Oligo dT Research Articles

Concise Affinity-Based Purification of Ligated mRNA for Structure-Activity Relationship Studies ofNucleosugar Modification Patterns.

Position-specific nucleoside sugar modifications have been shown to improve the translational activity and stability of chemically synthesized mRNA. For pharmaceutical applications of chemically modified mRNA, a rapid purification methodology is imperative to identify the optimal modification pattern. However, while the chemical synthesis of mRNAs can be accomplished by splint ligation of oligonucleotide fragments, the current purification method for ligated mRNAs based on denaturing polyacrylamide gel electrophoresis tends to be time consuming. In this study, we developed a two-step affinity purification method for rapid sample preparation. In this method, ligated mRNA is captured by oligo dT magnetic beads and streptavidin magnetic beads with 3'-biotinylated oligo DNA, which are complementary to the 3'-poly(A) and 5' terminal sequences of the target mRNA, respectively. Therefore, the target mRNA can be isolated from a complex mixture of splint ligations. Using this method, six sugar-modified mRNAs were simultaneously purified, and the translational activities of these mRNAs were evaluated immediately after purification. The results demonstrate that this methodology is suitable for the rapid preparation of various chemically synthesized mRNAs to identify their optimal modification patterns.

First report of lily mottle virus naturally infecting lily (Lilium asiatica) in Texas, USA.

Lily (Lilium asiatica) is an important plant grown for its range of flower colors and heavy scent. In March 2024, potyvirus-like symptoms consisting of light-yellow mottling and mosaic were noticed on 12/20 lily plants in a private property in Weslaco, Hidalgo County, Texas. Two symptomatic plants (WTX1 and WTX2) were sampled randomly for virus diagnosis. The leaf extracts of both samples were negative for the potyvirus group using Agdia's Poty ImmunoStrip® (Agdia, Inc., Elkhart, IN, USA). However, rub-inoculation of the extracts onto healthy Nicotiana benthamiana and Vigna unguiculata plants (n=4, each) induced mild mottle symptoms on the systemic leaves of both herbaceous test plants 20 to 28 days post inoculation, indicating the presence of a mechanically transmissible agent in the samples. No virus-like symptoms were observed on the mock-inoculated plants (n=1, each) of both species. To test for suspected potyvirus infection, 2-µg of total nucleic acid extracts (Dellaporta et al. 1983) from WTX1 and WTX2 were used for complimentary DNA (cDNA) synthesis with Oligo(dT) primer and the PrimeScript 1st strand cDNA synthesis kit (Takara Bio, USA). One microliter aliquot of each cDNA served as template in 12.5-µl conventional PCR reaction volumes with 5 U Taq polymerase (Roche Diagnostics, Indianapolis, IN), and two pairs of degenerate primers targeting the partial cylindrical inclusion (CI) gene and the helper component protease (HC-Pro) of potyviruses (Ha et al. 2008). The expected ~700-bp DNA product of each gene target was amplified from both samples. The amplicons were excised, gel eluted (Zymoclean™ Gel DNA Recovery kit) and cloned individually into the pJET1.2/Blunt vector (Life Technologies). Recombinant plasmids from two transformed Escherichia coli cells per cloned DNA insert were Sanger sequenced. BLASTn analysis of each gene-specific nucleotide (nt) sequence fragment revealed their identities (83 to 92 % nt) as lily mottle virus (LMoV; genus Potyvirus) at 93 to 98% query coverage. In pairwise comparison, the obtained sequences of LMoV isolates from TX specific to the CI (GenBank accession nos. PQ037260-61) and the HC-Pro (PQ037262-63) shared ~99% nt and 100% amino acid (aa) identities with each other and ~91-92% nt and 98-100% aa identities with the closest isolate, Bate5 (JN127341) from Australia. Based on these results, a pair of LMoV-specific primers (LM_v109-F: 5'-GGCCAGTAATGTGCACAAGC-3' and LM_c527-R: 5'-TCGCTGTAGCTAGCGACGTAC-3') was newly designed to target the partial CI gene, internal to the 700-bp fragment obtained above with the generic primers. The primer pair was used to screen each of the mechanically inoculated test plants by RT-PCR as previously described above. The expected 438-bp fragment of the LMoV CI gene was amplified from all the inoculated plants of both N. benthamiana and V. unguiculata; the results were confirmed by Sanger sequencing as described above. The mock-inoculated plant of each experimental host plant tested negative for LMoV. LMoV is a quarantine pest and was previously reported in the U.S. (Brierley and Smith, 1944) and other lily-producing parts of the world, such as the Netherlands, China, Korea, and Brazil. To our knowledge, this is the first confirmed report of the virus in Texas, thus expanding its geographical range. Testing of lily foundation stocks before propagation is essential to prevent further spread of LMoV via planting material.

