisuke Hashii, Mitsuaki Tabuchi, Núria S. Coll, Joaquín Ariño & Marc Valls

Sci. Rep. 6, 27058; doi: 10.1038/srep27058 (2016)

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

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Transcriptional profiling and muscle cross‐section analysis reveal signs of ischemia reperfusion injury following total knee arthroplasty with tourniquet

Jonathan B. Muyskens, Austin D. Hocker, Douglas W. Turnbull, Steven N. Shah, Brick A. Lantz, Brian A. Jewett, Hans C. Dreyer

Physiological Reports Published 5 January 2016 Vol. 4 no. e12671 DOI: 10.14814/phy2.12671

Total knee arthroplasty (TKA) is the most common and cost‐effective treatment for older adults with long‐standing osteoarthritis. Projections indicate that nearly 3.5 million older adults will undergo this procedure annually by the year 2030. Thus, understanding the factors that lead to optimal outcomes is of great clinical interest. In the majority of cases, tourniquet is applied during surgery to maintain a clear surgical field, however, there is debate as to whether this intervention is completely benign. In particular, muscle atrophy is a significant factor in preventing full functional recovery following surgery, and some evidence suggests that tourniquet application and the associated ischemia–reperfusion injury that results contributes to muscle atrophy. For this reason, we examined tissue level changes in muscle in TKA patients following surgery and found that there was a significant increase in cross‐sectional area of muscle fibers of all types. Furthermore, to detect changes not evident at the tissue level, we performed NextSeq analysis to assess the transcriptional landscape of quadriceps muscle cells following TKA with tourniquet and found 72 genes that were significantly upregulated. A large proportion of those genes regulate cell stress pathways, suggesting that muscle cells in our cohort of older adults were capable of mounting a significant response to cell stress. Furthermore, factors related to complement were upregulated, suggesting tourniquet may play a role in priming cells to ischemia reperfusion injury. Therefore, our analysis reveals potential harms of tourniquet during TKA, thus suggesting that surgeons should consider limiting its use.

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SPLIT

InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data

Konstantin Okonechnikov, Aki Imai-Matsushima, Lukas Paul, Alexander Seitz, Thomas F. Meyer, Fernando Garcia-Alcalde

PLoS One. 2016 Dec 1, doi:10.1371/journal.pone.0167417

Analysis of fusion transcripts has become increasingly important due to their link with cancer development. Since high-throughput sequencing approaches survey fusion events exhaustively, several computational methods for the detection of gene fusions from RNA-seq data have been developed. This kind of analysis, however, is complicated by native trans-splicing events, the splicing-induced complexity of the transcriptome and biases and artefacts introduced in experiments and data analysis. There are a number of tools available for the detection of fusions from RNA-seq data; however, certain differences in specificity and sensitivity between commonly used approaches have been found. The ability to detect gene fusions of different types, including isoform fusions and fusions involving non-coding regions, has not been thoroughly studied yet. Here, we propose a novel computational toolkit called InFusion for fusion gene detection from RNA-seq data. InFusion introduces several unique features, such as discovery of fusions involving intergenic regions, and detection of anti-sense transcription in chimeric RNAs based on strand-specificity. Our approach demonstrates superior detection accuracy on simulated data and several public RNA-seq datasets. This improved performance was also evident when evaluating data from RNA deep-sequencing of two well-established prostate cancer cell lines. InFusion identified 26 novel fusion events that were validated in vitro, including alternatively spliced gene fusion isoforms and chimeric transcripts that include intergenic regions. The toolkit is freely available to download from http:/bitbucket.org/kokonech/infusion.

Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit

Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line

Mireia Borràs-Fresneda, Joan-Francesc Barquinero, Maria Gomolka, Sabine Hornhardt, Ute Rössler, Gemma Armengol & Leonardo Barrios

Sci. Rep. 6, 27043; doi:10.1038/srep27043 (2016)

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

Features QuantSeq 3′ mRNA-Seq Library Prep Kits
Features SPLIT RNA Extraction Kit

Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia

Seham Ebrahim, Neil J. Ingham, Morag A. Lewis, Michael J.C. Rogers, Runjia Cui, Bechara Kachar, Johanna C. Pass, Karen P. Steel

doi:10.1016/j.celrep.2016.03.081

WHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.

