Alphaviruses are positive-sense RNA arboviruses that can cause either a chronic arthritis or a potentially lethal encephalitis. Like other RNA viruses, alphaviruses produce truncated, defective genomes featuring large deletions during replication. Defective RNAs (D-RNAs) have primarily been isolated from virions after high-multiplicity of infection passaging. Here, we aimed to characterize both intracellular and packaged viral D-RNA populations during early passage infections under the hypothesis that D-RNAs arise de novo intracellularly that may not be packaged and thus have remained undetected. To this end, we generated NGS libraries using RNA derived from passage 1 (P1) stock chikungunya virus (CHIKV) 181/clone 25, intracellular virus, and encapsidated P2 virus and analyzed samples for D-RNA expression, followed by diversity and differential expression analyses. We found that the diversity of D-RNA species is significantly higher for intracellular D-RNA populations than encapsidated and specific populations of D-RNAs are differentially expressed between intracellular and encapsidated compartments. Importantly, these trends were likewise observed in a murine model of CHIKV 15561 infection, as well as in vitro studies using related Mayaro, Sindbis, and Aura viruses. Additionally, we identified a novel subtype of subgenomic D-RNA that are conserved across arthritogenic alphaviruses. D-RNAs specific to intracellular populations were defined by recombination events specifically in the subgenomic region, which was confirmed by direct RNA nanopore sequencing of intracellular CHIKV RNAs. Together, these studies show that only a portion of D-RNAs generated intracellularly are packaged and D-RNAs readily arise de novo in the absence of transmitted template.

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

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

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

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

Results

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

Background

In cystic fibrosis (CF), impaired immune cell responses, driven by the dysfunctional CF transmembrane conductance regulator (CFTR) gene, may determine the disease severity but clinical heterogeneity remains a major therapeutic challenge. The characterization of molecular mechanisms underlying impaired immune responses in CF may reveal novel targets with therapeutic potential. Therefore, we utilized simultaneous RNA sequencing targeted at identifying differentially expressed genes, transcripts, and miRNAs that characterize impaired immune responses triggered by CF and its phenotypes.

Methods

Peripheral blood mononuclear cells (PBMCs) extracted from a healthy donor were stimulated with plasma from CF patients (n = 9) and healthy controls (n = 3). The PBMCs were cultured (1 × 10⁵ cells/well) for 9 h at 37 ° C in 5% CO₂. After culture, total RNA was extracted from each sample and used for simultaneous total RNA and miRNA sequencing.

Results

Analysis of expression signatures from peripheral blood mononuclear cells induced by plasma of CF patients and healthy controls identified 151 genes, 154 individual transcripts, and 41 miRNAs differentially expressed in CF compared to HC while the expression signatures of 285 genes, 241 individual transcripts, and seven miRNAs differed due to CF phenotypes. Top immune pathways influenced by CF included agranulocyte adhesion, diapedesis signaling, and IL17 signaling, while those influenced by CF phenotypes included natural killer cell signaling and PI3K signaling in B lymphocytes. Upstream regulator analysis indicated dysregulation of CCL5, NF-κB and IL1A due to CF while dysregulation of TREM1 and TP53 regulators were associated with CF phenotype. Five miRNAs showed inverse expression patterns with three target genes relevant in CF-associated impaired immune pathways while two miRNAs showed inverse expression patterns with two target genes relevant to a dysregulated immune pathway associated with CF phenotypes.

Conclusions

Our results indicate that miRNAs and individual transcript variants are relevant molecular targets contributing to impaired immune cell responses in CF.

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

Viral and cellular double-stranded RNA (dsRNA) is recognized by cytosolic innate immune sensors, including RIG-I-like receptors. Some cytoplasmic dsRNA is commonly present in cells, and one source is mitochondrial dsRNA, which results from bidirectional transcription of mitochondrial DNA (mtDNA). Here we demonstrate that Trp53 mutant mouse embryonic fibroblasts contain immune-stimulating endogenous dsRNA of mitochondrial origin. We show that the immune response induced by this dsRNA is mediated via RIG-I-like receptors and leads to the expression of type I interferon and proinflammatory cytokine genes. The mitochondrial dsRNA is cleaved by RNase L, which cleaves all cellular RNA including mitochondrial mRNAs, increasing activation of RIG-I-like receptors. When mitochondrial transcription is interrupted there is a subsequent decrease in this immune-stimulatory dsRNA. Our results reveal that the role of p53 in innate immunity is even more versatile and complex than previously anticipated. Our study, therefore, sheds new light on the role of endogenous RNA in diseases featuring aberrant immune responses.

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

Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a noncycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous population and perform RNA sequencing to delineate the transcriptome underlying the slow-cycling state. We show that cellular stress responses—the p53 transcriptional response and the integrated stress response (ISR)—are the most salient causes of spontaneous entry into the slow-cycling state. Finally, we show that cells’ ability to enter the slow-cycling state enhances their survival in stressful conditions. Thus, the slow-cycling state is hardwired to stress responses to promote cellular survival in unpredictable environments.

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

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

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

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

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

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

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

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

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

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

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

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

SIGNIFICANCE STATEMENT

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

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

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1’s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

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

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

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

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

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

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

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