RAG-seq: A NSR Primed and Transposase Tagmentation Mediated Strand-specific Total RNA Sequencing in Single Cell.

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular diversity with unprecedented resolution. However, many current methods are limited in capturing full-length transcripts and discerning strand orientation. We present RAG-seq, an innovative strand-specific total RNA sequencing technique that combines not-so-random (NSR) primers with Tn5 transposase-mediated tagmentation. RAG-seq overcomes previous limitations by delivering comprehensive transcript coverage and maintaining strand orientation, which is essential for accurate quantification of overlapping genes and detection of antisense transcripts. Through optimized reverse transcription with oligo dT primers, rRNA depletion via Depletion of Abundant Sequences by Hybridization (DASH), and linear amplification, RAG-seq enhances sensitivity and reproducibility, especially for low-input samples and single cells. Application to mouse oocytes and early embryos highlights RAG-seq's superior performance in identifying stage-specific antisense transcripts, shedding light on their regulatory roles during early development. This advancement represents a significant leap in transcriptome analysis within complex biological contexts.

CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5’ capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5’cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences.

Revealing the Arabidopsis AtGRP7 mRNA binding proteome by specific enhanced RNA interactome capture

BackgroundThe interaction of proteins with RNA in the cell is crucial to orchestrate all steps of RNA processing. RNA interactome capture (RIC) techniques have been implemented to catalogue RNA- binding proteins in the cell. In RIC, RNA-protein complexes are stabilized by UV crosslinking in vivo. Polyadenylated RNAs and associated proteins are pulled down from cell lysates using oligo(dT) beads and the RNA-binding proteome is identified by quantitative mass spectrometry. However, insights into the RNA-binding proteome of a single RNA that would yield mechanistic information on how RNA expression patterns are orchestrated, are scarce.ResultsHere, we explored RIC in Arabidopsis to identify proteins interacting with a single mRNA, using the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) transcript, one of the most abundant transcripts in Arabidopsis, as a showcase. Seedlings were treated with UV light to covalently crosslink RNA and proteins. The AtGRP7 transcript was captured from cell lysates with antisense oligonucleotides directed against the 5’untranslated region (UTR). The efficiency of RNA capture was greatly improved by using locked nucleic acid (LNA)/DNA oligonucleotides, as done in the enhanced RIC protocol. Furthermore, performing a tandem capture with two rounds of pulldown with the 5’UTR oligonucleotide increased the yield. In total, we identified 356 proteins enriched relative to a pulldown from atgrp7 mutant plants. These were benchmarked against proteins pulled down from nuclear lysates by AtGRP7 in vitro transcripts immobilized on beads. Among the proteins validated by in vitro interaction we found the family of Acetylation Lowers Binding Affinity (ALBA) proteins. Interaction of ALBA4 with the AtGRP7 RNA was independently validated via individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP). The expression of the AtGRP7 transcript in an alba loss-of-function mutant was slightly changed compared to wild-type, demonstrating the functional relevance of the interaction.ConclusionWe adapted specific RNA interactome capture with LNA/DNA oligonucleotides for use in plants using AtGRP7 as a showcase. We anticipate that with further optimization and up scaling the protocol should be applicable for less abundant transcripts.

Comparison of RNA-Sequencing Methods for Degraded RNA.

RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.

Abstract 7336: Exploring the impact of cancer predisposition gene variants by functional analysis and whole genome sequencing analysis in hereditary cancer patients

