RNA Fragments Research Articles

Mass-Spectrometry-Based Assay at Single-Base Resolution for Simultaneously Detecting m6A and m6Am in RNA.

The methylation modifications of adenosine, especially N6-methyladenosine (m6A) and N6, 2'-odimethyladenosine (m6Am), play vital roles in various biological, physiological, and pathological processes. However, current methods for detecting these modifications at single-base resolution have limitations. Mass spectrometry (MS), a highly accurate and sensitive technique, can be utilized to differentiate between m6A and m6Am by analyzing the molecular weight differences in their fragments during tandem MS analysis. In this study, we present an MS-based method that allows for the simultaneous determination of m6A and m6Am sites in targeted RNA fragments at single-nucleotide resolution. The approach involves the utilization of tandem MS in conjunction with targeted RNA enrichment and enzymatic digestion, eliminating the need for PCR amplification. By employing this strategy, we can accurately identify m6A and m6Am sites in targeted RNA fragments with high confidence. To evaluate the effectiveness of our method, we applied it to detect m6A and m6Am sites in cell and tissue samples. Furthermore, we verified the accuracy of our approach by performing CRISPR/Cas9-mediated knockout of the corresponding methyltransferases. Overall, our MS-based method offers a reliable and precise means for the simultaneous detection of m6A and m6Am modifications in targeted RNA fragments, providing valuable insights into the functional characterization of these modifications in various biological contexts.

Visualized lateral flow assay for logic determination of co-existing viral RNA fragments

Different types of pathogenic viruses that have common transmission path can be co-infected, inducing distinct disease procession in comparison to that infection of one. Also, in the post COVID-19 time, more types of respiratory infectious virus are becoming prevalent and are concurrent. Those bring an urgent need for detection of co-existing viruses. Here, we propose a visualized lateral flow assay for logic determination of co-existing viral RNA fragments. In the presence of specific viral RNA inputs, DNAzyme is de-blocked according to defined logic, and catalyzes the hydrolysis of hairpin-structural substrate. One of cleaved substrates contains DNAzyme domain to realize dual signal amplification, which obtains copious of the other cleaved substrates. The cleaved substrates act as linking strands for bridging DNA-modified gold nanoparticles onto lateral flow strip to induce coloration on test line. “AND”, “OR” and “INHIBIT” controlled lateral flow assays are respectively demonstrated for co-existing viral RNA detection, and the visual results can be obtained by the same kind of prepared strip, without need of re-fabricating strips according to logic systems. The work provides a flexible, convenient, visual and logic-processing strategy for simultaneous analysis of co-existing viruses.

Identification of individuals from low template blood samples using whole transcriptome shotgun sequencing

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5–4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10−9. Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid.

MDB-109. CHARACTERIZING DISTINCT SIGNATURES OF RNA TRANSLATIONAL REGULATION UPON MYC OVEREXPRESSION IN H9 NEURAL STEM CELLS

Abstract BACKGROUND Medulloblastoma (MB), a childhood brain tumor, emerges typically between 6-8 years, originating from cerebellar neuronal stem cells. Recurrent genetic alterations, notably MYC amplifications, are associated with poor therapy response. Subgrouped as WNT-MB, SHH-MB, Group 3 MB, and Group 4 MB, Group 3 MB is characterized by high-level MYC amplification in approximately 17% of cases. Patients with Group 3 MB and MYC activation present distinct transcriptional and proteomic signatures, including elevated levels of ribosomal proteins and ribosome assembly proteins. METHODS We conducted a comprehensive analysis of RNA-seq and Ribo-seq in human neural stem cells (H9 NSCs) with amplified MYC expression. H9-NSCs were cultured in tumor stem media and transduced with lentivirus for either GFP control or MYC cDNA. RNA and ribosome-protected fragments were isolated from cell lysates for RNA-seq and Ribo-seq in biological triplicates. Ribo-seq data was analyzed using ORFquant for coding sequence quantification and deltaTE for translational efficiency. RESULTS Overexpressing MYC in H9-NSCs led to significant differential expression of 4194 genes (p adj < 0.05) using RNA-seq and 2860 genes (p adj < 0.05) using Ribo-seq. While RNA-seq and Ribo-seq exhibited high correlation for most genes, we identified 364 differentially translated genes (p adj < 0.05) upon MYC overexpression. Genes regulated solely at the translational level were enriched for functions in cytoplasmic translation initiation, viral translation, and response to interleukin-4. Key genes highly regulated at the Ribo-seq level included CCND3, YBX1, and EEF2. Validating with proteomics data from the CPTAC medulloblastoma tissue set confirmed protein-specific upregulation of these targets in MYC-driven Group 3 medulloblastomas. CONCLUSIONS MYC overexpression revealed distinct behaviors in differentially expressed genes, either regulated at the level of translation alone, correlated with transcription, or independent of transcription. We anticipate that our findings will contribute to a refined understanding of the molecular landscape of MB, particularly in the context of Group 3 MB.