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Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice

Sathish K. Murali, Olena Andrukhova, Erica L. Clinkenbeard, Kenneth E. White, Reinhold G. Erben

PLoS Biol 14(4): e1002427. doi: 10.1371/journal.pbio.1002427

X-linked hypophosphatemia (XLH) is the most frequent form of inherited rickets in humans caused by mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). Hyp mice, a murine homologue of XLH, are characterized by hypophosphatemia, inappropriately low serum vitamin D levels, increased serum fibroblast growth factor-23 (Fgf23), and osteomalacia. Although Fgf23 is known to be responsible for hypophosphatemia and reduced vitamin D hormone levels in Hyp mice, its putative role as an auto-/paracrine osteomalacia-causing factor has not been explored. We recently reported that Fgf23 is a suppressor of tissue nonspecific alkaline phosphatase (Tnap) transcription via FGF receptor-3 (FGFR3) signaling, leading to inhibition of mineralization through accumulation of the TNAP substrate pyrophosphate. Here, we report that the pyrophosphate concentration is increased in Hyp bones, and that Tnap expression is decreased in Hyp-derived osteocyte-like cells but not in Hyp-derived osteoblasts ex vivo and in vitro. In situ mRNA expression profiling in bone cryosections revealed a ~70-fold up-regulation of Fgfr3 mRNA in osteocytes versus osteoblasts of Hyp mice. In addition, we show that blocking of increased Fgf23-FGFR3 signaling with anti-Fgf23 antibodies or an FGFR3 inhibitor partially restored the suppression of Tnap expression, phosphate production, and mineralization, and decreased pyrophosphate concentration in Hyp-derived osteocyte-like cells in vitro. In vivo, bone-specific deletion of Fgf23 in Hyp mice rescued the suppressed TNAP activity in osteocytes of Hyp mice. Moreover, treatment of wild-type osteoblasts or mice with recombinant FGF23 suppressed Tnap mRNA expression and increased pyrophosphate concentrations in the culture medium and in bone, respectively. In conclusion, we found that the cell autonomous increase in Fgf23 secretion in Hyp osteocytes drives the accumulation of pyrophosphate through auto-/paracrine suppression of TNAP. Hence, we have identified a novel mechanism contributing to the mineralization defect in Hyp mice.

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Amphiregulin lacks an essential role for the bone anabolic action of parathyroid hormone.

Freya F. Jay, Mithila Vaidya, Sabrina M. Porada, Olena Andrukhova, Marlon R. Schneider, Reinhold G. Erben

Mol Cell Endocrinol. 2015 Sep 28. pii: S0303-7207(15)30097-6. doi: 10.1016/j.mce.2015.09.031.

Although parathyroid hormone (PTH) has long been known to act as a bone anabolic agent when administered intermittently, the exact underlying mechanisms remain largely unknown. Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, has been identified to be a PTH target gene in vitro and in vivo. Here, we used female global AREG knockout (AREG-KO) mice to explore the role of AREG in mediating the bone anabolic effects of PTH. AREG-KO mice were characterized by unchanged distal femoral cancellous bone mass and only subtle decreases in bone mineral density (BMD) and cortical thickness at the femoral midshaft at 3 and 8 months of age, relative to wildtype controls. AREG deficiency was associated with complex changes in the mRNA expression of other EGFR ligands in femoral cancellous bone osteoblasts in situ in 3-week-old mice. To examine the bone anabolic effects of PTH in the absence and presence of AREG, we injected 3-month-old AREG-KO females and wildtype control littermates with 80 μg/kg PTH or vehicle 5 times per week over 4 weeks. Intermittent PTH treatment of AREG-KO mice led to increases in femoral trabecular and cortical BMD, cortical thickness, endocortical and periosteal bone formation, cancellous bone formation rate, and serum osteocalcin, comparable to those observed in wildtype control mice. In conclusion, our data indicate that the bone anabolic effects of PTH do not require AREG, at least in 3-month-old female mice.

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Nuclear accumulation of CDH1 mRNA in hepatocellular carcinoma cells.

Ghafoory S, Mehrabi A, Hafezi M, Cheng X, Breitkopf-Heinlein K, Hick M, Huichalaf M, Herbel V, Saffari A, Wölfl S.

Oncogenesis. 2015 Jun 1;4:e152. doi: 10.1038/oncsis.2015.11.

Expression of E-cadherin has a central role in maintaining epithelial morphology. In solid tumors, reduction of E-cadherin results in disruption of intercellular contacts. Consequently, cells lose adhesive properties and gain more invasive mesenchymal properties. Nevertheless, the mechanism of E-cadherin regulation is not completely elucidated. Here we analyzed the distribution of E-cadherin expression at the cell level in humanhepatocellular carcinoma, in which human liver paraffin blocks from 25 hepatocellular carcinoma patients were prepared from cancerous (CA) and noncancerous areas (NCA). In situ hybridization (ISH) was performed to detect E-cadherin and hypoxia-induced factor-1α (HIF1α) mRNAs and immunohistochemistry to stain E-cadherin protein. In parallel, RNA was extracted from CA and NCA, and E-cadherin and HIF1α were quantified by quantitative reverse transcription PCR. ISH revealed abundant E-cadherin mRNA in nuclei of hepatocellular carcinoma cells (HCCs), whereas immunohistochemistry showed depletion of E-cadherin protein from these areas. In sections of NCA, E-cadherin mRNA was also found in the cytosol, and E-cadherin protein was detected on the membrane of cells. Experiments in cell lines confirmed E-cadherin mRNA in nuclei of cells negative for E-cadherin protein. HIF1α expression is elevated in CAs, which is associated with a clear cytosolic staining for this mRNA. Our results demonstrate that E-caderhin mRNA is selectively retained in nuclei of HCCs, whereas other mRNAs are still exported, suggesting that translocation of E-cadherinmRNA from nuclei to cytoplasm has a role in regulating E-cadherin protein levels during epithelial mesenchymal transition.

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TeloPrime

Global survey of cis-regulation in mammalian translation * DISSERTATION

Jingyi Hou

Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin, Germany

This doctoral thesis consist of two parts: The first part describes a global survey of cisregulatory divergence in mammalian translation, where I applied mRNA sequencing and deep sequencing-based polysome profiling to quantify translational efficiency in F1 hybrid mice. The F1 progeny between Mus musculus C57BL/6J and Mus spretus SPRET/EiJ was chosen as a model system because the two have the largest number of genetic variants among all mouse strains with high-quality genome assemblies available. This large genomic divergence 1) provides a large number of potential regulatory variants between the two strains and 2) enables a sequencing-based approach to distinguish allelic RNA transcripts. The high quality of the data was demonstrated by employing two independent validation approaches, PacBio full-length sequencing and ribosome profiling. In total, 1008 genes (14.1%) were identified exhibiting significant allelic difference in translational efficiency. Several sequence features were associated with the observed allelic divergence in translation, including local RNA secondary structure near the start codon and proximal out-of-frame upstream AUGs. Finally, cis-effects are quantitatively comparable between transcriptional and translational regulation and these effects are more frequently compensatory between the two processes, suggesting a role of the translational regulation in buffering transcriptional noise and thereby maintaining the robustness of protein expression.

In the second part, I developed novel technology CAPTRE to measure the translational status of distinct mRNA TL isoforms. In mouse fibroblasts, a total of 22,357 TSSs derived from 10,875 protein-coding genes were identified. Among 4153 genes expressing multiple TSSs, 745 exhibited significant TE difference between their alternative TL isoforms. Longer isoforms were more frequently associated with lower TE and the global impact of several regulatory elements was also revisited, such as uORFs, cap-adjacent stable RNA secondary structures as well as 5′-terminal oligopyrimidine tract. In addition, several novel sequence motifs that can affect translation activity were identified and their effect was validated using two reporter systems. Finally, quantitative models combining different features identified in this study explained approximately 60% of the variance of the TE difference observed between TL isoforms.
This study provides novel mechanistic insights into translational regulation and characterizes the potential coupling between translational and transcriptional regulation in mammalian cells.