Abstract Background: Hereditary cancer confirmed by germline pathogenic variant has been increased with increasing next generation sequencing (NGS) panel tests in recent years. However, some cases who were suspected hereditary cancer represented having variant of uncertain significance (VUS) or no variants even though they had family members with many cancers. The aim of this study was to investigate variants function especially in splicing variants and to explore other candidate genes by whole genome sequencing (WGS) in suspected hereditary cancer patients. Methods: Total of 44 patients who clinically suspected hereditary cancer without defined pathogenic variants after NGS panel test were enrolled. Among them 11 patients had intronic variants and their variant effects were predicted by SpliceAI, a deep learning-based tool. To define alternative splicing, total RNA was extracted from patients buffy coats and cDNA was synthesized using oligo dT. Then the cDNA was amplified by specific primers and performed sequencing. Also, WGS was performed in 15 patients then comprehensive analysis encompassing 392 unique genes was performed including 282 well-established hereditary cancer predisposition genes and 228 DNA repair-related genes. We confirmed the variant by various type of studies and predicted impact on protein by analysis. Results: We analyzed intronic variants of 11 patients; ATM c.4611+3A>T(n=1). BRCA1 c.547+30A>G (n=2), BRCA1 c.5152+6T>C(n=1), MSH2 c.2635-7G>A(n=1), MUTYH c.934-2A>G(n=2), NF1 c.1527+4A>C(n=1), NF1 c.3871-14G>T(n=1), PALB2 c.3350+5G>A(n=1), and SMARCA4 c.3546+1G>A(n=1). Though two intronic variants (ATM c.4611+3A>T and SMARCA4:c.3546+1G>A) were observed a likelihood of aberrant splicing aggregate SpliceAI score more than 0.8, we observed three intronic variants induce alternative splicing (ATM c.4611+3A>T, NF1 c.1527+4A>C, and PALB2 c.3350+5G>A).In addition, WGS analysis demonstrated 6 out of 15 patients have germline variants in relevant genes. Four single nucleotide variants (SMARCAL1:c.1632delA, SETMAR:c.441dup, FAN1:c.590C>A, and PRF1:c.445G>A) were validated by sequencing and those were likely pathogenic variant (LPV) by ACMG/AMP criteria of ClinGen. Copy number variations were identified in 2 patients (PMS2 heterozygous deletion and RAD50 duplication) and structure variant of MSH2 was identified in one patient. Conclusion: Our analysis showed in silico analysis results might be different from functional analysis and rare variants in cancer predisposition genes could be confirmed WGS. However, large number of patients study would be needed to confirm necessity of WGS for hereditary cancer. This work was supported by National Cancer Center, Korea (No_NCC-2110181). Citation Format: Hyeji Kim, Jong Eun Park, Gi Yeon Lee, Jung-Ah Hwang, Jin-Sun Ryu, Ye-Ryeong Jung, Seeyoun Lee, Kum Hei Ryu, Dong Ock Lee, So-Youn Jung, Sang-Yoon Park, Wonyoung Choi, Sun-Young Kong. Exploring the impact of cancer predisposition gene variants by functional analysis and whole genome sequencing analysis in hereditary cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7336.

CircNUP50 is a novel therapeutic target that promotes cisplatin resistance in ovarian cancer by modulating p53 ubiquitination

BackgroundMost patients with ovarian cancer (OC) treated with platinum-based chemotherapy have a dismal prognosis owing to drug resistance. However, the regulatory mechanisms of circular RNA (circRNA) and p53 ubiquitination are unknown in platinum-resistant OC. We aimed to identify circRNAs associated with platinum-resistant OC to develop a novel treatment strategy.MethodsPlatinum-resistant circRNAs were screened through circRNA sequencing and validated using quantitative reverse-transcription PCR in OC cells and tissues. The characteristics of circNUP50 were analysed using Sanger sequencing, oligo (dT) primers, ribonuclease R and fluorescence in situ hybridisation assays. Functional experimental studies were performed in vitro and in vivo. The mechanism underlying circNUP50-mediated P53 ubiquitination was investigated through circRNA pull-down analysis and mass spectrometry, luciferase reporters, RNA binding protein immunoprecipitation, immunofluorescence assays, cycloheximide chase assays, and ubiquitination experiments. Finally, a platinum and si-circNUP50 co-delivery nanosystem (Psc@DPP) was constructed to treat platinum-resistant OC in an orthotopic animal model.ResultsWe found that circNUP50 contributes to platinum-resistant conditions in OC by promoting cell proliferation, affecting the cell cycle, and reducing apoptosis. The si-circNUP50 mRNA sequencing and circRNA pull-down analysis showed that circNUP50 mediates platinum resistance in OC by binding p53 and UBE2T, accelerating p53 ubiquitination. By contrast, miRNA sequencing and circRNA pull-down experiments indicated that circNUP50 could serve as a sponge for miR-197-3p, thereby upregulating G3BP1 to mediate p53 ubiquitination, promoting OC platinum resistance. Psc@DPP effectively overcame platinum resistance in an OC tumour model and provided a novel idea for treating platinum-resistant OC using si-circNUP50.ConclusionsThis study reveals a novel molecular mechanism by which circNUP50 mediates platinum resistance in OC by modulating p53 ubiquitination and provides new insights for developing effective therapeutic strategies for platinum resistance in OC.Graphical

