QuantSeq 3’ mRNA-Seq REV for Illumina
Inhibition of cytoplasmic cap methylation identifies 5′ TOP mRNAs as recapping targets and reveals recapping sites downstream of native 5′ ends
Daniel del Valle Morales, Jackson B Trotman, Ralf Bundschuh, Daniel R Schoenberg
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
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)
A transcriptome-wide antitermination mechanism sustaining identity of embryonic stem cells
Yaroslav A. Kainov & Eugene V. Makeyev
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
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
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
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
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 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
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
Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs
Margaret Rose Davis, Mildred Delaleau, Katherine L.B. Borden
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
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
Niclosamide Induces Epiboly Delay During Early Zebrafish Embryogenesis
Sara M Vliet, Subham Dasgupta, David C Volz
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
Transcriptomic data of Arabidopsis hypocotyl overexpressing a heterologous CsEXPA1 gene
Iqmal Asyraf Ilias, Othman Babul Airianah, Syarul Nataqain Baharum, Hoe-Han Goh
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
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and SPLIT RNA Extraction Kit
Transcriptome analysis of Carica papaya embryogenic callus upon De-etiolated 1 (DET1) gene suppression
Diyana Jamaluddin, Normah Mohd Noor, Hoe-Han Goh
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
Transcriptomic data of Arabidopsis thaliana hypocotyl upon suppression of expansin genes
Iqmal Asyraf Ilias, Othman Babul Airianah, Syarul Nataqain Baharum, Hoe-Han Goha
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
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
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
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina
Genome-wide transcriptome profiling of Carica papaya L. embryogenic callus
Jamaluddin, N.D., Mohd Noor, N. & Goh, HH.
Physiol Mol Biol Plants (2017). doi:10.1007/s12298-017-0429-8
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
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina