QuantSeq 3’ mRNA-Seq REV for Illumina

Cap homeostasis is the cyclical process of decapping and recapping that maintains the translation and stability of a subset of the transcriptome. Previous work showed levels of some recapping targets decline following transient expression of an inactive form of RNMT (ΔN-RNMT), likely due to degradation of mRNAs with improperly methylated caps. The current study examined transcriptome-wide changes following inhibition of cytoplasmic cap methylation. This identified mRNAs with 5′-terminal oligopyrimidine (TOP) sequences as the largest single class of recapping targets. Cap end mapping of several TOP mRNAs identified recapping events at native 5′ ends and downstream of the TOP sequence of EIF3K and EIF3D. This provides the first direct evidence for downstream recapping. Inhibition of cytoplasmic cap methylation was also associated with mRNA abundance increases for a number of transcription, splicing, and 3′ processing factors. Previous work suggested a role for alternative polyadenylation in target selection, but this proved not to be the case. However, inhibition of cytoplasmic cap methylation resulted in a shift of upstream polyadenylation sites to annotated 3′ ends. Together, these results solidify cap homeostasis as a fundamental process of gene expression control and show cytoplasmic recapping can impact regulatory elements present at the ends of mRNA molecules.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and TeloPrime Full-Length cDNA Amplification Kit

A Two-Layered Targeting Mechanism Underlies Nuclear RNA Sorting by the Human Exosome

Guifen Wu, Manfred Schmid, Leonor Rib, Patrik Polak, Nicola Meola, Albin Sandelin, Torben Heick Jensen

Cell Reports, doi:10.1016/j.celrep.2020.01.068

Degradation of transcripts in human nuclei is primarily facilitated by the RNA exosome. To obtain substrate specificity, the exosome is aided by adaptors; in the nucleoplasm, those adaptors are the nuclear exosome-targeting (NEXT) complex and the poly(A) (pA) exosome-targeting (PAXT) connection. How these adaptors guide exosome targeting remains enigmatic. Employing high-resolution 3′ end sequencing, we demonstrate that NEXT substrates arise from heterogenous and predominantly pA 3′ ends often covering kilobase-wide genomic regions. In contrast, PAXT targets harbor well-defined pA+ 3′ ends defined by canonical pA site use. Irrespective of this clear division, NEXT and PAXT act redundantly in two ways: (1) regional redundancy, where the majority of exosome-targeted transcription units produce NEXT- and PAXT-sensitive RNA isoforms, and (2) isoform redundancy, where the PAXT connection ensures fail-safe decay of post-transcriptionally polyadenylated NEXT targets. In conjunction, this provides a two-layered targeting mechanism for efficient nuclear sorting of the human transcriptome.

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

Eukaryotic gene expression relies on extensive crosstalk between transcription and RNA processing. Changes in this composite regulation network may provide an important means for shaping cell type-specific transcriptomes. Here we show that the RNA-associated protein Srrt/Ars2 sustains embryonic stem cell (ESC) identity by preventing premature termination of numerous transcripts at cryptic cleavage/polyadenylation sites in first introns. Srrt interacts with the nuclear cap-binding complex and facilitates recruitment of the spliceosome component U1 snRNP to cognate intronic positions. At least in some cases, U1 recruited in this manner inhibits downstream cleavage/polyadenylation events through a splicing-independent mechanism called telescripting. We further provide evidence that the naturally high expression of Srrt in ESCs offsets deleterious effects of retrotransposable sequences accumulating in its targets. Our work identifies Srrt as a molecular guardian of the pluripotent cell state.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

A Point Mutation in the RNA Recognition Motif of CSTF2 Associated with Intellectual Disability in Humans Causes Defects in 3′ End Processing