Enhanced adsorption performance of varying-length mRNA on Oligo dT affinity resins through optimal pore size and grafting

The enormous potential of mRNA-based therapeutics in vaccines and medicine has led to the consideration of various mRNA sizes, ranging from ∼1000 to 10,000 nucleotides (nt). However, the commonly used Oligo-deoxythymidine (Oligo dT) affinity resins are not initially designed for such a wide range of sizes, and present challenges in achieving optimal performance for the separation of a given mRNA. In this paper, two factors of the resin beads, pore size and grafting were focused. The effects of varying pore sizes, ranging from 150 to 850 nm, on both static and dynamic adsorption for varying-length mRNA (1000–8500 nt) were investigated. It was found that static adsorption capacity and uptake kinetics were correlated with the mRNA length. Interestingly, dynamic binding capacities (DBCs) showed an increase-then-decrease trend with increasing pore size. An optimal pore size of about 350 nm could lead to the highest DBCs for all tested mRNA. Additionally, the effects of polymer grafting on the adsorption performance were studied. The results revealed that polymer-grafted resin exhibited 24–55% higher DBCs than non-grafted resin. The enhanced DBCs could be attributed to the improved uptake rate facilitated by the polymer grafted. These investigations emphasize the importance of optimizing pore size and grafting for Oligo dT affinity resins, providing valuable guidance for selecting and designing new resins for different mRNA products.

Global Identification of RNA-Binding Proteins in Bacteria.

RNA-binding proteins (RBPs) are at the heart of many biological processes and are therefore essential for cellular life. Following identification of single RBPs by classical genetics and molecular biology methods, approaches for RBP discovery on a systems level have recently emerged. For instance, RNA interactome capture (RIC) enables the global purification of RBPs cross-linked to polyadenylated RNA using oligo(dT) probes. RIC was originally developed for eukaryotic organisms but was recently established for capturing RBPs in bacteria. In this chapter, we provide a detailed step-by-step protocol for performing RIC in bacteria. The protocol is based on its application to Escherichia coli but should be amenable for charting other genetically tractable bacterial species.

Magnetic particle separation using current-carrying plates: A novel geometry in magnetophoresis

This paper introduces a novel system designed for the separation and trapping of micro-magnetic particles in a microfluidic setup. The system employs two triangular-shaped current-carrying plates with different electrical currents beneath the microchannel to create a non-uniform magnetic field. This magnetic field efficiently separates and traps the magnetic particles, guiding them along distinct paths within the channel. The study includes a parametric analysis involving electrical current, particle size, magnetic susceptibility of particles, and carrier fluid, in order to identify optimal parameters for achieving maximum efficiency. Specifically, M-450 and Oligo(dT)25 magnetic particles are collected using current-carrying plates with electric currents of 50[mA] and 150[mA], respectively. Moreover, the system's efficiency is further boosted by setting the fluid velocity to 2[μm/s], leading to 99% efficiency in collecting and separating different types of particles. The proposed microchip demonstrates its versatility by enabling the trapping and separation of various particles, with the flexibility to adjust the electrical current value accordingly.

Critical view on oligo(dT)-based RNA-seq: bias arising, modeling, and mitigating.

The precise biological interpretation of oligo(dT)-based RNA sequencing (RNA-seq) datasets, particularly in single-cell RNA-seq (scRNA-seq), is invaluable for understanding complex biological systems. However, the presence of biases can lead to misleading results in downstream analysis. This study has now identified two additional biases that are not accounted for in established bias models: poly(A)-tail length bias and fixed-position GC-content bias. These biases have a significant negative impact on the overall quality of oligo(dT)-based RNA-seq data. To address these biases, we have developed a universal bias-mitigating method based on the lower-affinity binding of short and nonanchored oligo(dT) primers to poly(A) tails. This method significantly reduces poly(A) length bias and completely eliminates fixed-position GC bias. Furthermore, the use of short oligo(dT) with impartial binding behavior toward the diverse poly(A) tails renders RNA-seq with more reliable measurements. The findings of this study are particularly beneficial for scRNA-seq datasets, where accurate benchmarking is critical.

An RNA-Ligation-Based RACE-PAT Assay to Monitor Poly(A) Tail Length of mRNAs of Interest.

In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes during the lifetime of mRNAs and has been shown in many circumstances to be an important factor controlling transcript fates. Yet, the measure of the length of this homogenous nucleotide sequence is technically challenging, making it difficult to assess its dynamic variation. In this chapter, we describe an RNA-ligation-based RACE-PAT (Rapid Amplification of cDNA End-Poly(A) Tail) assay to monitor the poly(A) tail length of mRNAs. In the first step, an RNA oligonucleotide is ligated to mRNA 3' ends providing an anchoring site to prime cDNA synthesis, avoiding the bias introduced by oligo(dT)-derived primers. Afterward, reverse transcription is performed with an anchor primer with a unique 5' extension. The choice of the oligonucleotide 3' end at this step allows further flexibility to amplify modified tails, for example, by uridylation. Next, short DNA fragments encompassing the poly(A) tails are amplified by Polymerase Chain Reaction (PCR) using as forward primer, a transcript-specific primer hybridizing close to the transcript polyadenylation signal, and as reverse primer, an oligonucleotide corresponding to the 5' extension of the primer used for cDNA synthesis, ensuring that only cDNAs are amplified. The resulting DNA fragments are then visualized after size fractionation by electrophoresis. This method does not provide exact nucleotide count and composition but has the advantage of allowing the processing of many samples in parallel at a low cost.

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

CLT-seq as a universal homopolymer-sequencing concept reveals poly(A)-tail-tuned ncRNA regulation.

Dynamic tuning of the poly(A) tail is a crucial mechanism for controlling translation and stability of eukaryotic mRNA. Achieving a comprehensive understanding of how this regulation occurs requires unbiased abundance quantification of poly(A)-tail transcripts and simple poly(A)-length measurement using high-throughput sequencing platforms. Current methods have limitations due to complicated setups and elaborate library preparation plans. To address this, we introduce central limit theorem (CLT)-managed RNA-seq (CLT-seq), a simple and straightforward homopolymer-sequencing method. In CLT-seq, an anchor-free oligo(dT) primer rapidly binds to and unbinds from anywhere along the poly(A) tail string, leading to position-directed reverse transcription with equal probability. The CLT mechanism enables the synthesized poly(T) lengths, which correspond to the templated segment of the poly(A) tail, to distribute normally. Based on a well-fitted pseudogaussian-derived poly(A)-poly(T) conversion model, the actual poly(A)-tail profile is reconstructed from the acquired poly(T)-length profile through matrix operations. CLT-seq follows a simple procedure without requiring RNA-related pre-treatment, enrichment or selection, and the CLT-shortened poly(T) stretches are more compatible with existing sequencing platforms. This proof-of-concept approach facilitates direct homopolymer base-calling and features unbiased RNA-seq. Therefore, CLT-seq provides unbiased, robust and cost-efficient transcriptome-wide poly(A)-tail profiling. We demonstrate that CLT-seq on the most common Illumina platform delivers reliable poly(A)-tail profiling at a transcriptome-wide scale in human cellular contexts. We find that the poly(A)-tail-tuned ncRNA regulation undergoes a dynamic, complex process similar to mRNA regulation. Overall, CLT-seq offers a simplified, effective and economical approach to investigate poly(A)-tail regulation, with potential implications for understanding gene expression and identifying therapeutic targets.

Chemoproteomic Approaches to Studying RNA Modification-Associated Proteins.

The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for N6-methyladenosine (m6A) and N1-methyladenosine (m1A) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both m6A and m1A to mediate transcript destabilization. Furthermore, m6A also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.

Characterization of a novel psyllid-transmitted waikavirus in carrots

Carrots collected from the Western Negev region in Israel during the winter of 2019 showed disease symptoms of chlorosis, leaf curling, a loss of apical dominance, and multiple lateral roots that were not associated with known pathogens of the carrot yellows disease. Symptomatic carrots were studied for a possible involvement of plant viruses in disease manifestations using high throughput sequencing analyses. The results revealed the presence of a waikavirus, sharing a ∼70% nucleotide sequence identity with Waikavirus genus members. Virions purified from waikavirus-positive carrots were visualized by transmission electron microscopy, showing icosahedral particle diameter of ∼28 nm. The genome sequence was validated by overlapping amplicons by designed 12 primer sets. A complete genome sequence was achieved by rapid amplification of cDNA ends (RACE) for sequencing the 5′ end, and RT-PCR with oligo dT for sequencing the 3′ end. The genome encodes a single large ORF, characteristic of waikaviruses. Aligning the waikavirus-deduced amino-acid sequence with other waikavirus species at the Pro-Pol region, a conserved sequence between the putative proteinase and the RNA-dependent RNA polymerase, showed a ∼40% identity, indicating the identification of a new waikavirus species. The amino-acid sequence of the three coat proteins and cleavage sites were experimentally determined by liquid chromatography-mass spectrometry. A phylogenetic analysis based on the Pro-Pol region revealed that the new waikavirus clusters with persimmon waikavirus and actinidia yellowing virus 1. The new waikavirus genome was localized in the phloem of waikavirus-infected carrots. The virus was transmitted to carrot and coriander plants by the psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae).