Y4 RNA fragments from cardiosphere-derived cells ameliorate diabetic myocardial ischemia‒reperfusion injury by inhibiting protein kinase C β-mediated macrophage polarization

The specific pathophysiological pathways through which diabetes exacerbates myocardial ischemia/reperfusion (I/R) injury remain unclear; however, dysregulation of immune and inflammatory cells, potentially driven by abnormalities in their number and function due to diabetes, may play a significant role. In the present investigation, we simulated myocardial I/R injury by inducing ischemia through ligation of the left anterior descending coronary artery in mice for 40 min, followed by reperfusion for 24 h. Previous studies have indicated that protein kinase Cβ (PKCβ) is upregulated under hyperglycemic conditions and is implicated in the development of various diabetic complications. The Y4 RNA fragment is identified as the predominant small RNA component present in the extracellular vesicles of cardio sphere-derived cells (CDCs), exhibiting notable anti-inflammatory properties in the contexts of myocardial infarction and cardiac hypertrophy. Our investigation revealed that the administration of Y4 RNA into the ventricular cavity of db/db mice following myocardial I/R injury markedly enhanced cardiac function. Furthermore, Y4 RNA was observed to facilitate M2 macrophage polarization and interleukin-10 secretion through the suppression of PKCβ activation. The mechanism by which Y4 RNA affects PKCβ by regulating macrophage activation within the inflammatory environment involves the inhibition of ERK1/2 phosphorylation In our study, the role of PKCβ in regulating macrophage polarization during myocardial I/R injury was investigated through the use of PKCβ knockout mice. Our findings indicate that PKCβ plays a crucial role in modulating the inflammatory response associated with macrophage activation in db/db mice experiencing myocardial I/R, with a notable exacerbation of this response observed upon significant upregulation of PKCβ expression. In vitro studies further elucidated the protective mechanism by which Y4 RNA modulates the PKCβ/ERK1/2 signaling pathway to induce M2 macrophage activation. Overall, our findings suggest that Y4 RNA plays an anti-inflammatory role in diabetic I/R injury, suggesting a novel therapeutic approach for managing myocardial I/R injury in diabetic individuals.

Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides

ABSTRACT Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3’-5’ exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.

Episodic ozone exposure in Long-Evans rats has limited effects on cauda sperm motility and non-coding RNA populations

Epidemiological evidence suggests the potential for air pollutants to induce male reproductive toxicity. In experimental studies, exposure to ozone during sensitive windows in the sperm lifecycle has been associated with impaired sperm motility. Subsequently, we sought to investigate the effects of episodic exposure to ozone during sperm maturation in the rat. Long-Evans rats were exposed to either filtered air or ozone (0.4 or 0.8 ppm) for five non-consecutive days over two weeks. Ozone exposure did not impact male reproductive organ weights or sperm motility ∼24 hours following the final exposure. Furthermore, circulating sex hormones remained unchanged despite increased T3 and T4 in the 0.8 ppm group. While there was indication of altered adrenergic signaling attributable to ozone exposure in the testis, there were minimal impacts on small non-coding RNAs detected in cauda sperm. Only two piwi-interacting RNAs (piRNAs) were altered in the mature sperm of ozone-exposed rats (piR-rno-346434 and piR-rno-227431). Data across all rats were next analyzed to identify any non-coding RNAs that may be correlated with reduced sperm motility. A total of 7 microRNAs (miRNAs), 8 RNA fragments, and 1682 piRNAs correlated well with sperm motility. Utilizing our exposure paradigm herein, we were unable to substantiate the relationship between ozone exposure during maturation with sperm motility. However, these approaches served to identify a suite of non-coding RNAs that were associated with sperm motility in rats. With additional investigation, these RNAs may prove to have functional roles in the acquisition of motility or be unique biomarkers for male reproductive toxicity.

LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue

Whole transcriptome analysis (WTA) using RNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tissue is an invaluable tool to understand the molecular pathology of disease. RNA extracted from FFPE tissue is either degraded and/or in very low quantities hampering gene expression analysis. Earlier studies described protocols applied for cellular RNA using poly-A primer-based linear amplification. The current study describes a method, LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis). It employs random nonamer primer based method which can amplify short, fragmented RNA with high fidelity from as low as 5 ng to obtain enough material for WTA. The two-cycle method significantly amplified RNA at ∼1000 folds (p < 0.0001) improving the mean read lengths (p < 0.05) in WTA. Overall, increased mean read length positively correlated with on-target reads (Pearson's r = 0.71, p < 0.0001) in both amplified and unamplified RNA-seq analysis. Gene expression analysis compared between unamplified and amplified group displayed substantial overlap of the differentially expressed genes (DEGs) (log2 fold change cut-off < −2 and >2, p < 0.05) identified between lung cancer and asthma cohorts validating the method developed. This method is applicable in clinical molecular pathology field for both diagnostics and elucidation of disease mechanisms.

Small RNAs in the pathogenesis of preeclampsia

Preeclampsia is a major contributor to maternal and fetal morbidity and mortality. The disorder can be classified into early- and late-onset subtypes, both of which evolve in two stages. The first stage comprises the development of pre-clinical, utero-placental malperfusion. Early and late utero-placental malperfusion have different causes and time courses. Early-onset preeclampsia (20 % of cases) is driven by dysfunctional placentation in the first half of pregnancy. In late-onset preeclampsia (80 % of cases), malperfusion is a consequence of placental compression within the confines of a limited uterine cavity. In both subtypes, the malperfused placenta releases stress signals into the maternal circulation. These stress signals trigger onset of the clinical syndrome (the second stage). Small RNA molecules, which are implicated in cellular stress responses in general, may be involved at different stages. Micro RNAs contribute to abnormal trophoblast invasion, immune dysregulation, angiogenic imbalance, and syncytiotrophoblast-derived extracellular vesicle signalling in preeclampsia. Transfer RNA fragments are placental signals known to be specifically involved in cell stress responses. Disorder-specific differences in small nucleolar RNAs and piwi-interacting RNAs have also been reported. Here, we summarise key small RNA advances in preeclampsia pathogenesis. We propose that existing small RNA classifications are unhelpful and that non-biased assessment of RNA expression, incorporation of non-annotated molecules and consideration of chemical modifications to RNAs may be important in elucidating preeclampsia pathogenesis.

Structural Insights into the Rrp4 Subunit from the Crystal Structure of the Thermoplasma acidophilum Exosome.

The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the cytoplasm, the processing of short noncoding RNA, and the breakdown of RNA fragments. We determined two crystal structures of exosome components from Thermoplasma acidophilum (Taci): one with a resolution of 2.3 Å that reveals the central components (TaciRrp41 and TaciRrp42), and another with a resolution of 3.5 Å that displays the whole exosome (TaciRrp41, TaciRrp42, and TaciRrp4). The fundamental exosome structure revealed the presence of a heterodimeric complex consisting of TaciRrp41 and TaciRrp42. The structure comprises nine subunits, with TaciRrp41 and TaciRrp42 arranged in a circular configuration, while TaciRrp4 is located at the apex. The RNA degradation capabilities of the TaciRrp4:41:42 complex were verified by RNA degradation assays, consistent with prior findings in other archaeal exosomes. The resemblance between archaeal exosomes and bacterial PNPase suggests a common mechanism for RNA degradation. Despite sharing comparable topologies, the surface charge distributions of TaciRrp4 and other archaea structures are surprisingly distinct. Different RNA breakdown substrates may be responsible for this variation. These newfound structural findings enhance our comprehension of RNA processing and degradation in biological systems.

P.099 Transfer RNA fragments in patient plasma extracellular vesicles as biomarkers of high grade glioma

Background: High-grade gliomas (HGG) present challenges with short post-surgery survival and high progression rates. Extracellular vesicles (EVs) in the tumor microenvironment (TME) contribute to a pro-tumorigenic setting. Investigating Transfer RNA fragments (TfRNA) in HGG patient plasma EVs reveals potential biomarkers and therapeutic targets, shedding light on the molecular landscape for enhanced diagnostic and therapeutic strategies. This study examines TfRNA in 10 HGG patients at diagnosis, offering insights into the molecular landscape for improved management strategies. Methods: The study involved the collection of plasma samples from HGG patients and controls. EVs were isolated from these samples and subsequently analyzed for tfRNA. Results: Analysis of plasma EVs highlighted distinct differences in TfRNA fragments between High-Grade Glioma (HGG) and control samples. HGG EVs showed a global reduction in tRNA content, higher 5’ tfRNA proportions, and increased nuclear tfrna compared to controls. A notable biological marker, elevated in HGG, holds potential as a diagnostic indicator. Conclusions: Our study concludes that High-Grade Gliomas (HGG) demonstrate a global reduction in tfRNA content in plasma extracellular vesicles compared to non-cancer controls, echoing findings in other cancers. Despite this, specific tfRNA molecules in HGG show significant differential expression or sorting into EVs, indicating their potential as future biomarkers or therapeutic targets.

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

The role of catchment population size, data normalization, and chronology of public health interventions on wastewater-based COVID-19 viral trends

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic presented the most challenging global crisis in recent times. A pandemic caused by a novel pathogen such as SARS-CoV-2 necessitated the development of innovative techniques for the monitoring and surveillance of COVID-19 infections within communities. Wastewater surveillance (WWS) is recognized as a non-invasive, cost-effective, and valuable epidemiological tool to monitor the prevalence of COVID-19 infections in communities. Seven municipal wastewater sampling sites representing distinct sewershed communities were selected for the surveillance of the SARS-CoV-2 virus in Durham Region, Ontario, Canada over 8 months from March 2021 to October 2021. Viral RNA fragments of SARS-CoV-2 and the normalization target pepper mild mottle virus (PMMoV) were concentrated from wastewater influent using the PEG/NaCl superspeed centrifugation method and quantified using RT-qPCR. Strong significant correlations (Spearman's rs = 0.749 to 0.862, P < 0.001) were observed between SARS-CoV-2 gene copies/mL of wastewater and clinical cases reported in each delineated sewershed by onset date. Although raw wastewater offered higher correlation coefficients with clinical cases by onset date compared to PMMoV normalized data, only one site had a statistically significantly higher Spearman's correlation coefficient value for raw data than normalized data. Implementation of community stay-at-home orders and vaccinations over the course of the study period in 2021 were found to strongly correspond to decreasing SARS-CoV-2 wastewater trends in the wastewater treatment plants and upstream pumping stations.

Tectorigenin improves metabolic dysfunction-associated steatohepatitis by down-regulating tRF-3040b and promoting mitophagy to inhibit pyroptosis pathway

Tectorigenin (TEC) as a plant extract has the advantage of low side effects on metabolic dysfunction-associated steatohepatitis (MASH) treatment. Our previous study have shown that tRNA-derived RNA fragments (tRFs) associated with autophagy and pyroptosis in MASH, but whether TEC can mitigate MASH through tRFs-mediated mitophagy is not fully understood. This study aims to investigate whether TEC relies on tRFs to adjust the crosstalk of hepatocyte mitophagy with pyroptosis in MASH. Immunofluorescence results of PINK1 and PRKN with MitoTracker Green-labeled mitochondria verified that TEC enhanced mitophagy. Additionally, TEC inhibited pyroptosis, as reflected by the level of GSDME, NLRP3, IL-1β, and IL-18 decreased after TEC treatment, while the effect of pyroptosis inhibition by TEC was abrogated by Pink1 silencing. We found that the upregulation expression of tRF-3040b caused by MASH was suppressed by TEC. The promotion of mitophagy and the suppression of pyroptosis induced by TEC were abrogated by tRF-3040b mimics. TEC reduced lipid deposition, inflammation, and pyroptosis, and promoted mitophagy in mice, but tRF-3040b agomir inhibited these effects. In summary, our findings provided that TEC significantly reduced the expression of tRF-3040b to enhance mitophagy, thereby inhibiting pyroptosis in MASH. We elucidated a powerful theoretical basis and provided safe and effective potential drugs for MASH with the prevention and treatment.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

CoCoNuTs are a diverse subclass of Type IV restriction systems predicted to target RNA.

A comprehensive census of McrBC systems, among the most common forms of prokaryotic Type IV restriction systems, followed by phylogenetic analysis, reveals their enormous abundance in diverse prokaryotes and a plethora of genomic associations. We focus on a previously uncharacterized branch, which we denote coiled-coil nuclease tandems (CoCoNuTs) for their salient features: the presence of extensive coiled-coil structures and tandem nucleases. The CoCoNuTs alone show extraordinary variety, with three distinct types and multiple subtypes. All CoCoNuTs contain domains predicted to interact with translation system components, such as OB-folds resembling the SmpB protein that binds bacterial transfer-messenger RNA (tmRNA), YTH-like domains that might recognize methylated tmRNA, tRNA, or rRNA, and RNA-binding Hsp70 chaperone homologs, along with RNases, such as HEPN domains, all suggesting that the CoCoNuTs target RNA. Many CoCoNuTs might additionally target DNA, via McrC nuclease homologs. Additional restriction systems, such as Type I RM, BREX, and Druantia Type III, are frequently encoded in the same predicted superoperons. In many of these superoperons, CoCoNuTs are likely regulated by cyclic nucleotides, possibly, RNA fragments with cyclic termini, that bind associated CARF (CRISPR-Associated Rossmann Fold) domains. We hypothesize that the CoCoNuTs, together with the ancillary restriction factors, employ an echeloned defense strategy analogous to that of Type III CRISPR-Cas systems, in which an immune response eliminating virus DNA and/or RNA is launched first, but then, if it fails, an abortive infection response leading to PCD/dormancy via host RNA cleavage takes over.

The mitochondrial tRNA-derived fragment, mt-tRF-LeuTAA, couples mitochondrial metabolism to insulin secretion

ObjectiveThe contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. MethodsWe used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. ResultsOur study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic β-cells. ConclusionsOur findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.

Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Identification of long non-coding RNAs in response to microsporidia infection in Silkworm, Bombyx mori.

Microsporidia Nosema bombycis (Nb) is a cellular parasite responsible for pébrine disease in silkworms, significantly impacting the sericulture industry. Long non-coding RNAs (lncRNAs), which are RNA fragments longer than 200 nucleotides, are pivotal in a range of cellular and physiological functions. However, the potential role of silkworm lncRNAs in response to Nb infection remains unknown. This study conducted transcriptome sequencing on both larvae and Nb-infected midguts of silkworms, identifying 1,440 lncRNAs across all examined midgut samples. Within the Nb-infected group, 42 differentially expressed lncRNAs (DElncRNAs) and 305 differentially expressed mRNAs (DEmRNAs) were detected. Functional annotation and pathway analysis showed that these DEmRNAs are mostly involved in metabolism, apoptosis, autophagy, and other key pathways. The co-expression network of DEmRNAs and DElncRNAs illustrates that 1 gene could be regulated by multiple lncRNAs and 1 lncRNA may target multiple genes, indicating that the regulation of lncRNA is intricate and networked. In addition, the DElncRNA-miRNA-mRNA network showed that some DElncRNAs may be involved in the immune response and metabolism through miRNA. Notably, the study observed an increase in lncRNA MSTRG857.1 following Nb infection, which may promote Nb proliferation. These findings offer insights into the complex interplay between insects and microsporidia.

Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction.

Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.