Petar N. Grozdanov, Elahe Masoumzadeh, Vera M. Kalscheuer, Thierry Bienvenu, Pierre Billuart, Marie-Ange Delrue, Michael P. Latham, Clinton C. MacDonald

bioRxiv, doi:10.1101/2020.01.02.893107

CSTF2 encodes an RNA-binding protein that is essential for mRNA cleavage and polyadenylation (C/P). No disease-associated mutations have been described for this gene. Here, we report a mutation in the RNA recognition motif (RRM) of CSTF2 that changes an aspartic acid at position 50 to alanine (p.D50A), resulting in intellectual disability in male patients. In mice, this mutation was sufficient to alter polyadenylation sites in over 1,000 genes critical for brain development. Using a reporter gene assay, we demonstrated that C/P efficiency of CSTF2D50A was lower than wild type. To account for this, we determined that p.D50A changed locations of amino acid side chains altering RNA binding sites in the RRM. The changes modified the electrostatic potential of the RRM leading to a greater affinity for RNA. These results highlight the importance of 3′ end mRNA processing in correct expression of genes important for brain plasticity and neuronal development.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Genetic deletion of Sphk2 confers protection against Pseudomonas aeruginosa mediated differential expression of genes related to virulent infection and inflammation in mouse lung

David L. Ebenezer, Panfeng Fu, Yashaswin Krishnan, Mark Maienschein-Cline, Hong Hu, Segun Jung, Ravi Madduri, Zarema Arbieva, Anantha Harijith & Viswanathan Natarajan

BMC Genomics, doi:10.1186/s12864-019-6367-9

Background

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative bacterium that causes serious life threatening and nosocomial infections including pneumonia. PA has the ability to alter host genome to facilitate its invasion, thus increasing the virulence of the organism. Sphingosine-1- phosphate (S1P), a bioactive lipid, is known to play a key role in facilitating infection. Sphingosine kinases (SPHK) 1&2 phosphorylate sphingosine to generate S1P in mammalian cells. We reported earlier that Sphk2−/− mice offered significant protection against lung inflammation, compared to wild type (WT) animals. Therefore, we profiled the differential expression of genes between the protected group of Sphk2−/− and the wild type controls to better understand the underlying protective mechanisms related to the Sphk2 deletion in lung inflammatory injury. Whole transcriptome shotgun sequencing (RNA-Seq) was performed on mouse lung tissue using NextSeq 500 sequencing system.

Results

Two-way analysis of variance (ANOVA) analysis was performed and differentially expressed genes following PA infection were identified using whole transcriptome of Sphk2−/− mice and their WT counterparts. Pathway (PW) enrichment analyses of the RNA seq data identified several signaling pathways that are likely to play a crucial role in pneumonia caused by PA such as those involved in: 1. Immune response to PA infection and NF-κB signal transduction; 2. PKC signal transduction; 3. Impact on epigenetic regulation; 4. Epithelial sodium channel pathway; 5. Mucin expression; and 6. Bacterial infection related pathways.

Our genomic data suggests a potential role for SPHK2 in PA-induced pneumonia through elevated expression of inflammatory genes in lung tissue. Further, validation by RT-PCR on 10 differentially expressed genes showed 100% concordance in terms of vectoral changes as well as significant fold change.