Simultaneous separation of different magnetic particles by sputtering magnetic wires at the bottom of a microchip: Novel geometry in magnetophoresis

In this paper, sputtered magnetic electrodes at the bottom of the microchannel is used to separate different magnetic particles in a microfluidic device, simultaneously. The proposed design consists of a microchannel with a length of 30 mm with three different outlets. The Nickel electrodes with different angles and thicknesses are used to separate different particles. A permanent magnet is used to produce a uniform magnetic field and the presence of the magnetic wires disturbs the uniformity of the magnetic field which leads to magnetic force applied to the magnetic particles in the channel’s environment. In order to validate the results, numerical solution was compared with analytical relations. Important parameters such as microparticle size and characteristic (M−450, Myone, Oligo (dT)25), wire dimensions, wire spacing, wire angle and the flow rate were analyzed. The results show that M−450 particles are affected by greater force in comparison to Myone and Oligo (dT)25 microparticles due to their higher magnetism property and size. It was shown that by increasing the angle of the sputtered wires (at the bottom of the channel), the particle deviation was reduced. Also, the effect of wire thickness of the electrodes is investigated for thicknesses of 10, 15 and 20 μm. The maximum deviation was related to the thickness of 20 μm, thus, the particles were not able to pass off over the electrodes and moves along the wire path. By increasing the distance between the wires from 400 μm to 700 μm, the microparticle deviation was increased. As the flow rate of the inlet fluid increases (from 25 to 75 mm/s), the hydrodynamic force on the microparticles increases and therefore, the particle deviation decreases when crossing off over the electrodes. It is observed that for speed of 50 mm/s, the best separation efficiency (94%) was obtained for the proposed microchip design.

RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins.

RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.

Circ_0007706 downregulation ameliorates neonatal hypoxic ischemic encephalopathy via regulating the miR-579-3p/TRAF6 axis

BackgroundNeonatal hypoxic ischemic encephalopathy (HIE) is a main factor of neonatal death and permanent neurologic deficit. This study sought to investigate the functional role of hsa_circ_0007706 (circ_0007706) in modulating neonatal HIE. MethodsIn vitro HIE cell model was established in hBMVECs under the condition of oxygen‑glucose deprivation/reperfusion (OGD/R) treatment. qRT-PCR analysis was utilized for detecting the level of circ_0007706, microRNA-579–3p (miR-579–3p) and TNF receptor-associated factor 6 (TRAF6). RNase R treatment and Oligo (dT) 18 primers were employed to verify the features of circ_0007706, and nucleocytoplasmic separation was conducted for determining the location of circ_0007706. CCK-8 assay, EdU assay, and flow cytometry were carried out to measure cell proliferation and apoptosis, respectively. The protein expression of Bax, Bcl-2 and TRAF6 was detected using western blot. Meanwhile, the levels of the pro-inflammatory factors were determined via ELISA. SOD activity and MDA level were assessed via the respective kits. Besides, dual-luciferase reporter assay and RNA pull-down were used to identify the association between miR-579–3p and circ_0007706 or TRAF6. ResultsCirc_0007706 was elevated in HIE newborns and OGD/R cell model. Knockdown of circ_0007706 greatly alleviated OGD/R-induced injury, inflammatory response and oxidative stress. We found that miR-579–3p was a direct target of circ_0007706, and miR-579–3p inhibitor could reverse the impact of circ_0007706 knockdown on OGD/R-caused cell damage in hBMVECs. In addition, miR-579–3p directly interacted with TRAF6, and the protective effects of miR-579–3p on OGD/R-induced injury in hBMVECs were harbored by TRAF6 overexpression. Our data indicated that circ_0007706 knockdown could downregulate the expression of TRAF6 by sponging miR-579–3p in OGD/R-treated hBMVECs. ConclusionThis study demonstrated that circ_0007706 knockdown assuaged HIE-induced injury by decreasing TRAF6 expression via targeting miR-579–3p.