RNA Fragments Research Articles

Mapping protein-RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2).

Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA-protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events ( https://github.com/malewins/Plant-iCLIPseq ), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites ( https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html ).

Role of miRNA in Cardiovascular Diseases in Children-Systematic Review.

The number of children suffering from cardiovascular diseases (CVDs) is rising globally. Therefore, there is an urgent need to acquire a better understanding of the genetic factors and molecular mechanisms related to the pathogenesis of CVDs in order to develop new prevention and treatment strategies for the future. MicroRNAs (miRNAs) constitute a class of small non-coding RNA fragments that range from 17 to 25 nucleotides in length and play an essential role in regulating gene expression, controlling an abundance of biological aspects of cell life, such as proliferation, differentiation, and apoptosis, thus affecting immune response, stem cell growth, ageing and haematopoiesis. In recent years, the concept of miRNAs as diagnostic markers allowing discrimination between healthy individuals and those affected by CVDs entered the purview of academic debate. In this review, we aimed to systematise available information regarding miRNAs associated with arrhythmias, cardiomyopathies, myocarditis and congenital heart diseases in children. We focused on the targeted genes and metabolic pathways influenced by those particular miRNAs, and finally, tried to determine the future of miRNAs as novel biomarkers of CVD.

Timely Monitoring of SARS-CoV-2 RNA Fragments in Wastewater Shows the Emergence of JN.1 (BA.2.86.1.1, Clade 23I) in Berlin, Germany.

The importance of COVID-19 surveillance from wastewater continues to grow since case-based surveillance in the general population has been scaled back world-wide. In Berlin, Germany, quantitative and genomic wastewater monitoring for SARS-CoV-2 is performed in three wastewater treatment plants (WWTP) covering 84% of the population since December 2021. The SARS-CoV-2 Omicron sublineage JN.1 (B.2.86.1.1), was first identified from wastewater on 22 October 2023 and rapidly became the dominant sublineage. This change was accompanied by a parallel and still ongoing increase in the notification-based 7-day-hospitalization incidence of COVID-19 and COVID-19 ICU utilization, indicating increasing COVID-19 activity in the (hospital-prone) population and a higher strain on the healthcare system. In retrospect, unique mutations of JN.1 could be identified in wastewater as early as September 2023 but were of unknown relevance at the time. The timely detection of new sublineages in wastewater therefore depends on the availability of new sequences from GISAID and updates to Pango lineage definitions and Nextclade. We show that genomic wastewater surveillance provides timely public health evidence on a regional level, complementing the existing indicators.

DsRNA sequencing revealed a previously missed terminal sequence of a +ssRNA virus that infects dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5' terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5' Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Synthesis of short DNA and RNA fragments by resonant acoustic mixing (RAM)

We demonstrate the first use of Resonant Acoustic Mixing (RAM) without bulk solvent for the synthesis of short oligonucleotide fragments.

A high-fidelity DNAzyme-assisted CRISPR/Cas13a system with single-nucleotide resolved specificity.

A CRISPR/Cas system represents an innovative tool for developing a new-generation biosensing and diagnostic strategy. However, the off-target issue (i.e., mistaken cleavage of nucleic acid targets and reporters) remains a great challenge for its practical applications. We hypothesize that this issue can be overcome by taking advantage of the site-specific cleavage ability of RNA-cleaving DNAzymes. To test this idea, we propose a DNAzyme Operation Enhances the Specificity of CRISPR/Cas13a strategy (termed DOES-CRISPR) to overcome the problem of relatively poor specificity that is typical of the traditional CRISPR/Cas13a system. The key to the design is that the partial hybridization of the CRISPR RNA (crRNA) with the cleavage fragment of off-target RNA was not able to activate the collateral cleavage activity of Cas13a. We showed that DOES-CRISPR can significantly improve the specificity of traditional CRISPR/Cas13a-based molecular detection by up to ∼43-fold. The broad utility of the strategy is illustrated through engineering three different systems for the detection of microRNAs (miR-17 and let-7e), CYP2C19*17 gene, SARS-Cov-2 variants (Gamma, Delta, and Omicron) and Omicron subtypes (BQ.1 and XBB.1) with single-nucleotide resolved specificity. Finally, clinical evaluation of this assay using 10 patient blood samples demonstrated a clinical sensitivity of 100% and specificity of 100% for genotyping CYP2C19*17, and analyzing 20 throat swab samples provided a diagnostic sensitivity of 95% and specificity of 100% for Omicron detection, and a clinical sensitivity of 92% and specificity of 100% for XBB.1 detection.