Conclusion

Using Sphk2−/− mice and differential gene expression analysis, we have shown here that S1P/SPHK2 signaling could play a key role in promoting PA pneumonia. The identified genes promote inflammation and suppress others that naturally inhibit inflammation and host defense. Thus, targeting SPHK2/S1P signaling in PA-induced lung inflammation could serve as a potential therapy to combat PA-induced pneumonia.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Alternative polyadenylation (APA), which is regulated by both cis-elements and trans-factors, is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. It could change the 3’UTR of an mRNA transcript affecting its stability, translation efficiency, nuclear export and mRNA or translated protein localization, or, if an exonic/intronic polyadenylation site (PAS) upstream of the stop codon is used, it could affect a gene’s coding region to produce different protein isoforms with distinct properties. Accumulating evidence suggests that global APA-mediated 3’UTR length change might play an important role in oncogenic transformation, pluripotency, lymphocyte activation, neuronal stimulation and in embryonic development and differentiation. However, recent studies found limited effects of 3’UTRs in most genes compared to other regulatory elements located in 5’UTRs or coding sequence. APA as a molecular trait is a low-level phenotype in the hierarchy of biological organization, and might only exert very limited effects on organismal fitness. Therefore, some researchers proposed the “error hypothesis”, stating that most observed APA is noise and that APA diversity within and between tissues is generally neutral or deleterious, and not functional. Similarly, it has been suggested that APA divergence between species is largely non-adaptive. This scenario would be consistent with the (nearly) neutral theory of molecular evolution, which predicts that genes under relaxed selective constraints accumulate neutral (or slightly deleterious) changes at a faster rate than those under stronger purifying selection. In order to clarify the general and tissue-dependent function and regulation of APA and its evolution in mammals, we applied 3’mRNA sequencing for multiple tissues of an F1 hybrid between the C57BL/6J (Mus musculus) and SPRET/EiJ (Mus spretus) mouse strains. We analyzed the factors regulating APA diversity and addressed the question whether APA is generally non-adaptive as proposed by the error hypothesis. In this study, we quantified all annotated PASs in nine tissues of the F1 hybrid mouse and comprehensively characterized different features of single-PAS genes and multi-PAS genes. Next, we checked the positional effects on PAS strength and discussed the functional difference between rank 1 and rank 2 PASs among distinct gene groups. By quantifying PAS usage in each allele, we studied the genes with divergent major PAS expression level and dN/dS ratio difference, and unveiled different evolutionary patterns between APA patterns and gene expression (mRNA levels). We found that in general APA of multi-PAS genes is consistent with the error hypothesis, and that most APA diversity within and between tissues appears to reflect noise, resulting from molecular error due to weak cis-regulation. However, we did not find different selective constraint in dN/dS between genes with high and with low APA diversity, but found strong correlation between mRNA abundance and APA accuracy. The minor and major relative PAS usage is also affected by PAS position. In addition to most major PAS, many minor PASs appear to have functional importance. They are highly conserved and can compete with the major PASs. Last, we found a small fraction of genes exhibits strongly tissue-regulated APA patterns. In these genes, PAS usage is under intensive trans-regulation between the C57BL/6J and SPRET/EiJ alleles in the F1 hybrid mouse. Whereas many divergent PASs exist between the two alleles in genes with low expression level and under relax selective constraints, comparing these with genes showing allelic mRNA transcript level differences, we unveiled different evolutionary patterns between APA and gene expression.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

PolyASite 2.0: a consolidated atlas of polyadenylation sites from 3′ end sequencing

Christina J Herrmann, Ralf Schmidt, Alexander Kanitz, Panu Artimo, Andreas J Gruber, Mihaela Zavolan

Nucleic Acids Research, doi:10.1093/nar/gkz918

Generated by 3′ end cleavage and polyadenylation at alternative polyadenylation (poly(A)) sites, alternative terminal exons account for much of the variation between human transcript isoforms. More than a dozen protocols have been developed so far for capturing and sequencing RNA 3′ ends from a variety of cell types and species. In previous studies, we have used these data to uncover novel regulatory signals and cell type-specific isoforms. Here we present an update of the PolyASite (https://polyasite.unibas.ch) resource of poly(A) sites, constructed from publicly available human, mouse and worm 3′ end sequencing datasets by enforcing uniform quality measures, including the flagging of putative internal priming sites. Through integrated processing of all data, we identified and clustered sites that are closely spaced and share polyadenylation signals, as these are likely the result of stochastic variations in processing. For each cluster, we identified the representative – most frequently processed – site and estimated the relative use in the transcriptome across all samples. We have established a modern web portal for efficient finding, exploration and export of data. Database generation is fully automated, greatly facilitating incorporation of new datasets and the updating of underlying genome resources.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Suboptimal RNA–RNA interaction limits U1 snRNP inhibition of canonical mRNA 3’ processing

Junjie Shi, Yanhui Deng, Shanshan Huang, Chunliu Huang, Jinkai Wang, Andy Peng Xiang & Chengguo Yao

RNA Biology, doi:10.1080/15476286.2019.1636596

It is increasingly appreciated that U1 snRNP transcriptomically suppresses the usage of intronic polyadenylation site (PAS) of mRNAs, an outstanding question is why frequently used PASs are not suppressed. Here we found that U1 snRNP could be transiently associated with sequences upstream of actionable PASs in human cells, and RNA–RNA interaction might contribute to the association. By focusing on individual PAS, we showed that the stable assembly of U1 snRNP near PAS might be generally required for U1 inhibition of mRNA 3ʹ processing. Therefore, actionable PASs that often lack optimal U1 snRNP docking site nearby is free from U1 inhibitory effect. Consistently, natural 5ʹ splicing site (5ʹ-SS) is moderately enriched ~250 nt upstream of intronic PASs whose usage is sensitive to functional knockdown of U1 snRNA. Collectively, our results provided an insight into how U1 snRNP selectively inhibits the usage of PASs in a cellular context, and supported a prevailing model that U1 snRNP scans pre-mRNA through RNA–RNA interaction to find a stable interaction site to exercise its function in pre-mRNA processing, including repressing the usage of cryptic PASs.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

A specialised SKI complex assists the cytoplasmic RNA exosome in the absence of direct association with ribosomes

Elodie Zhang, Varun Khanna, Estelle Dacheux, Abdelkader Namane, Antonia Doyen, Maïté Gomard, Bernard Turcotte, Alain Jacquier, Micheline Fromont‐Racine

The EMBO Journal, doi:10.15252/embj.2018100640

The Ski2‐Ski3‐Ski8 (SKI ) complex assists the RNA exosome during the 3′ to 5′ degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3′ to 5′ degradation of mRNA s, including 3′ untranslated regions (UTR s) and devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co‐translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterised a new SKI ‐associated factor, Ska1, that associates with a subpopulation of the SKI complex. We showed that Ska1 is specifically involved in the degradation of long 3′UTR ‐containing mRNA s, poorly translated mRNA s as well as other RNA regions not associated with ribosomes, such as cytoplasmic lncRNA s. We further show that the overexpression of SKA 1 antagonises the SKI‐ribosome association. We propose that the Ska1‐SKI complex assists the cytoplasmic exosome in the absence of direct association of the SKI complex with ribosomes.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination

Kinga Kamieniarz-Gdula, Michal R. Gdula, Karin Panser, Takayuki Nojima, Joan Monks, Jacek R. Wiśniewski, Joey Riepsaame, Neil Brockdorff, Andrea Pauli, Nick J. Proudfoot

Molecular Cell, doi:10.1016/j.molcel.2019.01.027

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3′ mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

3′-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3′ ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3′ tails instead of the canonical CCA tail, and globally identify 3′ ends of splicing intermediates arising from inefficiently spliced transcripts.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

The eukaryotic translation initiation factor eIF4E is nuclear and cytoplasmic where it plays roles in export and translation of specific transcripts, respectively. When we were studying its mRNA export activity, we unexpectedly discovered that eIF4E drives the protein expression of elements of the 3′-end core cleavage complex involved in cleavage and polyadenylation (CPA), including CPSF3, the enzyme responsible for cleavage, as well as its co-factors CPSF1, CPSF2, CPSF4, Symplekin, WDR33, and FIP1L1. Using multiple strategies, we demonstrate that eIF4E stimulates 3′-end cleavage of selected RNAs. eIF4E physically interacts with CPSF3, CPSF1, and uncleaved target RNA, suggesting it acts directly and indirectly on the pathway. Through these effects, eIF4E can generate better substrates for its mRNA export and translation activities. Thus, we identified an unanticipated function for eIF4E in 3′-end processing of specific target RNAs, and this function could potentially affect the expression of a broad range of oncoproteins.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have been developed to measure transcription and RNA decay rates. However, to our knowledge, none of those techniques can simultaneously interrogate transcription and RNA decay. The presented protocol allows this and provides a simple approach to simultaneously estimate total RNA levels, transcription and decay rates from the same RNA sample. It is based on brief metabolic labeling of RNA and subsequent concurrent sequencing of polyA+ and polyA RNA 3′ ends. The protocol was developed in S. cerevisiae and should be broadly applicable.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence

Takayuki Nojima, Michael Tellier, Jonathan Foxwell, Claudia Ribeiro de Almeida, Sue Mei Tan-Wong, Somdutta Dhir, Gwendal Dujardin, Ashish Dhir, Shona Murphy, Nick J. Proudfoot