Features TeloPrime Full-Length cDNA Amplification Kit

Thyroglobulin Represents a Novel Molecular Architecture of Vertebrates

Guillaume Holzer, Yoshiaki Morishita, Jean-Baptiste Fini, Thibault Lorin, Benjamin Gillet, Sandrine Hughes, Marie Tohmé, Gilbert Deléage, Barbara Demeneix, Peter Arvan and Vincent Laudet

JBC.M116.719047. doi: 10.1074/jbc.M116.719047

Thyroid hormones modulate not only multiple functions in vertebrates (energy metabolism, central nervous system function, seasonal changes in physiology and behavior), but also in some non-vertebrates where they control critical post-embryonic developmental transitions such as metamorphosis. Despite their obvious biological importance, the thyroid hormone precursor protein, thyroglobulin (Tg), has been experimentally investigated only in mammals. This may bias our view of how thyroid hormones are produced in other organisms. In this study, we searched genomic databases and found Tg orthologs in all vertebrates including the sea lamprey (Petromyzon marinus). We cloned a full-size Tg coding sequence from western clawed frog (Xenopus tropicalis) and zebrafish (Dano rerio). Comparisons between the representative mammal, amphibian, teleost fish, and basal vertebrate indicate that all of the different domains of Tg, as well as Tg regional structure, are conserved throughout the vertebrates. Indeed, in Xenopus, zebrafish and lamprey Tgs, key residues, including the hormonogenic tyrosines and the disulfide bond-forming cysteines critical for Tg function are well conserved, despite overall divergence of amino acid sequences. We uncovered upstream sequences that include start codons of zebrafish and Xenopus Tgs, and experimentally proved that these are full-length secreted proteins, which are specifically recognized by antibodies against rat Tg. By contrast, we have not been able to find any orthologs of Tg among non-vertebrate species. Thus, Tg appears to be a novel protein elaborated as a single event at the base of vertebrates and virtually unchanged thereafter.

Features TeloPrime Full-Length cDNA Amplification Kit

cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing

Maria Cartolano, Bruno Huettel, Benjamin Hartwig, Richard Reinhardt, Korbinian Schneeberger

PLoS ONE 11(6):e0157779. doi:10.1371/journal.pone.0157779

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

Features TeloPrime Full-Length cDNA Amplification Kit

Pervasive isoform‐specific translational regulation via alternative transcription start sites in mammals

Xi Wang, Jingyi Hou, Claudia Quedenau, Wei Chen

Molecular Systems Biology (2016) 12, 875, DOI 10.15252/msb.20166941

Transcription initiated at alternative sites can produce mRNA isoforms with different 5ʹUTRs, which are potentially subjected to differential translational regulation. However, the prevalence of such isoform‐specific translational control across mammalian genomes is currently unknown. By combining polysome profiling with high‐throughput mRNA 5ʹ end sequencing, we directly measured the translational status of mRNA isoforms with distinct start sites. Among 9,951 genes expressed in mouse fibroblasts, we identified 4,153 showed significant initiation at multiple sites, of which 745 genes exhibited significant isoform‐divergent translation. Systematic analyses of the isoform‐specific translation revealed that isoforms with longer 5ʹUTRs tended to translate less efficiently. Further investigation of cis‐elements within 5ʹUTRs not only provided novel insights into the regulation by known sequence features, but also led to the discovery of novel regulatory sequence motifs. Quantitative models integrating all these features explained over half of the variance in the observed isoform‐divergent translation. Overall, our study demonstrated the extensive translational regulation by usage of alternative transcription start sites and offered comprehensive understanding of translational regulation by diverse sequence features embedded in 5ʹUTRs.

Features TeloPrime Full-Length cDNA Amplification Kit

Mix2 Software

Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution

Vitaly Sedlyarov, Jörg Fallmann, Florian Ebner, Jakob Huemer, Lucy Sneezum, Masa Ivin, Kristina Kreiner, Andrea Tanzer, Claus Vogl, Ivo Hofacker, Pavel Kovarik

doi: 10.15252/msb.20156628

Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation‐associated mRNA decay remains to be established. Using time‐resolved high‐resolution RNA binding analysis of the mRNA‐destabilizing protein tristetraprolin (TTP), an inflammation‐limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non‐destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP‐controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA‐stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co‐regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome‐wide atlas (http://ttp-atlas.univie.ac.at) of cis‐acting elements controlling mRNA decay in inflammation.

Features SENSE mRNA‐Seq Library Prep Kit
Features Mix2 RNA-Seq Data Analysis Software