In Silico Identification of tRNA Fragments, Novel Candidates for Cancer Biomarkers, and Therapeutic Targets.

The identification of a wide variety of RNA molecules using high-throughput sequencing techniques in the transcriptome pool of living organisms has revealed hidden regulatory insights in the cell. The class of non-coding RNA fragments produced from transfer RNA, or tRFs, is one such example. They are heterogeneously sized molecules with lengths ranging between 15 and 50nt. They have a history of being dysregulated in human malignancies and other illnesses. The detection of these molecules has been made easier by a variety of bioinformatics techniques. The various types of tRFs and how they relate to cancer are covered in this chapter. It also provides a summary of the biological significance of tRFs reported in human cancer. Additionally, it emphasizes the utilities of databases and computational tools that have been created by different research teams for the investigation of tRFs. This will further aid the exploration and analysis of tRFs in cancer research and will support future advancement and a better comprehension of these molecules.

New plasma diagnostic markers for colorectal cancer: transporter fragments of glutamate tRNA origin.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Early diagnosis of the disease can greatly improve the clinical prognosis for patients with CRC. Unfortunately, there are no current simple and effective early diagnostic markers available. The transfer RNA (tRNA)-derived RNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs), which have been shown to play an important role in the development and prognosis of CRC. However, only a few studies on tRFs as early diagnostic markers in CRC have been conducted. In this study, previously ignored tRFs expression data were extracted from six paired small RNA sequencing data in the Sequence Read Archive (SRA) database using MINTmap. Three i-tRFs, derived from the tRNA that transports glutamate (i-tRF-Glu), were identified and used to construct a random forest diagnostic model. The model performance was evaluated using the receiver operating characteristic (ROC) curve and precision-recall (PR) curve. The area under the curves (AUC) for the ROC and PR was 0.941 and 0.944, respectively. We further verified the differences in expression of the these i-tRF-Glu in the tissue and plasma of both CRC patients and healthy subjects using quantitative real-time PCR (qRT-PCR). We found that the ROC-AUC of the three was greater than traditional plasma tumor markers such as CEA and CA199. Our bioinformatics analysis suggested that the these i-tRF-Glu are associated with cancer development and glutamate (Glu)-glutamine (Gln) metabolism. Overall, our study uncovered these i-tRF-Glu that have early diagnostic significance and therapeutic potential for CRC, this warrants further investigation into the diagnostic and therapeutic potential of these i-tRF-Glu in CRC.

A novel viral RNA detection method based on the combined use of trans-acting ribozymes and HCR-FRET analyses.

The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis.

Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via β-Klotho and perilipin 2

ObjectiveNon-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5′tRF transfer RNA fragments and microRNA miR-194-5p. MethodsCombined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5′tRF and miR-194-5p. ResultsUnlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5′tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5′tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5′tRF mimic elevated mRNA levels of the metabolic regulator β-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5′tRF levels. ConclusionOur findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5′tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.

First Report of Vacuiphoma oculihominis Causing Fruit Rot on Navel Orange (Citrus sinensis) in China.

Newhall navel orange [Citrus sinensis (L.) Osbeck] is an economically important agricultural product in China. In February 2022, a rare lesion symptom was observed on Newhall navel oranges that were harvested from an orchard Ganzhou city, Jiangxi province, China (25.53° N, 114.79° E) and stored for 90 days (18±2℃, 80 to 90% RH) at the Jiangxi Key Laboratory for Postharvest Technology and Non-destructive Testing of Fruits and Vegetables (28.68° N, 115.85° E). Approximately 2% (15/750) of the oranges exhibited symptoms, with normal appearance but ink-black flesh and juice, yellowish lesions on edges of the symptoms, and no unusual odor. To isolate the pathogen, three 5 × 5 mm pieces of symptomatic tissue from a diseased orange were disinfected in 75% ethanol for 30 s, rinsed three times with sterile water, and inoculated on potato dextrose agar (PDA) at 25±1℃ and a 12:12 h photoperiod for 7 days. A pure isolate named ND-hsp was obtained. The colony was light yellow center with pale edge on the top and brown on the bottom. Conidia and pycnidia were observed on PDA medium after 2 months. Conidia were long oval, no septa, 2.9 × 3.4 μm (n = 50), and pycnidia were solitary, 39.4 × 43.9 μm (n = 20), with one or no orifice, brown to dark brown. The morphological characteristics of ND-hsp strain on PDA, oatmeal agar and malt extract agar were similar to those of the Didymellaceae (Aveskamp et al. 2010). Ulteriorly, the genomic DNA of the ND-hsp isolate was extracted from its mycelia using a fungal genomic DNA extraction kit (Solarbio, Beijing, China) for subsequent phylogenetic analyses. Four primer sets, LR0R (Rehner and Samuels 1994) /LR7 (Vilgalys and Hester 1990), V9G (Hoog and Gerrits 1998) /ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/RPB2-7cR (Liu et al. 1999) were used to amplify the corresponding DNA fragments of large subunit ribosomal RNA (LSU), internal transcribed spacer region (ITS), beta-tubulin gene (TUB2) and RNA polymerase Ⅱ second largest subunit (RPB2), respectively. The obtained sequences were assigned GenBank accession numbers and showed 99 to 100% identity with their counterparts of Vacuiphoma oculihominis UTHSC DI16-308. A phylogenetic tree was constructed in MEGA 7.0 using the concatenated sequences, placing the isolate within the V. oculihominis clade by 100% bootstrap support. Pathogenicity experiments were performed in triplicate. Ten Newhall navel oranges were surface sterilized with 75% ethanol and inoculated with 15μL of a spore suspension (2×106 spores/ml) into a 3 mm-diameter wound on the equator. The control group received sterile water instead of the spore suspension. Treated and control oranges were incubated at 25±1 ℃ and about 90% relative humidity for 20 days. All oranges were cut longitudinally or transversely through the inoculated wound and examined internally. The oranges inoculated with ND-hsp exhibited ink-black flesh and juice symptoms consistent with the initial oranges. The control oranges remained asymptomatic. Under the Koch's rule, V. oculihominis was reisolated from diseased oranges and kept in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. GenBank database analysis confirms that V. oculihominis has been found in human eye secretions and decayed trees. This is the first report of V. oculihominis as a pathogen on navel oranges in China. Our findings contribute to understanding of citrus fruit pathogens.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

Light-Triggered Signal Enhancement Strategy Integrated with a CRISPR/Cas13a-Based Assay for Ultrasensitive and Specific miRNA Detection.

The development of facile, accurate, and affordable assays for microRNAs (miRNAs) in early cancer is greatly desirable but encounters an obstacle due to low cellular abundance in biofuids. In this study, we present a novel approach called a light-triggered exponential amplification strategy coupled with a CRISPR/Cas13a-based diagnostic system (LEXPA-CRISPR), which directly transduces rare miRNA targets into photocontrolled signal enhancement response. This innovative platform leverages trans-cleavage of CRISPR/Cas13a, activated by the miRNA target, to cleave specific RNA fragments within the MB@PC-NAC assembly, thus releasing free PC-single-stranded DNA (PC-ssDNA) that is modified by a photocleavable linker (PC linker). UV irradiation is further employed toward the photoresponsive PC-ssDNA, resulting in instantaneous generation of oligo with a new 5' phosphate group (Pho-ssDNA). The Pho-ssDNA serves as a trigger for rolling circle amplification (RCA) reaction, which generates thousands of long ssDNA repeats of diverse lengths with a strong fluorescence signal. Through optimization, we achieved a detection limit of 1 fM for miR21 without the need for target amplification. Moreover, the programmable versatility of LEXPA-CRISPR is also demonstrated for miR17 determination only with simple modification of CRISPR RNA (crRNA) sequences. This proposed biosensor successfully monitored the levels of miR21 and miR17 in tumor cells, showing a satisfactory consistency with the standard qRT-PCR method. Conclusively, LEXPA-CRISPR represents a promising strategy for ultrasensitive miRNA detection. It combines the advantages of light-triggered signal amplification and robust collateral cleavage activity of Cas13a, making it an attractive tool for practical CRISPR-based diagnostics.

Structure and energetics guide dynamic behaviour in a T = 3 icosahedral virus capsid

Although virus capsids appear as rigid, symmetric particles in experimentally determined structures; biochemical studies suggest a significant degree of structural flexibility in the particles. We carried out all-atom simulations on the icosahedral capsid of an insect virus, Flock House Virus, which show intriguing differences in the degree of flexibility of quasi-equivalent capsid subunits consistent with previously described biological behaviour. The flexibility of all the β and γ subunits of the protein and RNA fragments is analysed and compared. Both γA subunit and RNA fragment exhibit higher flexibility than the γB and γC subunits. The capsid shell is permeable to the bidirectional movement of water molecules, and the movement is heavily influenced by the geometry of the capsid shell along specific symmetry axes. In comparison to the symmetry axes along I5 and I3, the I2 axis exhibits a slightly higher water content. This enriched water environment along I2 could play a pivotal role in facilitating the structural transitions necessary for RNA release, shedding some light on the intricate and dynamic processes underlying the viral life cycle. Our study suggests that the physical characterization of whole virus capsids is the key to identifying biologically relevant transition states in the virus life cycle and understanding the basis of virus infectivity.

Examining Cell-Specific Localization inAedes aegyptiTissues with Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in theAedes aegyptimosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs. The methods presented highlight the synthesis of RNA probes and describe the 2-d process of incubating the tissues/organs with the RNA probes. We also describe tyramide signal amplification for improved signal detection.

Alzheimer’s Disease‐impacted Expression of tRNA‐derived Fragments in Body Fluid: Implication for New Disease Biomarkers

AbstractBackgroundAlzheimer’s disease (AD), the most common type of dementia, affects over six million Americans aged 65 years and older. Although numerous research efforts were made on AD, the knowledge of the mechanisms and therapeutic strategies is still very limited. At present, identifying the prodromal stages of AD remains a significant challenge. tRNA‐derived RNA fragments (tRFs), a family of recently discovered small non‐coding RNAs (sncRNAs), express high in both human and primate hippocampus. Our recent discovery also revealed that some hippocampus tRF expression is much higher in AD patients compared with age‐matched health individuals.MethodHerein, we further characterized the expression of tRFs in CSF and serum from healthy, mild cognitive impairment (MCI), and AD patients, using a combination of T4 polynucleotide kinase (T4 PNK)‐RNA‐seq and qRT‐PCR specific to tRF validation.ResultOur results indicated tRF5‐ProAGG altered by AD in both CSF and serum samples, and serum AD‐impacted tRF5‐ProAGG showed a correlation to the disease severity. Notably, serum tRF5‐ProAGG showed a change tendency when MCI progresses to AD, suggesting that it is a promising biomarker for AD. We also predicted putative targets of tRF5‐ProAGG using our in‐house computational program and confirmed that some targets, such as SV2B, CDH8, and NEFL, were decreased in AD’s hippocampus. Interestingly, SV2B and tRF5‐ProAGG showed a negative correlation.ConclusiontRF5‐ProAGG shows the potential as an AD biomarker and plays a vital role in disease progression.

Utilizing low‐volume PBMC samples from Alzheimer’s disease clinical research cohorts for immune cell RNA‐sequencing

AbstractBackgroundStudies using peripheral blood mononuclear cells (PBMCs) from individuals with Alzheimer’s disease (AD) biomarker information has the potential to clarify the role of peripheral immune systems in AD. However, PBMC collection often requires immediate labor‐intensive processing and large‐volume collection, making it difficult to implement in clinical AD research. Here, we present our optimized protocol to collect, cryopreserve, and process PBMC samples for monocyte and lymphocyte RNA‐sequencing (RNA‐Seq) studies in large clinical AD research programs.MethodWe used 8 mL Cell Preparation Tubes (CPT) to collect blood samples, and each CPT was processed and cryopreserved into two aliquots per the manufacturer’s protocol. Later, one PBMC vial ( = 4 mL blood) was thawed and washed in RPMI (+10% FBS, +1% PS). After recording cell numbers and viability, 2.0×106 cells from each sample were stained with anti‐CD4, CD8, CD14, CD20 antibodies, and Sytox blue (viability) in PBS (+2% FBS). The cells were washed and resuspended in 500 uL PBS (+2% FBS; target concentration 4.0×106/mL) for flow cytometry. We sorted and collected CD4+ T cells, CD8+ T cells, CD14+ monocytes, and CD20+ B cells into RNA lysis‐buffer (TCL + 1% BME), aiming for 5,000 or 10,000 cells/cell type/donor. We used the RNeasy micro kit for RNA extraction and Fragment Analyzer for quality control.ResultWe collected and cryopreserved PBMC samples from 436 Memory and Aging Cohort (MAC) participants. We used 28 samples for this pilot study (mean age = 68.2 ± 8.1, 21 females). From one cryopreserved aliquot ( = 4 mL of blood), we recovered an average of 3.40×106 ± 1.43×106 viable cells (mean viability 90.4% ± 5.8). Average quantity of RNA was 12.2 ± 4.95 ng/104 cells. The RNA integrity (RIN) score was 8.89 ± 1.7, consistent with excellent RNA quality (Fig. 1).ConclusionOur low‐volume protocol (4 mL of blood per participant) deployed in a large clinical AD study yielded excellent‐quality RNA for cell‐type‐specific bulk RNA‐Seq (using SMART‐Seq2 library). This protocol can be readily adapted for cell‐type‐specific proteomics or single‐cell multiome projects and is a promising approach to deeply phenotype the peripheral immune system in the context of aging and AD.

Deciphering Antigen Specificity of Protective Regulatory T Cell Clones in Patients after Allogeneic Stem Cell Transplantation

Introduction: Clonal expansion of donor regulatory T cells (Tregs) in recipients of allogeneic stem cell transplantations (allo-SCT) is associated with the absence of acute graft-versus-host disease (aGVHD). Therefore, we hypothesize that antigen-specific donor Tregs prevent aGVHD. In this project, we aim to identify the cognate antigen of expanded Treg clones in patients after allo-SCT. Methods: We recruited 177 consecutive patients undergoing allo-SCT for acute myeloid leukemia without severe infection and CMV-reactivation for this study. Healthy adults matching patients' sex and age served as the control group. We sorted CD3 +CD4 +CD25 hiCD127 low Tregs from peripheral blood mononuclear cell samples collected early after transplantation (21 to 36 days, median 27 days), and we performed single-cell RNA sequencing (scRNAseq) to profile Treg transcriptome including both TCR alpha and beta chain. To determine the antigen specificity of a given TCR, we adapted a cytokine-capturing antigen-screening system (Lee and Meyerson, Sci Immunol, 2021). We built the system by 1) expressing designated TCRαβ chains in Jurkat cells, and 2) expressing cell-surface interleukin-2 capture mAb, corresponding MHC-II molecules, and CD74-fused epitope libraries in HEK293T cells serving as antigen-presenting cells (APCs). By co-culturing both modified cell lines, we can perform high-throughput screening of pooled candidates. To obtain epitope-encoding minigenes, we either used RNA fragmentation to generate candidates randomly or customized oligonucleotide synthesis to encode the tiled human proteome. To scale down the diversity of the synthetic library, we utilized NetMHCIIpan (DTU Health Tech) for MHC-II binding affinity prediction. By sequencing the CD74-fused minigene after screening out APCs with the highest cytokine intensity on the cell surface, we can decode the peptide sequence recognized by the given TCR. We have validated the cytokine-capturing antigen-screening system using a CMV-specific TCR and its cognate antigen. Results: In total, we analyzed six patients with and six patients without aGVHD. ScRNAseq showed that the FOXP3 + Treg population in patients without aGVHD shared more transcriptional similarities with healthy control as compared to patients with aGVHD. Due to lymphocytopenia, Tregs are extremely low in numbers early after allo-SCT, but clustering analysis revealed highly clonal expanded populations of Tregs with various degrees of activation. Notably, one patient without aGVHD was found to have a distinct Treg-dominant CD4 population, and a markedly abundant Treg clone (>15% of the entire Treg repertoire) was found to be parts of both FOXP3 + and FOXP3 - clusters. We then transduced the most abundant TCR into JM-T, the TCR-deficient Jurkat cell. We used an RNA-fragmented randomized library for the first screening, in which we identified one shared epitope in three independent screenings of high frequency. We then moved on to perform screenings for synthesized candidate pools predicted by NetMHCIIpan, representing human proteome and common post-allo-SCT infections. Currently, we are analyzing sequencing reads from parallel screenings and attempting direct expansion of IL-2 + sort-outs. Conclusion: By expanding our established screening system, we aim to elucidate the antigens of multiple clonal expanded TCR candidates from the same patient. Identification of specific antigens leading to clonal Treg expansion and protection from GVHD holds the potential for the development of targeted cell therapies.