Molecular Cell, doi:10.1016/j.molcel.2018.10.011

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have revealed that niclosamide exhibits diverse mechanisms of action and, as a result, demonstrates promise for a number of applications, including the treatment of cancer, bacterial infections, and Zika virus. As new applications of niclosamide will require non-oral delivery routes that may lead to exposure in utero, the objective of this study was to investigate the mechanism of niclosamide toxicity during early stages of embryonic development. Using zebrafish as a model, we found that niclosamide induced a concentration-dependent delay in epiboly progression during late-blastula and early-gastrula, an effect that was dependent on exposure during the maternal-to-zygotic transition – a period characterized by degradation of maternally-derived transcripts, zygotic genome activation, and initiation of cell motility. Moreover, we found that niclosamide did not affect embryonic oxygen consumption, suggesting that oxidative phosphorylation – a well-established target for niclosamide within intestinal parasites – may not play a role in niclosamide-induced epiboly delay. However, mRNA-sequencing revealed that niclosamide exposure during blastula and early-gastrula significantly impacted the timing of zygotic genome activation as well as the abundance of cytoskeleton- and cell cycle regulation-specific transcripts. In addition, we found that niclosamide inhibited tubulin polymerization in vitro, suggesting that niclosamide-induced delays in epiboly progression may, in part, be driven by disruption of microtubule formation and cell motility within the developing embryo.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Expansin increases cell wall extensibility to allow cell wall loosening and cell expansion even in the absence of hydrolytic activity. Previous studies showed that excessive overexpression of expansin gene resulted in defective growth (Goh et al., 2014; Rochange et al., 2001) [1,2] and altered cell wall chemical composition (Zenoni et al., 2011) [3]. However, the molecular mechanism on how the overexpression of non-enzymatic cell wall protein expansin can result in widespread effects on plant cell wall and organ growth remains unclear. We acquired transcriptomic data on previously reported transgenic Arabidopsis line (Goh et al., 2014) [1] to investigate the effects of overexpressing a heterologus cucumber expansin gene (CsEXPA1) on the global gene expression pattern during early and late phases of etiolated hypocotyl growth.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Alzheimer’s brains show inter-related changes in RNA and lipid metabolism

Shahar Barbash, Benjamin P. Garfinkel, Rotem Maoz, Alon Simchovitz, Bettina Nadorp, Alessandro Guffanti, Estelle R. Bennett, Courtney Nadeau, Andreas Türk, Lukas Paul, Torsten Reda, Yan Li, Aron S. Buchman, David S. Greenberg, Alexander Seitz, David A. Bennett, Patrick Giavalisco, Hermona Soreq

Neurobiology of Disease, doi: 10.1016/j.nbd.2017.06.008

Alzheimer’s disease (AD) involves changes in both lipid and RNA metabolism, but it remained unknown if these differences associate with AD’s cognition and/or post-mortem neuropathology indices. Here, we report RNA-sequencing evidence of inter-related associations between lipid processing, cognition level, and AD neuropathology. In two unrelated cohorts, we identified pathway-enriched facilitation of lipid processing and alternative splicing genes, including the neuronal-enriched NOVA1 and hnRNPA1. Specifically, this association emerged in temporal lobe tissue samples from donors where postmortem evidence demonstrated AD neuropathology, but who presented normal cognition proximate to death. The observed changes further associated with modified ATP synthesis and mitochondrial transcripts, indicating metabolic relevance; accordingly, mass-spectrometry-derived lipidomic profiles distinguished between individuals with and without cognitive impairment prior to death. In spite of the limited group sizes, tissues from persons with both cognitive impairment and AD pathology showed elevation in several drug-targeted genes of other brain, vascular and autoimmune disorders, accompanied by pathology-related increases in distinct lipid processing transcripts, and in the RNA metabolism genes hnRNPH2, TARDBP, CLP1 and EWSR1. To further detect 3′-polyadenylation variants, we employed multiple cDNA primer pairs. This identified variants that showed limited differences in scope and length between the tested cohorts, yet enabled superior clustering of demented and non-demented AD brains versus controls compared to total mRNA expression values. Our findings indicate inter-related cognition-associated differences in AD’s lipid processing, alternative splicing and 3′-polyadenylation, calling for pursuing the underlying psychological and therapeutics implications.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and SPLIT RNA Extraction Kit