Long-range RNA structures in the human transcriptome beyond evolutionarily conserved regions.

RNA structure has been increasingly recognized as a critical player in the biogenesis and turnover of many transcripts classes. In eukaryotes, the prediction of RNA structure by thermodynamic modeling meets fundamental limitations due to the large sizes and complex, discontinuous organization of eukaryotic genes. Signatures of functional RNA structures can be found by detecting compensatory substitutions in homologous sequences, but a comparative approach is applicable only within conserved sequence blocks. Here, we developed a computational pipeline called PHRIC, which is not limited to conserved regions and relies on RNA contacts derived from RNA in situ conformation sequencing (RIC-seq) experiments. It extracts pairs of short RNA fragments surrounded by nested clusters of RNA contacts and predicts long, nearly perfect complementary base pairings formed between these fragments. In application to a panel of RIC-seq experiments in seven human cell lines, PHRIC predicted ~12,000 stable long-range RNA structures with equilibrium free energy below -15 kcal/mol, the vast majority of which fall outside of regions annotated as conserved among vertebrates. These structures, nevertheless, show some level of sequence conservation and remarkable compensatory substitution patterns in other clades. Furthermore, we found that introns have a higher propensity to form stable long-range RNA structures between each other, and moreover that RNA structures tend to concentrate within the same intron rather than connect adjacent introns. These results for the first time extend the application of proximity ligation assays to RNA structure prediction beyond conserved regions.

Symptomology, prevalence, and impact of Hop latent viroid on greenhouse-grown cannabis (Cannabis sativa L.) plants in Canada

The incidence of Hop latent viroid (HLVd) affecting cannabis plants in licenced production facilities in Canada during 2020–2023 was determined by RT-PCR analyses of 15 947 samples from nine provinces. Positive detection ranged from 5.3% to 92% of samples submitted, depending on province and year of sampling. The average country-wide HLVd infection incidence was 25.6%. Symptoms on affected plants varied with plant growth stage, and ranged from asymptomatic to mild leaf curl and mottling on stock (mother) plants and on vegetative plants, to extreme stunting and reduced inflorescence development on flowering plants. All symptomatic and some asymptomatic plants contained a 256 nucleotide RNA fragment with 100% sequence homology to HLVd from GenBank. The viroid was detected in various tissues of stock, vegetative, and flowering plants, including the uppermost leaves, at middle and lower positions in the canopy, and in the roots. The incidence of infection varied with the cannabis genotype. Flowering plants displayed yellowing or darkening of inflorescence leaves surrounding the pistillate flowers. Reductions of 12–42% in inflorescence stem lengths, fresh weights, and plant heights were observed in infected plants compared to noninfected plants, depending on the genotype. Levels of tetrahydrocannabinol (THC) and terpenes in diseased inflorescences were also significantly lower. The development of glandular trichomes which produce and store cannabinoids and terpenes was greatly reduced by HLVd infection. The affected trichomes had shorter stalk lengths and smaller glandular head sizes, and appeared shrivelled upon drying of the inflorescences. Hop latent viroid poses a significant economic threat to the cannabis industry.