Papaya is considered to be one of the most nutritional fruits. It is rich in vitamins, carotenoids, flavonoids and other phytonutrient which function as antioxidant in our body [1]. Previous studies revealed that the suppression of a negative regulator gene in photomorphogenesis, De-etiolated 1 (DET1) can improve the phytonutrient in tomato and canola without affecting the fruit quality [2] ; [3]. This report contains the experimental data on high-throughput 3′ mRNA sequencing of transformed papaya callus upon DET1 gene suppression.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Expansin is a cell wall loosening protein without hydrolytic activity, which allows cell expansion by influencing cell wall extensibility. Previous studies showed that the suppression of expansin genes (EXPA1, EXPA3, EXPA5 and EXPA10) resulted in defective organ growth and altered cell wall chemical composition [1,2]. However, the molecular mechanism on how the suppression of non-enzymatic expansin expression can result in widespread effects on plant cell wall and organ growth is still unclear. In this study, we performed transcriptomic analysis on the hypocotyls of previously reported transgenic Arabidopsis line [1] to investigate the effects of expansin gene suppression on the global gene expression pattern, particularly on the cell wall related genes.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

High-Resolution RNA Maps Suggest Common Principles of Splicing and Polyadenylation Regulation by TDP-43

Gregor Rot, Zhen Wang, Ina Huppertz, Miha Modic, Tina Lenče, Martina Hallegger, Nejc Haberman, Tomaž Curk, Christian von Mering, Jernej Ule

Cell Reports 19, 1056–1067, doi: 10.1016/j.celrep.2017.04.028

Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3′ mRNA sequencing. This reveals at nucleotide resolution the “RNA maps” describing how the RNA binding positions of RBPs relate to their regulatory functions. We use this approach to examine how TDP-43, an RBP involved in several neurodegenerative diseases, binds around its regulated poly(A) sites. Binding close to the poly(A) site generally represses, whereas binding further downstream enhances use of the site, which is similar to TDP-43 binding around regulated exons. Our RNAmotifs2 software also identifies sequence motifs that cluster together with the binding motifs of TDP-43. We conclude that TDP-43 directly regulates diverse types of pre-mRNA processing according to common position-dependent principles.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

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 REV for Illumina

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.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Genome-wide transcriptome profiling is a powerful tool to study global gene expression patterns in plant development. We report the first transcriptome profile analysis of papaya embryogenic callus to improve our understanding on genes associated with somatic embryogenesis. By using 3′ mRNA-sequencing, we generated 6,190,687 processed reads and 47.0% were aligned to papaya genome reference, in which 21,170 (75.4%) of 27,082 annotated genes were found to be expressed but only 41% was expressed at functionally high levels. The top 10% of genes with high transcript abundance were significantly enriched in biological processes related to cell proliferation, stress response, and metabolism. Genes functioning in somatic embryogenesis such as SERK and LEA, hormone-related genes, stress-related genes, and genes involved in secondary metabolite biosynthesis pathways were highly expressed. Transcription factors such as NAC, WRKY, MYB, WUSCHEL, Agamous-like MADS-box protein and bHLH important in somatic embryos of other plants species were found to be expressed in papaya embryogenic callus. Abundant expression of enolase and ADH is consistent with proteome study of papaya somatic embryo. Our study highlights that some genes related to secondary metabolite biosynthesis, especially phenylpropanoid biosynthesis, were highly expressed in papaya embryogenic callus, which might have implication for cell factory applications. The discovery of all genes expressed in papaya embryogenic callus provides an important information into early biological processes during the induction of embryogenesis and useful for future research in other plant species.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina

Dysregulation of Alternative Poly-adenylation as a Potential Player in Autism Spectrum Disorder

Krzysztof J. Szkop, Peter I. C. Cooke, Joanne A. Humphries, Viktoria Kalna, David S. Moss, Eugene F. Schuster and Irene Nobeli

Frontiers in Molecular Neuroscience, doi:10.3389/fnmol.2017.00279

We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3′ untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3′ UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3′ sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain.

Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina