While the prognostic role of immunoglobulin heavy chain locus (IGH) rearrangement in minimal residual disease (MRD) in pediatric B-acute lymphoblastic leukemia (B-ALL) has been reported, the contribution of light chain loci (IGK/IGL) remains elusive. This study is to evaluate the prognosis of IGH and IGK/IGL rearrangement-based MRD detected by next-generation sequencing in B-ALL at the end of induction (EOI) and end of consolidation (EOC). IGK/IGL rearrangements identify 5.5% of patients without trackable IGH clones. Concordance rates for IGH and IGK/IGL are 79.9% (cutoff 0.01%) at EOI and 81.0% (cutoff 0.0001%) at EOC, respectively. Patients with NGS-MRD < 0.01% at EOI or <0.0001% at EOC present excellent outcome, with 3-year event-free survival rates higher than 95%. IGH-MRD is prognostic at EOI/EOC, while IGK-MRD at EOI/EOC and IGL-MRD at EOI are not. At EOI, NGS identifies 26.2% of higher risk patients whose MRD < 0.01% by flow cytometry. However, analyzing IGK/IGL along with IGH fails to identify additional higher risk patients both at EOI and at EOC. In conclusion, IGH is crucial for MRD monitoring while IGK and IGL have relatively limited value. While the prognostic role of immunoglobulin heavy chain locus (IGH) rearrangement in minimal residual disease (MRD) in pediatric B-acute lymphoblastic leukemia (B-ALL) has been reported, the contribution of light chain loci (IGK/IGL) remains elusive. Here, the authors investigate it using a next generation sequencing approach. Risk-adapted chemotherapy has improved the prognosis of childhood ALL remarkably for the past four decades4. By determining the patients’ risk of relapse based on a reliable method, patients can be stratified and treated according to their risk. Minimal/measurable residual disease (MRD), defined as the presence of residual leukemic cells not detected by conventional tools, is a strong prognostic factor5, and treatment strategies based on MRD status improve outcomes in childhood ALL6. There is compelling evidence associated with MRD negativity and the beneficial therapeutic outcome of long-term survival7. Treatment modalities can be tailored, and novel treatments may be administered based on MRD risk stratification to enhance the curative rate9.Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children. It involves chromosomal abnormalities and genetic alterations at an early stage of differentiation of lymphoid precursor cells11. MFC-based MRD measurements depend on the surface immunophenotype of the leukemia cells, and MRD measurements by PCR are based on the identification of leukemia-specific fusion genes/associated target genes. However, some leukemic clones may have antigenic changes after therapy, and tumor heterogeneity may lead to false negativity, thereby limiting the application of MFC12. Recent studies applying next-generation sequencing (NGS) to monitor MRD have reported higher sensitivity and precision than MFC or quantitative PCR (qPCR) in hematological malignancies16. MRD monitoring using sensitive and robust NGS approach enhances the identification of relapse risk post-hematopoietic stem cell transplantation or chimeric antigen receptor modified T-cell therapy18, and allows the identification of new emerging clones at each timepoint of the monitoring19.Multiparameter flow cytometry (MFC) or polymerase chain reaction (PCR) is widely applied for detecting MRD and essential in determining disease risks and directing therapeutic approaches for children with ALL20. Previous clinical studies have mostly focused on the prognostic significance of NGS of the immunoglobulin heavy chain locus (IGH)21, but there is a knowledge gap regarding the prognostic value of the immunoglobulin kappa (IGK) and lambda (IGL) light chain loci.Immunoglobulin (Ig) gene rearrangements are a promising patient-specific target for the detection of MRD in B-lineage ALL (B-ALL) to identify the frequency and distribution of clonal sequences associated with a malignant lymphocyte populationIn this study, we aim to assess the prognostic value of both IGH and light-chain (IGK/IGL) MRD measurements in childhood B-ALL. To address this, we utilize a multiplex PCR and NGS-based technique to detect multiple B and T cell receptor sequences which encompass rearranged IGH (VDJ), IGH (DJ), IGK, IGKDE, and IGL. Herein, sequential detection of Ig gene rearrangements using NGS is conducted both at the time of diagnosis and during chemotherapy, and the prognostic values of IGH, IGK and IGL gene rearrangements are investigated.A total of 430 pediatric patients with B-ALL were enrolled between November 2018 and April 2022. As a part of the initial diagnostics, an Ig-/T-cell receptor (TCR) immune panel was analyzed to identify prognostic markers. Among these pediatric patients, 399 children (92.8%) had at least one trackable Ig clonal rearrangement and were included for further analyses. This cohort included 221 male and 178 female patients, with a median age of 4.4 (range: 0.3–17.8) years. The baseline characteristics are presented in Table To statistically assess the data quality and stability across different time points and patient samples, the data for 1) number of input cells for sequencing, 2) size of sequencing raw data, (3) Q30 value of the FASTQ data, (4) IGK counts, and (5) IGL counts were subjected to data quality assessment using a 3-sigma (3-σ) test principle (Supplementary Tables  number o size of P < 0.001) . In contrast, of the IGK and IGL categories, majority of patients aged 1-10 years exhibited a dominance of one clone rearrangement which was comparable with those aged over 10 years old .Among 399 patients with B-ALL, 724 IGH clonal (including IGH-DJ) rearrangements in 377 children, 266 IGK clonal (including IGKDE) rearrangements in 176 children, and 83 IGL clonal rearrangements in 68 children were detected. Clonal rearrangements of ≥2 were found in 70.5% of children. The distribution of clones is shown in Fig. 01) Fig. . Among 301) Fig. . Twenty-01) Fig. . SpecifiThe clearance of Ig clones was assessed at sequential timepoints of chemotherapy. As shown in Fig. P < 0.001, Fig. P = 0.624; Fig. P = 0.019; Fig. P = 0.170; Fig. We then investigated the clearance patterns of clones with different sizes. The clearance rate of IGH clones with frequencies <10% was significantly faster compared with the clearance rate of clones with frequencies ≥50%, and the MRD clearance rate was higher at EOI . At a higher sensitivity level (0.0001%), IGH-MRD and IGK/IGL-MRD had a concordance rate of 68.9%   at the level of 0.0001%  Fig. . At EOC,01% Fig. .Fig. 3CoMLL-rearrangements had higher NGS-MRD levels compared to other fusion genes, including ETV6/RUNX1, BCR/ABL1, and TCF3/PBX1 at EOC  in patients whose NGS-MRD were <0.0001%, ≥0.0001% and <0.01%, and ≥0.01% at EOI, respectively , respectively   in 95.2% of patients at EOI  Fig. . In contP = 0.034; Fig. P < 0.001; Fig. P = 0.081, Fig. P = 0.740, Fig. P = 0.080, Fig. P = 0.017, Fig. When evaluating the MRD level solely based on the change of IGH alone, 351 children were included at EOI, and the 3-year EFS rates of children with IGH-MRD < 0.0001%, ≥0.0001% and <0.01%, and ≥0.01% were 94.7% ± 2.5%, 95.0% ± 3.7%, and 88.0% ± 4.4%, respectively , (2) IGH negative and IGK/IGL negative (IGH−IGK/IGL−), and (3) IGH negative but IGK/IGL positive (IGH−IGK/IGL+). Among the 351 children at EOI, 271 had IGH <0.01% (IGH negative), 18 had IGH <0.01% but IGK/IGL ≥0.01% (IGH−IGK/IGL+), and two of the 18 patients relapsed. The EFS of children with IGH−IGK/IGL+ was comparable with that of those with IGH−IGK/IGL− . At EOC, when 0.0001% of IGH, IGK, and IGL was taken as the cutoff value for MRD negativity, the 3 groups were also compared. The results showed that 20 of the 312 IGH-negative children were IGK/IGL positive, and one child relapsed. The EFS of IGH−IGK/IGL+ children was comparable to that of IGH−IGK/IGL− children as well .Next, we probed whether IGK/IGL tracking can further improve the accuracy of MRD by Ig NGS reports Fig. . We cate23. In addition to serving as an independent prognostic factor, MRD helps guide risk stratification and treatment decisions based on its dynamic change25. However, most conventional MRD techniques are insufficiently standardized or sensitive in detecting low levels of MRD. While MFC and qPCR are the most commonly used techniques, they also have limitations. MFC is readily available and fast, but due to the heterogeneity of ALL, it requires cell surface markers that may not be sensitive or specific enough26. On the other hand, while qPCR of Ig/TCR offers high sensitivity and specificity, it is time-consuming and has limitations related to the choice of primers and changes in clones, which can lead to false negative results27. In contrast, NGS with its high-throughput sequencing and ultra-sensitivity overcomes these limitations, with sensitivity levels that can reach up to 10−6. This high sensitivity allows for the identification of patients in the very-low-risk group and expands the intervention window for those at risk of relapse14.Monitoring minimal residual disease (MRD) is a standardized and well-accepted method for measuring disease status in patients with ALL, and it has become an integral part of diagnostic patient care29. Thus, two MRD markers with high sensitivity were recommended to reduce the false-negative results arising from clonal evolution in previous studies around or before the year 200020. In contrast, NGS provides comprehensive analysis of all kinds of clonal rearrangements30. Whether the combination of IGH targets with other Ig rearrangements like IGK and IGL is a more suitable strategy for sensitive MRD detection in the era of NGS should be clarified.Ig and TCR gene rearrangements are clone-specific molecular markers used in MRD analysis. The use of oligoclonal Ig/TCR targets in MRD analysis may result in underestimation of the ALL load due to continuous and secondary rearrangements31. In this cohort of pediatric patients with B-ALL, it was observed that 7.4% of the cases did not have dominant Ig clonal rearrangement for MRD tracking, which was similar to a previous report in adult ALL32. The possible reasons for this could be attributed to the following two aspects: First, malignant cells transformation may occur prior to Ig gene rearrangements, rendering them undetectable through this method33. Secondly, mutations at the primer binding sites of the assay may result in ineffective and nonspecific amplification35.Studies have shown that monitoring B-ALL patients at different timepoints using IGH, IGK (VK-KDE), TCRG, and TCRD gene rearrangements achieves acceptable sensitivity levels16, studies on IGK and IGL are limited, and most previous studies have tracked IGK and IGL rearrangements by qPCR, resulting in controversial results4. In this study, we investigated the distribution pattern of IGK and IGL and assessed whether the monitoring of IGH along with IGK and IGL had any complementary significance. Our cohort showed that IGH, IGK, and IGL clonal rearrangements were found in 94.5%, 44.1%, and 17.0% of children, respectively, which was similar to previous reports10. However, we found that IGK and IGL were not independent factors to monitor MRD, and IGH was the only marker with strong prognostic significance at both EOI and EOC, which was quite similar to another study conducted in adult ALL patients32. The cutoff value of its application was set at different sensitivities. MRD less than 0.01% was the most important indicator for excellent outcome, while it would be beneficial if MRD was negative or less than 0.0001% at EOC. Unfortunately, at EOI, we did not find any correlation between IGK or IGL clearance and EFS in children with residual IGK and IGL. As IGK and IGL can only cover a small population and their independent prognostic significance is not clearly noticeable, they are not suitable for the evaluation of MRD independently.Although clinical studies have used NGS to monitor IGH rearrangement in B-ALL+ MRD presented similar EFS to those with IGH-IGK/IGL- MRD both at EOI and EOC. This indicates that the detection of IGK and IGL presented limited value to IGH monitoring. Unlike NGS, which could identify 26.2% of patients with intermediate-risk from those with negative MFC-MRD, analyzing IGK and IGL along with IGH identified only another 7% of patients with positive light-chain but similar outcome to IGH−IGK/IGL− patients. Thus, we considered that integrating IGK and IGL levels into the IGH MRD added relatively limited value to the IGH-based risk stratification system.As IGK and IGL alone were not sufficient indicators of MRD, whether they can be combined with IGH to further improve the evaluation efficiency of IGH was investigated. In our study, among the children who were negative for IGH at EOI, 6.6% (18/271) and 7.1% (22/312) were positive for the light-chain clones IGK and IGL, respectively. Patients with IGH-IGK/IGL16. While the EOI-MRD measurement is important for prognosis and risk stratification, assessing MRD at later timepoints can add value for clinical management36. In our study, children with MRD levels <0.01% at EOI had a good prognosis. However, at the EOC, the MRD detection limit must be <0.0001% to show a favorable outcome. At EOC, 82.3% of patients achieved MRD below 0.0001%. For the remaining 17.7% of children whose leukemia clones were not cleared, even if their MRD is below 0.01%, advanced therapeutic intervention such as adding immunotherapy modalities, such as blinatumomab, is necessary, given its superior efficacy in MRD eradication compared to traditional chemotherapy. In the adult ALL E1910 study, the addition of blinatumomab to consolidation therapy also showed good survival benefits for MRD <0.01% (detected by MFC) patients, suggesting that blinatumomab may further improve survival by deepening the depth of MRD remission37. In another clinical trial conducted in children with high-risk or first-relapsed B-ALL, even patients with MRD less than 0.01% showed a trend of benefiting from blinatumomab when compared with traditional chemotherapy, indicating that deeper MRD must be eradicated to prevent relapse9. As our study established that NGS is both sensitive and high throughput, detecting deeper MRD remission by NGS is clinically significant in guiding the application of immunotherapy.Previous studies on B-ALL have generally evaluated the prognostic significance of NGS-MRD at EOI timepoints but have not focused much on EOC−6. Seq-MRD and Clonoseq target the IGH (VDJ), IGH(DJ), IGK, and IGL loci to identify clonotypes using sequencing data, while Invivoscribe focuses on the IGH and IGK loci. Moreover, the definition of the dominant clone for following tracking was the same between Seq-MRD and Clonoseq, which is different from that of Invivoscribe39.In addition to Seq-MRD testing method used in this study, there are other available MRD assays that capture the immunoglobulin heavy chain (IGH) and kappa/lambda light chain (K/L) loci, such as Adaptive Clonoseq and Invivoscribe. These three methods share a similar overall objective of detecting and monitoring MRD in B-cell malignancies, and they all demonstrate high sensitivity reaching 1041. It plays a crucial role in improving risk stratification, treatment response monitoring, and prognostic outcomes42. NGS enables more sensitive and specific detection of MRD, allowing for precise risk assessment and identification of patients at higher risk of relapse43. It facilitates personalized treatment strategies, such as targeted therapies and transplantation, based on MRD status44. NGS-based MRD assessment provides valuable information for clinical decision-making and enhances the management of these diseases.NGS for MRD assessment is applicable to various B-lineage malignancies except for ALL, such as chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and lymphomaOur present study has certain limitations. First, the follow-up time of the cohort was relatively short. Although our result of the prognosis was consistent with previous research, the exact conclusion needs to be observed for a longer period. Second, the small sample size of patients with IGK and IGL limited our ability to draw definitive conclusions. Future studies with extended observation periods and larger population will provide a more comprehensive understanding of the prognostic significance of MRD monitoring in this patient population.In conclusion, the results of our study indicate that NGS of Ig is an improved analytic platform for the measurement of post-treatment MRD in pediatric B-ALL. Patients with NGS-MRD <0.01% at EOI or <0.0001% at EOC present a nearly curable disease. With high availability and sensitivity, the clonal rearrangement of IGH is the most important for MRD monitoring, whereas the clonal rearrangement of IGK and IGL was found only in approximately half of the cohort with very limited independent prognostic significance. Thus, we recommend continuing to pursue IGH monitoring but not IGK/IGL monitoring according to our experience.This study was approved by the Institutional Review Board of Children’s Hospital, Zhejiang University School of Medicine and was conducted in accordance with the Declaration of Helsinki regulations and the International Conference on Harmonization and Good Clinical Practice guidelines . Informed consent was obtained from the parents/guardians of each patient. No participant compensation was provided. Sex and/or gender of participants was not considered in the study design; more than half of participants were male (240 of 430). Sex of participants was determined based on physical examination and chromosome karyotype.This was a single-center, observational study conducted in children with ALL between November 2018 and June 2022. Children ≤18 years with newly diagnosed B-ALL who undergone NGS of B-cell receptors at the Children’s Hospital of Zhejiang University School of Medicine were included in this study. Patients with extramedullary leukemia at diagnosis were excluded. The European Group for the Immunological Characterization of Leukemias (EGIL) criteria were applied to diagnose and classify ALL in this study. All enrolled patients were treated according to the ZJCH-ALL-2019 protocol detailed in the Supplementary Fig. 6 mononuclear cells (MNCs). To amplify all possible rearranged sequences in the samples, a specific set of primers was used, including 23 primers for the IGH gene, 9 primers for the incomplete IGH gene, 15 primers for the immunoglobulin kappa locus (IGK), including KDE, and 17 primers for the immunoglobulin lambda locus (IGL). These primers targeted the V, D, and J regions of the IGH, IGK, and IGL genes, respectively. For sample identification, primers with sample identifiers were used in the second round of PCR amplification. Subsequently, the PCR products with identifiers were purified using AMPure XP beads  and combined into a sequencing library. The mixed library was then sequenced using the Illumina® Novaseq PE150 platform.DNA was extracted from bone marrow samples and sent for sequencing using Seq-MRD® technology . The details of the sequencing method are as follows. The input DNA for diagnostic samples  was 500 ng, while for post-treatment samples (MRD tracking test), it was at least 7 μg, which corresponded to 1 × 1045.The Ig-Blast algorithm was used to determine Ig clonotypes, and the obtained CDR3 sequences were compared with the IMGT database. Clonotype frequencies were calculated by dividing the number of sequencing reads for each clonotype by the total number of passed sequencing reads within a sample. A frequency threshold of 3% was used to define dominant clone gene rearrangements. If a sequence accounted for at least 3% of all sequences at a given locus, represented at least 0.02% of total nucleated cells in the sample, significantly separated from the background clone repertoire, and had sufficient uniqueness for tracking, it was considered suitable for trackingFor enhanced result accuracy, each testing batch included an internal standard. This standard comprised a positive control with known rearrangements spike-in DNA, which allowed assessment of PCR amplification efficiency. Furthermore, during the development of the NGS MRD methodology, comprehensive optimization and validation processes were carried out to ensure reliability and validity. To mitigate primer amplification bias, primers were optimized using over 200 spike-in sequences synthesized from plasmids. Adjustments were made to primers with low amplification efficiency by varying PCR enzymes, buffers, temperatures, primer melting temperatures (Tm), and reaction cycle numbers. Mixed known DNA samples served as positive controls during sample testing to assess batch-specific amplification performance.The linearity of the NGS MRD method was assessed by generating samples with known absolute copy numbers and rearrangements. Cell line DNA samples at different concentrations were mixed with healthy individuals’ PBMC DNA samples. The detected results were compared to the expected results to confirm the linear relationship.The uniqueness of the dominant clone sequences was assessed by establishing a healthy donor Ig database. The probability of occurrence of the detected dominant clone in the healthy donor Ig database was investigated, and each dominant clone sequence was assigned a uniqueness index. This index provided an evaluation of the likelihood of the sequence being present in the non-ALL repertoire. Clones with low uniqueness indices were excluded from MRD tracking. Different clone sequences in the tested samples have varying limit of detection (LoD). When determining the LoD for trackable sequences, several factors were taken into account, including the amount of DNA input, sequence uniqueness, as well as the diversity and mutation of those sequences.χ2 test, the correlation between quantitative variables was measured using Pearson correlation and tested using Student’s t distribution, and ANOVA was used to compare quantitative variables. Event-free survival (EFS) and overall survival (OS) curves were estimated using the Kaplan–Meier method and compared according to the log-rank test. Death during induction, abandonment before complete remission, death in continuous complete remission, relapse, and secondary malignancies were considered as events in the calculation of EFS probability. The EFS time was calculated from the date of diagnosis to the last date of follow-up or the first event. The OS was calculated from the date of diagnosis to death from any causes with censoring the patients alive at the time of data analysis. The final date for follow-up was August 30, 2022. Data visualization was performed in R version 4.0.3 using the following packages: ggplot2, survival, survminer and networkD3 package and GraphPad Prism 9.0.0. Statistical analysis was performed in Graphpad Prism 9.0.0 and SPSS software (version 23.0.0.0). A P value < 0.05 (two tailed) was considered to be statistically significant. Bonferroni adjustment was performed for multiple comparisons.The association between categorical variables was tested using Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Emerging evidence highlights the multifaceted roles of the RNA epitranscriptome during viral infections. By modulating the modification landscape of viral and host RNAs, viruses enhance their propagation and elude host surveillance mechanisms. Here, we discuss how specific RNA modifications, in either host or viral RNA molecules, impact the virus-life cycle and host antiviral responses, highlighting the potential of targeting the RNA epitranscriptome for novel antiviral therapies. Viruses are completely dependent on their host cells’ machinery to replicate and form new infectious virus particles, employing different strategies to co-opt, hijack, or inhibit cellular processes. To restrict viral propagation and coordinate the immune response, different host cell receptors sense viral cues and trigger distinct antiviral signaling pathways driving the production of interferon (IFN), IFN-stimulated genes (ISG’s), and proinflammatory cytokines . To usurTo facilitate their replication and elude host immune surveillance, viruses evolved several tactics, including the modification of its own viral RNA molecules. The repertoire of epitranscriptomic modifications in viral mRNAs is expanding and, in the next sections, we discuss how viruses introduce epitranscriptomic marks into their own mRNA to potentiate their replication.7GpppN, cap1), along with an 2′-O-methyl group (cap2) within the first and second nucleosides downstream of the m7G cap  marks are found within tRNA, rRNA, and mRNA molecules (4C is introduced onto RNA molecules under the actions of N-acetyltransferase 10 (NAT10), which requires the assistance of THUMP domain-containing 1 (THUMPD1)   and box THUMPD1) , to intrTHUMPD1) .4C was identified in the genomes of Zika virus (ZIKV), DENV, hepatitis C virus (HCV), poliovirus (PV), HIV-1, enterovirus-71 (EV71) and IAV -binding protein 2 (PCBP2) to the EV71 IRES, enhancing transcript stability and the interaction with RNA-dependent RNA polymerase (3D) (ac and IAV . HIV-1 a and IAV . The ac4ase (3D) .Ψ) is one of the most prevalent RNA modifications, and a substantial amount of this modification has been found in the genomes of several viruses, including ZIKV, DENV, HCV, PV, IAV and HIV-1  infections, with one of its non-coding RNAs, EBV-encoded RNA 2 (EBER2), being significantly marked by pseudouridylation  is found within multiple RNA classes and is catalyzed by enzymes belonging to the NOL1/NOP2/SUN domain (NSUN) family and DNA methyltransferase family protein (DNMT2) in eukaryotes (5-methycytidine (mkaryotes .5C on their genomic RNA (5C marks on murine leukemia virus (MLV) transcripts are recognized by the m5C reader ALYREF to promote their nuclear export , a transcriptional activator that, upon methylation, contributes to the metabolic reprogramming of HCV-infected cells   . NSUN1 w TAR RNA .6-methyladenosine (m6A) plays a role in regulating mRNA stability, splicing, and translation  was also suggested to facilitate EBV viral RNA decapping and promote its RNA decay by recruiting RNA degradation complexes  to inosine (I) within RNA transcripts  by a fam6A methylations are a known mark used by pattern recognition receptors (PRRs), to differentiate between host and foreign/pathogenic RNA. m6A-methylated RNA binds poorly to RIG-I, in contrast to RNA with Ψ which binds with high affinity, with both failing to initiate RIG-I downstream immune activation. In DNA viruses infections, disturbing hnRNPA2B1-FT0 interaction elevated the levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and interferon gamma inducible protein 16 (IFI16) m6A-modified mRNA to bolster the antiviral response (6-Methyladenosine RNA Binding Protein F2 (YTHDF2) reader enzyme binds to methylated RNA, reducing RIG-I binding, blocking RIG-I conformational changes and IFN transcription , appear to have their translation enhanced by m6A and its machinery, in addition to the fact that YTHDF1 also increases the expression of IFITM1 in an m6A-dependent manner  exploits its US3 protein to reduce the m6A methylome as part of the PRV-induced metabolic dysfunction  ORF50 RNA enabled its binding to the m6A reader SND1 for ORF50 transcript stability  modified host mRNAs by degrading the phosphorylated CTD Interacting Factor 1 (PCIF1), an inhibitor of HIV-1 transcription (a (eIF2α) phosphorylation and IFN during immune responses , as this protein exhibits a higher affinity for the modified hASLLys3UUU compared to the unmodified human tRNA  modification at the wobble position of the corresponding tRNATyrUAC. This suggests a potential correlation between phage codon bias and tRNA modification content  that serve a variety of cellular regulatory functions . Alterat5C in the EBV ncRNA 7SL has been shown to enhance its binding to RIG-I and, thus the IFN response , likely indicating that this ncRNA is an Angiogenin target for m5C-dependent cleavage , which associated with polysomes and was translated into the E7 oncoprotein (Circular RNAs (circRNAs) have recently emerged as a class of ncRNAs involved in viral infection. Several viruses can alter the circRNA landscape of infected cells to modulate host gene expression . Notablye escape . The HBVogenesis . Ultimatogenesis . Besidesoprotein .5C via NSUN7 depletion leads to hypomethylation of eRNAs, a transcriptional coactivator that interacts with proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α) to modulate cellular metabolic responses. As the metabolic response is reprogrammed during HCV infection, and accompanied by PGC-1α induction, NSUN7 may be involved in this process  , piwi-in process .The RNA epitranscriptome plays a critical role in shaping viral infections and host antiviral responses. It affects viral RNA stability, translation, and recognition by the host, hindering the host’s ability to detect viruses and mount a response . Convers6A by 3-DAA significantly decreased viral replication (6A-modified EBV transcripts, suppresses EBV infection, showing that this enzyme induces RNA deterioration (A comprehensive understanding of the enzymes responsible for writing and erasing RNA modifications, as well as the reader proteins that interpret these modifications, presents exciting opportunities for developing more effective antiviral therapies. The development of small-molecule inhibitors, RNA-targeting therapies, and epitranscriptomic editing tools, holds promise in defeating viral resistance to antiviral therapies. While the field of RNA modification-targeted antiviral drugs is still in its early stages, several studies have shown promising results in potential targets. For example, in HSV-1 infection, inhibition of mlication . Conversioration . NeverthTo grasp the crosstalk between viruses and the RNA epitranscriptome, a detailed mapping and characterization of RNA modifications during virus infection is required. Additionally, unraveling the crosstalk between different RNA modifications, their modifying enzymes, and their interplay with viruses will deepen our understanding of the complex dynamics at the virus-host cell interface. Further research efforts and technological advancements are crucial for fully harnessing the therapeutical potential of the RNA epitranscriptome.


Most of the resources that support the early development of the embryo are stored in the oocyte. Clearing of maternal resources and activation of the embryonic genome to produce its own mRNA transcripts marks the maternal-to-embryo transition. Dependence on stored mRNA can last from a few hours to several days, depending on animal species. The mechanisms regulating stabilization and recruitment of stored maternal transcripts have not yet been described in full detail but are known to involve reversible polyadenylation and modulation of 3’UTR-mediated elements. RNA epigenetic modifications, new players in this field, have an important role in RNA regulation and stabilization.6A, known to be the most prevalent and involved in various aspects of RNA metabolism and physiological functions, is particularly abundant in porcine oocyte mRNA compared to liver used as a somatic tissue reference. The second objective was to compare the epitranscriptome machinery, such as methyltransferases (“writers”), binding proteins (“readers”) and demethylases (“erasers”) catalyzing the different process, in follicles and oocytes of different mammalian species by immunofluorescence and confocal microscopy. The expression and localization patterns of these proteins differ between mice, pigs and cows ovaries and oocytes. m5C-associated proteins were generally less abundant. In contrast, m6A-associated proteins were expressed strongly during the early and late stages of folliculogenesis. Transzonal projections were found to contain more granules bearing the m5C mark in mice but both m5C and m6A methylation marks in association with mature oocytes of pigs and cows. Eraser proteins showed the greatest interspecies diversity in terms of distribution in the germinal tissues.The objectives of this study were first to determine if some of post-transcriptional methylation of stored mRNA is greater in oocytes than in somatic cells. We found that mSo far, few studies have looked at the oocyte and ovarian epitranscriptomic profile. Our findings indicate that a hitherto unrecognized species-specific layer of transcript regulation occurs at the RNA level and might be consequential during the oocyte transcriptional silencing period.The online version contains supplementary material available at 10.1186/s13048-023-01172-8. A central tenet of gene expression theory is that DNA-encoded information is first transcribed to RNA, an intermediary, transitory and short-lived molecule that is translated into protein as needed. However, oocytes display atypical RNA management, stocking molecules synthesized during oogenesis to support early development until embryonic genome activation . MessengMaternal RNA reserves ensure protein synthesis in the absence of transcriptional activity prior to genome activation , 5. TranThe lifespan of maternal transcripts has not been measured directly but could in theory extend from final synthesis prior to DNA compaction in the oocyte to genome activation in the embryo. Compacted DNA is seen in oocytes still in the follicle. Based on the mean time for ovulation from this point plus fertilization and activation of embryo development, this lifespan can be estimated at 30 h in mice, 7 days in pigs and 10 days in cattle.It is known that stabilization of maternal RNA involves removal of the poly(A) tail and packaging into ribonucleoprotein complexes . In addi6-Methyladenosine (m6A), occurring mainly on mRNA and long non-coding RNA [Another type of post-transcriptional modification is RNA editing. Analogous to the epigenome, which is based primarily on 5-methylation of DNA cytidine groups  , transcrding RNA . Enzymes6A, were found abundantly in maternal RNA, as was the contingent of different RMPs, in some cases with species specific distributions.We posit that some transcript labelling mechanism must exist and we hypothesized that different RNA methylation marks and process are involved in stabilizing stored maternal RNA. The aim of this study was therefore to compare the type and the abundance of RNA modifications in oocytes and somatic cells and to investigate if a relationship exists between the types of RMP present in oocytes of animals with different length of the transcriptional silence period. Modifications, namely mAll animals used in this study were handled according to the guidelines of the Canadian council on animal care and manipulation of animals used in research. Mouse experiments were carried out on 8-weeks old animals in accordance with protocols approved by the Comité de Protection des Animaux du Centre Hospitalier Universitaire de Québec. Bovine ovaries from 2–5 years old dairy cows and porcine ovaries and liver tissues from 5 months old gilts were collected from a local slaughterhouse.All chemicals and enzymes, unless otherwise specified, were purchased from Sigma-Aldrich .Total RNA from oocytes, cumulus and granulosa cells were extracted with the Picopure RNA Isolation Kit  including on-column DNAse I digestion  in accordance with the manufacturer’s instructions. Cumulus and granulosa cells were homogenized beforehand using QIAshredder spin-columns (Qiagen) and ovarian and liver tissues were homogenized using a Ruptor bead mill as recommended by the manufacturer . Total RNA was extracted from homogenates using TRIzol (Thermo Fisher Scientific) then isolated using a Picopure kit. Total RNA integrity and concentration were evaluated on a 2100-Bioanalyzer  using the RNA 6000 Nano Kit (Agilent Technologies). Samples with a RIN greater than 7 were chosen.p < 0.05.RNA was converted to mono-ribonucleotides as described previously , 22. BriOvarian tissue  was fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline  overnight at room temperature then dehydrated in ethanol at increasing concentrations and embedded in paraffin. Sections 7 µm thick were cut on a microtome, mounted in series on positively charged glass slides, dewaxed with xylene and rehydrated by washing with ethanol at concentrations decreasing to zero in distilled water. The slides were then processed in heat-induced epitope retrieval buffer  in a pressure cooker at 95 °C for 20 min as per the manufacturer’s instructions . Slides were cooled for about 5 min at room temperature then washed 3 times in PBS for 5 min each. Non‐specific binding was prevented by blocking with 5% donkey serum diluted in Tris-buffered saline with Tween 20 (TBST) for 1 h before applying the primary anti-rabbit or anti-chicken antibody diluted in TBST or the corresponding secondary antibody as a negative control  paraformaldehyde in PBS-polyvinyl alcohol (PBS-PVA) for 15 min followed by three 5-min washes in PBS-PVA then permeabilized in Triton X-100 in TBST 1:200) for 15 min, blocked for 1 h with donkey serum (Sigma-Aldrich) diluted in TBST (1:20) and contacted with the primary antibody (in TBST with 20% BSA) overnight at 4 °C. Application of secondary antibody alone served as a negative control  fitted with a 40X water-immersion Zeiss objective and Zen Black acquisition software. The images were analyzed using Fiji ImageJ . Seconda1-methyladenosine, N6-methyladenosine, 5-methylcytosine and 7-methylguanosine  in total RNA of oocytes, cumulus cells, granulosa cells, ovarian and liver tissue from pigs were detected quantitatively using LC–MS/MS. Liver was used as a somatic cell reference since mRNA is rarely stable in this tissue. As shown in Fig. 1A was less abundant in oocytes than in liver, ovary and granulosa cells whereas m6A was more abundant in oocyte compared to liver. In the case of m5C and m7G, cumulus cells stand out with a low abundance of these molecules  or tRNA aspartic acid methyltransferase 1 catalyzes methylation of cytosine. It is also slightly active as a DNA methyl transferase. This writer protein was barely detectable in early antral follicles of mice, pigs and cows and in mouse and cow oocytes , which produces m5C-modified RNA [A family of proteins binds to methyl-CpG domains on DNA . One of fied RNA , 26. Thi6A (N6-methyladenosine) to regulate mRNA stability. YTHDF2 was distributed throughout the oocyte cytoplasm in all three species  member of the YTH domain family, recognize and bind to m6A [The RNA-binding protein FMRP  is involved in shuttling transcripts and regulating their docking on ribosomes , 28. Its6A . It was Chemical marks imprinted on RNA by writer proteins can be erased by the action of demethylases such as AlkB homolog 5, RNA Demethylase (ALKBH5) or by FTO Alpha-Ketoglutarate Dependent Dioxygenase (FTO). In early follicles of all three species, ALKBH5 was found mostly in the oocyte nucleus Fig. A. In antDrosophila and Xenopus have shown clearly that mRNA is not stored randomly in the oocyte cytoplasm [Oocytes accumulate RNA reserves to sustain protein synthesis during the transcriptional silence that reigns from the moment the genome condenses in the immature oocyte until activation of the embryonic genome , 30. Theytoplasm , 32. Furytoplasm , 34. The6A modification of RNA is more frequent in oocytes than in liver cells at least in pigs has interesting implications for maternal transcripts, since it has been demonstrated that an abundance of m6A near the stop codon in 3’UTRs affects polyadenylation site selection [6A marks in the 5’ and 3’ UTR gene coding regions [6A is abundant in granulosa cells and this mark is greater in small (< 3 mm) compared to large follicles (> 5 mm) demonstrating a potential role of m6A during folliculogenesis through the modulation of specific transcript [6A associated gene in granulosa cells from aging women, an increased number of m6A methylated genes compared to younger female group [6A marks throughout the long non-coding RNA of XIST [6A in female fertility and its implication in various functions from transcript stability to folliculogenesis and ovarian functions through aging.The finding that melection . Another regions . Moreoveanscript . Since rle group . Differele group . Transcr of XIST . Taken t5C modifications [5C by mass spectrometry, expression of TRDMT1 was very low in all species except for some in the nucleus of porcine oocytes. The presence of TRDMT1 in the nucleus was expected since it has CpG DNA methylation activity, which plays a key role in oogenesis and embryonic development [Drosophila, 5-hydroxymethylcytosine is found on polyadenylated RNA and promotes mRNA translation in different physiological processes including embryogenesis [6A writer is known to play a role in embryogenesis and tissue differentiation in the zebra fish [wtap knock-out becomes lethal during early embryogenesis [Just like on DNA, epigenomic marks on RNA are due to enzymatic activities that transfer, recognize or remove them. Some of these enzymes have been associated with infertility of one type or another. TRDMT1 (DNMT2) combined with NOL1/NOP2/SUN as partner enzymes has been shown to catalyze writing of mications . Althougelopment , 42. Cytelopment . In Drosogenesis . In the bra fish  through bra fish , 46. In ogenesis .FMR1 is located on the X chromosome and codes for an RNA-binding protein [FMR1) is considered as an m6A reader since it controls the export, the stability and the translation of methylated RNAs [6A appears to be sequence-dependent, and possibly involve interaction with YTHDF [FMR1 dysregulation is known to increase the risk of primary ovarian insufficiency leading to premature menopause [The reader proteins MBD2, YTHDF2 and FMRP were detected in the cytoplasm of early and mature oocytes in all three species. MBD2 proteins offer different localization in mice and bovine early embryo stages and MD2 knockout mice is not known to lead to gross abnormalities and animals remain fertile , 48. How protein . FMRP , a post-transcriptional modification that occurs on the first nucleotide on mRNA following the m7G cap [6Am mark stabilizes mRNA [6A during oogenesis. The differences between species in terms of FTO are consistent with the timing required for the breakdown of maternal reserves.The greatest differences between species were observed for eraser enzymes, both in terms of abundance and localisation. FTO was the most abundant in the oocyte cytoplasm and most abundant in mice. ALKBH5 and FTO are both essential for fertility, the former being involved in mRNA export and RNA metabolism as well as important for spermatogenesis  and the  m7G cap . The revzes mRNA . Our obs6A writer WTAP is detectable in transzonal projections of both large mammals but not in those of mice. The three readers were found inside projections in all three species, whereas FTO was only in the case of cattle. This is consistent with observed cross-species differences in the duration of transzonal attachment to the oolemma, for example, persisting during maturation in mice, rats, rabbits and humans but disconnecting and retracting immediately after germinal vesicle breakdown in cattle [We have reported previously that in cattle, transzonal projections connecting cumulus cells to the oocyte act like synapses and can harbor ribonucleoprotein granules or transfer these and other complex materials to the oocyte , 62. RNAn cattle .6-methyladenosine pulldown has allowed identification of FMRP, FXR1P and FXR2P as new readers [6A residues [The mechanisms underlying RNA shuttling through transzonal projections are still largely unknown, but we and others have described the role played by FMRP in RNA granules transport in mouse neurons . N6-meth readers . Moreoveresidues , 64. The6A eraser FTO. Increased presence of RNA modifying enzymes in transzonal projections was associated with greater dependence on RNA stability. This study provides support for an important role played by the epitranscriptome in the management of maternal RNA stored in mammalian oocytes.Three animal species were compared in an attempt to reveal the role of methylation in the persistence of maternal mRNA during mammalian early development. The longest transcriptional silence period and the earliest disconnection of transzonal projections following meiosis resumption are observed in bovine oocytes. Murine oocytes have the shortest transcriptional silence and are the least dependent on transcript stability and storage. Known modifiers of RNA nucleoside bases were very abundant in oocytes of all three species from the early to antral follicle stages. Species-associated differences in the distribution of these enzymes were observed, notably for the mAdditional file 1:Table S1. Description of the antibodies used for immunofluorescence in this study.Additional file 2:Figure S1. Ovarian section in paraffin, negative control without primary antibody, showing no specific staining by the secondary antibody. DNA is stained with Hoescht 33342 (blue). Bar = 20 µm.Additional file 3:Figure S2. Oocyte whole mount, negative control without primary antibody, showing no specific staining by the secondary antibody. DNA is stained with Hoescht 33342 (blue) and actin with Acti-stain 555 phalloidin (red). Bar = 20 µm (same magnification was used for swine and bovine).Additional file 4:Stage and diameter of the follicles used in this study and the corresponding chromatin stage and transcription activity reported in the literature[iterature-68.


Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg Compact RNA structural motifs control many aspects of gene expression, but methods for their identification are lacking. Here the authors present a sequencing-based terbium probing approach to detect complex 3D structural elements, which can be used to pinpoint potential riboregulatory elements. It has recently become clear that many types of multi-kilobase RNA transcripts contain regions of tertiary structure that, either alone or in concert with protein partners, carry out biological function7. However, identifying these regions of complex RNA structure remains challenging. Current structure prediction methods on long RNAs are unable to pinpoint regions containing stable RNA tertiary structure modules or complex protein binding sites from sequence alone8. While biophysical techniques such as NMR9, x-ray crystallography10 and cryo-EM11 are invaluable tools for the observation of RNA structure, they are time-consuming and difficult to perform on a multikilobase-length RNA that contains a mixture of both structured and flexible regions. As our understanding of their biological functions becomes increasingly important, and interest in small molecule targeting of RNAs grows, it is vital to develop tools for identifying regions of tertiary structure in long RNA molecules.RNAs can adopt complex folded motifs and higher-order 3-D structures that are essential across a variety of specific cellular processes16, and these data have primarily been used to infer secondary but not tertiary structures of RNA. Fewer methods have been developed to detect higher order structure and these protocols are limited to an assessment of solvent accessible regions19 or identification of long-range RNA-RNA base-pairs by cross-linking methods21 or statistical correlations in mutational profiling23. The field would benefit from a readily adaptable, high throughput approach for identifying regions of local tertiary structure, which are often hallmarks of functional RNA motifs and riboregulatory elements.In recent years, chemical probing has become a powerful tool for studying RNA structure. Many important advances have improved our ability to identify single- versus double-stranded nucleotides in RNA25  normally occurs on a slow timescale that ranges from hours to days28, the same phenomenon is greatly accelerated by lanthanide ions such as terbium (Tb3+) and europium (Eu3+)29. Tb3+ and Mg2+ share similar ionic radii 26 and coordination geometry preferences for oxygen30, but lanthanide ions have an additional positive charge and the pKa of coordinated water molecules is much lower for ions such as Tb3+, (pKa ~7.9 for Tb3+-H2O versus ~11.4 for Mg2+-H2O)31. Therefore, at low ion concentrations and neutral pH, Tb3+ coordinates with structured RNA binding sites in a manner that is similar to that of Mg2+ 35, but the more potent Tb3+ general base rapidly facilitates RNA backbone cleavage at sites of metal ion binding31  with a processive reverse transcriptase, MarathonRT37. We used the D135 ribozyme derived from yeast group II intron aI5γ, which has been extensively characterized using the classical version of Tb3+ probing36. We found that reverse transcription stops  that has been well characterized biochemically and crystallographically39. First, reagent concentrations and reaction times were optimized to obtain an ideal reactivity signal and ensure the RNA is not over-cleaved , non-specific cleavage is observed . Second, these sites must be observed in two independent replicates to demonstrate reproducibility. Third, selected sites must show a dependence of signal on Tb3+ concentration to ensure that stop signals are not due to spontaneous RT termination events. Nucleotide sites that satisfy these criteria are highlighted in red in the secondary structure diagram of the O.i. intron 41. Here, a single G236 residue in D1 flips out of a sharp backbone turn and base-stacks with A370 in D5  internal ribosome entry site (IRES), specifically focusing on domain II, which has well-characterized structural features identified by both cryoEM7. We decided to conduct the first experiments on a structurally well-defined cellular RNA with known protein binding sites. To this end, we probed human RNase P in order to understand how terbium can be used to reveal higher-order RNA structural motifs in that stable RNP. To circumvent the issues of introducing lanthanide ions into cells, we developed an approach for gently lysing mammalian cells in a way that maintains intact RNA-Protein (RNP) complexes , form networks of hydrogen bonds with proteins Rpp20 and Rpp30b , which strips proteins from RNA.  and compared these data to the corresponding distances in a simple helical structure within domain 4 of the intron  relative to the same distances calculated from a simple helix (9.6-12.5 Å), indicating local compression of the RNA backbone. We speculate that additional data will allow for the refinement of these parameters. For example, while some Tb3+ cleavage sites, such as those in region 7, are not characterized by small Pn -> Pn+2 values, visual inspection of the structure shows that these same nucleotides are part of a larger motif in 3-D space that contains adjacent pinched backbones that are characterized by strong Tb-seq signatures and small Pn -> Pn+2 values (in region 5). Therefore, the same bound metal ion may be catalyzing both cleavage events. Additional data and analysis will enable a more quantitative description of specific structural features that are recognized and cleaved by Tb3+.Performing Tb-seq on RNAs with known structures provided a useful starting point for assessing the types of RNA motifs that are recognized and cleaved by Tb3+ cleavage signals in RNA probing studies. First of all, metal ions like Tb3+ cannot stimulate the cleavage of an RNA backbone unless the 2’-hydroxyl nucleophile adopts a precisely in-line geometry capable of phosphodiester cleavage. Certain architectural environments may constrain local structure such that in-line attack is not possible, and it is therefore likely that some Tb3+-bound sites remain uncleaved by the probe, resulting in false-negative data. Furthermore, it is not possible to interpret a Tb3+ cleavage site as reflective of a particular type of RNA structural motif. This is because there is simply not enough information on site-bound metal ions in the available RNA structural database. Although it is hoped that this may grow with time, the emergence of RNA cryo-EM structure determination  and the decline in the prevalence of crystallographically-determined structures , indicates that progress will be slow, making it all the more important that additional orthogonal information on metal sites in RNA is obtained using other methods. As discussed in the previous paragraph, additional data will be necessary to understand the precise structural features that result in cleavage by Tb3+. That said, it is reasonable to claim that Tb3+ cleavages reflect metal ion binding to sites of high electronegative potential that result from the close-packed RNA backbone atoms that are prevalent within RNA tertiary structures and RNP complexes.There are limitations in our ability to interpret the structural significance of TbThe human transcriptome contains a vast set of large, complex RNA molecules, and until recently, we have lacked the tools to assess their 3-D structural content. However, the biochemical methods that were initially developed to study tRNAs, riboswitches and ribozymes are being gradually being adapted to explore the growing repertoire of multi-kilobase RNAs that are central to gene expression and pathogenicity. Here we present a much-needed expansion of the RNA probing toolbox that allows investigators to rapidly pinpoint candidate RNA tertiary structures efficiently and precisely, paving the way for downstream mechanistic study and therapeutic targeting.59 and Oceanobacillus iheyensis (O.i.) group II intron D1-539, RNA were carried using T7 RNA polymerase60 in a buffer containing 12 mM MgCl2, 40 mM Tris-Cl pH8, 2 mM Spermidine, 10 mM NaCl, 0.01% Triton X-100, 10 mM DTT, 5 μl SUPERase-In and 3.6 mM of each NTP, in a procedure adapted from previous work61. The reactions were incubated at 37 °C for 2 hours. Thereafter, 4U of TURBO DNase was added and the mixture was incubated at 37 °C for 30 min. To chelate excess divalent ions, 5 μl of 0.5 M EDTA was added. Transcription products were gel purified on a denaturing 5% polyacrylamide gel and eluted overnight at 4 °C in a gel elution buffer . The RNA was ethanol precipitated and resuspended in ME buffer . The in vitro transcription of the full-length HCV genome (JC1)62 was performed as described above. The transcribed RNA was buffer exchanged into a filtration buffer  was added and folded at 37 °C for 30 min. Subsequently, probing was performed by incubating 18 μl of the folded RNA with 10X TbCl3 stocks prepared in the monovalent buffer  or 2 μl of monovalent buffer (negative control) for 40 min at 25 °C. For the time-course experiments, probing was performed at the indicated times. All reactions were quenched with the addition of 3 μl of 50 mM EDTA pH 8 and precipitated by adding 1/10 volume of Na-Acetate , 0.5 μl of glycogen (Invitrogen) and three volumes of ethanol. RNAs were resuspended in 4 μl of loading buffer (82 % (v/v) deionized formamide, 0.16 % (w/v) xylene cyanol (XC), 0.16 % (w/v) bromophenol blue (BB), 10 mM EDTA, pH 8.0) and resolved on a denaturing 5% polyacrylamide gel. The gel was dried, exposed to phosphor screens overnight and scanned using a Typhoon FLA9500 phosphorimager  or Typhoon RGB Biomolecular imager (Cytiva).For D135, Tb3+ probing of O.i. and HCV, 1 μg of RNA was diluted in their respective monovalent ion buffers (50 mM HEPES pH7 and 150 mM KCl for O.i. or 50 mM HEPES pH7.2 and 150 mM KCl for HCV) to a final volume of 18 μl. Thereafter, 2 μl of 100 mM MgCl2  was added and incubated at 37 °C for 30 min. Subsequently, probing was performed by incubating 18 μl of the folded RNA with 2 μl of 10X TbCl3 stocks prepared in their respective monovalent ion buffers  or 2 μl of respective monovalent buffer (negative control) for 10 min at 25 °C. Reactivities were compared under conditions where 0.5 mM TbCl3 was employed and used in all figures unless indicated otherwise. All reactions were quenched with the addition of 3 μl of 50 mM EDTA pH 8. For the denaturing control, RNA was folded as described above but afterward, deionized formamide was added to a final concentration of 50%. The denatured RNA was probed with a final concentration of 0.5 mM TbCl3. For the secondary structure control, the RNA was incubated in a monovalent buffer in the absence of MgCl2 and probing was carried out at the indicated TbCl3 concentrations. All RNA samples were cleaned up using a Zymo RNA clean and concentrator column according to the manufacturer’s instructions.For Tb40 were prepared as described above except, 1 mM UTP and 50 μCi of [α-32P-UTP] were added to the transcription. The reaction was incubated at 37 °C for 2 h followed by purification on a denaturing 5% polyacrylamide gel. 5 nM of the radiolabeled transcript was spiked into the folding reaction described above. Reactions were mixed with 10% (v/v) glycerol containing, 0.16 % (w/v) xylene cyanol, 0.16 % (w/v) bromophenol blue and loaded onto a native 5% polyacrylamide gel.Radiolabeled transcripts6 cells) in a 150 cm tissue culture-treated dish.For in-cell studies of RNase P RNA structure, Huh7.5 cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM w/o sodium pyruvate) that was supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1 mM non-essential amino acids. Cells were cultured to ~80% confluency  of SARS-Related Coronavirus 2 Isolate USA/WA2020 (BEI Resources #NR-52281) was added to each flask in fresh media. Cells were incubated with virus inocula until three days post-infection (dpi).For studies of SARS-CoV-2 RNA, Huh7.5 cells were cultured in DMEM supplemented with 10% FBS and 1% Penn/Strep. Approximately 5 × 102, 1 mM CaCl2, 30ul SUPERase-In (20U/μl) and 1x cOmplete Protease Inhibitor Cocktail EDTA-free. To 250 μl of resuspended cells, 50/μl of Turbo DNase (2U/μl) was added and the mixture was incubated at 37 °C for 20 min. For cell lysis+Proteinase-K probing experiments, cells were prepared, lysed and DNase digested as described above, but the lysis buffer did not contain protease inhibitor. Subsequently, 25 μl of 20 mg/ml Proteinase-K was added to each 250 μl of lysed cells and the mixture was incubated at 37 °C for an additional 20 min.For all flasks the media was aspirated, cells were washed once with cold wash buffer , and then dislodged in 2 ml of cold wash buffer with a cell scraper. The cells were collected and centrifuged at 200 g x 5 min at 4 °C. The supernatant was removed and the cells were resuspended in 2 ml lysis buffer . The reactions were immediately placed on a rocker and allowed to incubate at 25 °C for 10 min before quenching with 20 μl of 0.1 M EDTA. RNA was extracted using Trizol according to the manufacturer’s instructions. For experiments involving RNase P, total RNA was ribosome depleted using a Ribominus kit that was used according to the manufacturer’s protocol with the following exception: the ribodepleted supernatant was purified using a Zymo RNA clean and a concentrator to retain RNAs that are greater than 17 nucleotides in size. For experiments involving SARS-CoV-2, total RNA was cleaned using a Zymo RNA clean and concentrator column.All reactions were centrifuged at 200 g x 15 sec. Probing was performed by incubating 225 μl of supernatant with 25 μl of freshly made 10x TbCl37 (can be obtained from Kerafast), 10 μl of 2x MarathonRT buffer , 1 μl of 10 mM dNTP mix (NEB) were added and incubated at 42 °C for 30 min. RNA was degraded with the addition of 1 μl of 3 M KOH, heated to 95 °C for 5 min and snap cooled to 4 °C for 5 min. Thereafter, 1 μl of 3 M HCl was added to neutralize the reaction. For primer extension reactions that would be visualized using electrophoresis, reverse transcription was carried out as described, but using a 32P-labeled primer. The primer was labeled at the 5-end using T4 PNK according to manufactures instructions and purified on a denaturing 12% polyacrylamide gel. After reverse transcription, the cDNA was ethanol precipitated at −20 °C overnight. The cDNA pellets were dissolved in 5 μl of loading buffer  and resolved on a denaturing 5% polyacrylamide gel. The gel was dried, exposed to phosphor screens overnight and scanned using a Typhoon RGB Biomolecular imager (Cytiva). For ladder generation, RT was carried out using a Thermo Sequenase cycling kit according to the manufacturer’s instructions with an input of 500 ng of the template.For each probing condition, 1-4 μg of in vitro transcribed or cellular RNA was mixed with 1-2pmol of gene-specific primers .All FASTQ files were processed using Cutadapt v1.9.1) to remove Illumina adapter sequences and then aligned to the respective RNA sequence using HISAT2 (v2.10). Stop information was extracted using RTEventsCounter.py script.9.1 to r3 (in-vitro transcribed RNAs) or 1 mM TbCl3 (cell lysate RNAs) was employed and used in all figures unless indicated otherwise. For the Δ Tb analysis, the reactivity obtained from cell lysate + Proteinase-K probing experiments was subtracted from the reactivity obtained from cell lysate probing experiments (Eq. (Reactivities were compared under conditions where 0.5 mM TbCl65. All three-dimensional structure renderings were done using PyMOL Molecular Graphics System, v1.2r3pre, Schrödinger, LLC. Graphical displays were made using GraphPad Prism 8 or RStudio Version 1.2.5001. All gels were visualized using ImageJ 1.52a or ImageQuant TL v8.2.0.0.All secondary structures were visualized and drawn using Structure Editor, v1.0n = 2 was chosen for most experiments. A sample size in this case corresponds to biological replicates undergoing of full chemical probing data on intact RNAs. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.Sample size of as Further information on research design is available in the Supplementary Information FileReporting Summary


USB1, Dyskeratosis Congenita (DC) for PARN and Pontocerebellar Hypoplasia type 7 (PCH7) for TOE1. Herein, we review the biochemical properties of USB1, PARN, and TOE1, how they modulate ncRNA levels, and their roles in human diseases.The levels of non-coding RNAs (ncRNAs) are regulated by transcription, RNA processing, and RNA degradation pathways. One mechanism for the degradation of ncRNAs involves the addition of oligo(A) tails by non-canonical poly(A) polymerases, which then recruit processive sequence-independent 3′ to 5′ exonucleases for RNA degradation. This pathway of decay is also regulated by three 3′ to 5′ exoribonucleases, USB1, PARN, and TOE1, which remove oligo(A) tails and thereby can protect ncRNAs from decay in a manner analogous to the deubiquitination of proteins. Loss-of-function mutations in these genes lead to premature degradation of some ncRNAs and lead to specific human diseases such as Poikiloderma with Neutropenia (PN) for  Gene expression in eukaryotes is regulated at multiple levels. While transcription regulation plays an important role, the regulation of RNA processing and degradation are also important in modulating the levels of both mRNAs and non-coding RNAs (ncRNAs) .After transcription, 3′-end modifications of mRNA molecules can modulate the fate of newly synthesized RNAs. The 3′ ends of mRNAs are dynamically changed by the opposing effects of poly(A) polymerases and exonucleases, which can affect all aspects of mRNA metabolism . For exaSimilarly, 3′-end modifications of ncRNAs can regulate RNA processing and/or degradation. For instance, the addition of CCA to tRNA and uridylation of U6 small nuclear RNA (snRNA) are involved in RNA maturation , 10, 11,Oligo(A) tail addition can also promote the degradation of some ncRNAs. For example, oligoadenylation by PAPD5/PAPD7 of miRNAs, human telomerase RNA (hTR), Y RNAs, ribosomal RNAs (rRNAs), small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs (scaRNAs), and small nuclear RNAs (snRNAs) can recruit exonucleases to degrade the RNAs , 29, 30.As oligo(A) tails can promote RNA degradation, it is not surprising that a set of deadenylases can remove oligo(A) tails and thereby stabilize some ncRNAs. To date, there are three such ncRNA deadenylases that can regulate ncRNAs in this manner including the poly(A) specific ribonuclease (PARN), USB1 , and Target of Erg1 . Recent studies have shown that these enzymes regulate the stability of several ncRNAs in mammalian cells, such as hTR, Y RNAs, piwi-interacting RNAs (piRNAs), and miRNAs by removing poly(A) tails added by PAPD5/7. The poly(A) tail removal limits the recruitment of exonucleases DIS3L, DIS3L2, and/or the nuclease exosome , 36, 37.The process of oligo(A) tail addition and removal regulating the degradation of ncRNAs can be considered analogous to the control of protein degradation by ubiquination and deubiquination . In thisUSB1 (USB1 mutants) lead to the genetic disorder Poikiloderma with Neutropenia (PN), an autosomal-recessive bone marrow failure (BMF) syndrome with marked clinical overlap with Dyskeratosis Congenita (DC) (PARN (PARN mutants) cause a severe form of DC called Hoyeraal-Hreidarsson syndrome, which causes abnormally short telomeres and congenital defects (TOE1 (TOE1 mutants) cause Pontocerebellar Hypoplasia type 7 (PCH7), a unique recessive syndrome characterized by neurodegeneration with ambiguous genitalia  . In cont defects , 40, 41 enitalia , 45.Herein, we review the properties of USB1, PARN, and TOE1, and their roles in RNA regulation. As mutations in these enzymes lead to human diseases, understanding their functions and targets may provide insights into treatments for these diseases.6D30 and 6D2Z) (S. pombe) USB1, and His133 and His231 in budding yeast (Saccharomyces cerevisiae) (PDB ID 5UQJ). Although the overall sequence conservation can be rather low between family members, all known crystal structures of 2H family members, including USB1, display a characteristic fold with conserved terminal and transit lobes and the H x S/T motifs centrally positioned in a substrate binding cleft  , His109 ng cleft , 65, 66.In contrast, PARN and TOE1 belong to the DEDD protein superfamily, one of the six exoribonuclease superfamilies , 68. ExoPARN consists of three domains: an RNA recognition motif domain (RRM), a nuclease domain, and an R3H domain . Mutatioin vitro, demonstrating these residues are important for TOE1’s enzymatic activity  (S. cerevisiae USB1’s 2′-CPDase activity remained poorly understood. By comparing S. cerevisiae USB1 (PDB ID 5UQJ) and K. marxianus USB1 structures (PDB ID 6PFQ), it has been suggested that the CPDase activity comes from a loop structure that is conserved in yeast and forms a distinct penultimate (n-1) nucleotide binding site , humans and nd 5UQJ) , 64. Mornd 5UQJ) , have prnd 5UQJ) , 64. Howing site . This suUSB1 deletion leads to cell death  show global defects in pre-mRNA splicing, and this can be reversed by over-expressing U6   tails in vitro  , 58. Theto hESCs . First, in vitro , 57. TheUSB1 expression in hemopoietic tissue  specific nuclease, first identified in extracts of  oocytes . PARN re oocytes . PARN ha oocytes , 90. Thi oocytes . This obin vitro, suggesting that 3′-end poly(A) removal is linked to 5′ end cap structure of the RNA substrates  tail during deadenylation , 94, 95.bstrates , 91, 93.bstrates , 90. Howbstrates . Interesbstrates . This suXenopus oocyte  shortening during s oocyte , 99, 100s oocyte   shortening , an age-related disease featuring processive lung scarring (6B)  , 29, 111PARN requires binding partners for proper regulation in cells. The binding partners can either repress or activate the enzyme’s activities. For example, the interaction of the poly(A) tail with poly(A) binding proteins (PABPs) and/or the interaction of cap-binding proteins such as eIF4E and CBP80 with the 5′ cap structure has been shown to repress PARN and protect RNA substrates , 114. OnTOE1 is a deadenylase that was first identified as a target of Erg1, an immediate early transcription factor. One function of Erg1 is to decrease the growth and tumorigenic potential of several tumor cell types , 119 andTOE1 exhibits deadenylation on RNA substrates and shows a preference for poly(A) . HoweverTOE1 has been shown to deadenylate several ncRNAs, including snRNAs . More spTOE1 expression also shows effects on development. Over-expression of TOE1 negatively affects the growth of HEK293 and H4 cells by altering the cell cycle through the induction of p21 , 74. On C). This was first suggested by the identification of various TOE1 mutations in PCH7 patients , and spliceosomal proteins , 34, 77;USB1, PARN, and TOE1 are all involved in human diseases featuring abnormal 3′ end extensions of ncRNAs . To date, there are 38 PN patients reported with 19 different mutations in C16orf57 , a poikiloderma that is sometimes confused with PN . A previous study showed that lymphoblastoid cells from PN patients do not exhibit reduced U6 snRNA levels and have normal pre-mRNA splicing , and the catalytic subunit TERC . Several studies have shown that PARN inhibition leads to a failure to remove the poly(A) tail of hTR, leading to the recruitment of nuclear 3′ to 5′ exonuclease EXOSC10, which leads to hTR degradation . TOE1 is associated with pre-snRNAs and snRNAs associated with TOE1 catalytically inactive mutant, U1, U2, and U5 snRNAs contained longer tails than those associated with WT TOE1, suggesting that TOE1 plays a role as a 3′-5′ exonuclease for specific snRNA processing . It is interesting to note that cerebellum hypoplasia also manifests in patients with telomere-related diseases, such as Hoyeraal-Hreidarsson and Revesz syndromes  tails can serve as a common pathway for regulating the stability of some ncRNAs. In this context, the poly(A) specific exonucleases USB1, PARN, and TOE1 have emerged as playing important roles in controlling the stability of ncRNAs. Looking forward there are outstanding issues to address. First, it will be important to understand why these enzymes are specifically required for some developmental transitions in both plants and mammals. Understanding these areas should shed new light on the roles of their biological roles and may identify new RNA substrates for these enzymes. Second, it will be important to determine whether therapeutic intervention in the ncRNA adenylation/deadenylation dynamic can be useful in therapeutic contexts. Currently, inhibition of adenylation by PAPD5/7 inhibitor has been suggested as possible therapy for DC and PN , 145. AlThe authors declared no conflict of interest for this review.


RNA can fold back on itself to adopt a wide range of structures. These range from relatively simple hairpins to intricate 3D folds and can be accompanied by regulatory interactions with both metabolites and macromolecules. The last 50 yr have witnessed elucidation of an astonishing array of RNA structures including transfer RNAs, ribozymes, riboswitches, the ribosome, the spliceosome, and most recently entire RNA structuromes. These advances in RNA structural biology have deepened insight into fundamental biological processes including gene editing, transcription, translation, and structure-based detection and response to temperature and other environmental signals. These discoveries reveal that RNA can be relatively static, like a rock; that it can have catalytic functions of cutting bonds, like scissors; and that it can adopt myriad functional shapes, like paper. We relate these extraordinary discoveries in the biology of RNA structure to the plant way of life. We trace plant-specific discovery of ribozymes and riboswitches, alternative splicing, organellar ribosomes, thermometers, whole-transcriptome structuromes and pan-structuromes, and conclude that plants have a special set of RNA structures that confer unique types of gene regulation. We finish with a consideration of future directions for the RNA structure–function field. As in all living systems, RNA in plants has multifarious functions, the majority of which are predicated on the proper folding of the RNA. Like proteins, RNA possesses a structure that can be described at 5 different levels: primary, secondary, tertiary, quaternary, and quinary. The primary structure is the RNA sequence itself. Secondary structure describes the paired and unpaired elements of stems, loops, and bulges that arise as the single-stranded RNA molecule folds back on itself and interacts via hydrogen bonding and stacking. Tertiary or 3D structure typically compacts the RNA and is achieved by longer distance Watson–Crick and non-Watson–Crick interactions of elements within the pre-formed secondary structures. These interactions give rise to tertiary structural elements, including pseudoknots, which lock together 2 stem-loops (SLs) by base pairing and sugar-phosphate interactions, often in a so-called kissing interaction. Quaternary structure results from a folded RNA's interaction with other biopolymers such as proteins and other RNAs, and quinary structure  arises fA given protein typically has a more limited number of closely related conformations it can take on and remain functional, and this “rock-like” property largely holds for some highly structured RNAs, such as the ribosome and transfer RNAs (tRNAs). However, unlike most proteins, many RNAs populate 2 or more distinct conformations with different functions , while fThe sections of this review are organized to introduce each RNA class with a brief historical context regarding structural aspects , followetransfers information from mRNA to the growing polypeptide chain. The sequence and secondary structure of tRNA were worked out in 1965 by Robert Holley , which can fold into a cloverleaf structure. As a point of nomenclature, we retain the conventions in the field of “tRFs” and “TLSs,” even though “tRNA” is represented as “tR” in the acronym “tRFs” and as “T” in “TLSs.” In this section, we highlight some of the key recent advances regarding the roles of plant tRFs and TLSs in RNA trafficking, translation, gene silencing, and abiotic stress response .Plant tRFs are generated in response to numerous abiotic stresses, including phosphate starvation, temperature, oxidative stress, and UV light , althougCucurbita maxima) inhibited in vitro translation in a wheat germ extract system. Interestingly, this inhibition was lost if the RNA was extracted using a denaturing protocol, suggesting an essential role of RNA structure. Although phloem sap contains a diversity of RNA species, a specific role for tRFs was demonstrated in targeted experiments wherein tRFs generated from yeast tRNA, but not intact tRNA or nucleotides, were shown to inhibit the in vitro translation of FLOWERING LOCUS T, luciferase, and AtMPB2C. tRFs’ function in translation suppression was further validated by a mimic study using a brome mosaic virus RNA in vitro translation system with yeast artificial tRNA fragments (Ala(AGC) and tRNAAsn(GUU), but not tRFs known to be associated with AGO complexes (see below), inhibited green fluorescent protein synthesis in a wheat germ extract system, and the G18 and G19 residues of the D loop were found to be essential for this repression  T-DNA mutants , derived from mature tRNAs of the majority of codon types, were reduced significantly in  mutants . In thisBradyrhizobium japonicum, tRFs produced by the bacteria enter root cells via an unknown mechanism, where they are loaded into the AGO1 (Argonaute) complex and target sequence-homologous host genes for suppression, thereby promoting nodule initiation and development  or a stem-bulge-SL-forming tRNA partial sequence  and LIGHT-HARVESTING COMPLEX GENE 3 , were investigated and both RNAs possess conserved 3WJ and MWJs (multi-way junctions) in cloverleaf-like structures as revealed by RNA structural profiling using dimethyl sulfate (DMS)-MaPseq , microRNAs (miRNAs), rRNAs, mRNAs, and tRNAs also can move locally in plants via plasmodesmata from cell to cell and/or over long distances by entering the phloem vascular system . In 2015sequence . In partsequence , again s)-MaPseq   known as telomerase, which comprises telomerase RNA (TR) and telomerase reverse transcriptase (TERT). The C-rich template for the repeats is contained within TR, which folds into a complex structure involving a conserved pseudoknot . Other sThe first publication on the discovery of telomerase was in 1985  Greider and subsn is templated by AtTR's 9 nt stretch of 5′-CUAAACCCU. They also identified TRs in diverse land plants, including crop and model plants. Dorothy Shippen and colleagues   and incrCytosolic, chloroplastic, and mitochondrial ribosomes in plants have both canonical and specialized aspects. For example, recent work suggests that the plant cytoplasmic ribosome can function as a metabolite sensor. This function was first established in bacteria  for sensThere are 2 plant-specific structures of the ribosome, 1 in chloroplasts and 1 in mitochondria. First, we consider the chloroplast ribosome structures, focusing on the pY translation factor complex from spinach leaves, which was solved by cryoEM to near-atomic resolution at ≥3.4 Å  Bieri e. ChloropTrypanosoma, wherein mitoribosomes obtained more novel ribosomal proteins and drastically decreased rRNA size, plant mitoribosomes lost few proteins and instead gained extended rRNA that binds novel ribosomal proteins. The expansions of the 5S, 18S, and 26S rRNAs make the plant mitochondria SSU and LSU rRNAs significantly larger than prokaryotic rRNAs—20% and 9% larger, respectively. Yaser Hashem and co-workers solved a 78S mitoribosome from cauliflower at near-atomic resolution  contains over 169,000 distinct transcripts representing 24,344 genes and should greatly facilitate analysis of stress- and tissue-specific splicing , one possibility is that stress sometimes leads to melting of snRNA-containing base pairs  , which wLETHAL UNLESS CBC7 (LUC7) protein was identified from RNA immunoprecipitation experiments and transcriptome analyses as a crucial component of U1 snRNP in mediating constitutive and alternative splicing events . luc7 triple mutants exhibit impaired development and hypersensitivity to ABA, cold, and salinity relative to the wild type. Both exon skipping and intron retention, particularly of terminal introns, are elevated in the luc7 triple mutant. Strikingly, transcripts retaining LUC7-dependent introns in the luc7 triple mutant escaped export from the nucleus, thereby avoiding NMD, which occurs in the cytoplasm. Interestingly, retention of the terminal intron is also enhanced in wild-type plants by cold and salinity suggesting an interplay between stress and LUC7 functionality. Finally, we note that a lncRNA has been shown to modulate alternative splicing regulators in plants (AS COMPETITOR lncRNA (or ASCO-lncRNA), interacts with a family of nuclear speckle RNA-binding proteins to control splicing patterns of several mRNA targets. The mechanism appears to involve competition between the ASCO-lncRNA and an alternative splicing mRNA target for binding these proteins and may be the tip of the iceberg for lncRNA modulation of alternative spicing  accelerating the rate of a chemical reaction; (ii) being highly specific for their substrates; (iii) undergoing multiple turnovers; and (iv) being unchanged in the reaction. The first enzyme discovered, in 1833, was diastase, which breaks down starch into maltose . For thenditions . This soch bonds . Althougch bonds , the nat2+ concentrations (prorp1 knockdown lines show defects in photosynthesis and drastic reductions in levels of mature plastid tRNA-Phe(GAA) and tRNA-Arg(ACG)  , while pe lethal , as are  mutants .Group II introns comprise another large RNP complex with ribozyme characteristics. Group II introns, which are evolutionarily related to the spliceosomal RNAs in mechanism (see above), can operate as ribozymes without proteins in vitro, although they tend to associate with proteins in vivo . Like GrThe above ribozyme examples are of relatively large RNAs, typically with lengths of 300 or more nucleotides. It is convenient, in fact, to separate ribozymes based on size. The smaller ribozymes, which typically range from ∼50 to 100 nts, encompass 10 subclasses: hammerhead, HDV, twister, glmS, twister sister, pistol, CPEB3, hairpin, pistol, and VS . Each suNowadays, once a new small ribozyme is discovered, its 3D structure and chemical mechanism are typically solved in less than a year. While much has been learned about the chemistry of ribozymes , relativcox 1 (cytochorome oxidase subunit 1) mitochondrial gene of green algae and the nonvascular land plant, Marchantia (a liverwort), but were originally detected in only 1 flowering plant genus, Peperomia. With the rise of molecular phylogenetics, however, this sequence was found in 1998 to be present in cox1 genes in 48 out of 341 angiosperms surveyed by Southern blot analysis  and Ara2 (At4g30870). Ara1 expression was detected by RT-PCR in leaves, stems, and flowers, but not siliques, while Ara2 expression was not detected. These ribozyme sequences were shown by in vitro assays to be catalytically active  that do not thermoregulate . RNA theperature .Synechocyistis hsp17 gene, in which the G•U wobbles are replaced by canonical GC base pairs (Two of the most common RNA thermometers are the 4U element and ROSE (repression of heat shock gene expression) thermometers. In the 4U element, the UUUU sequence base pairs with an AGGA SD sequence, with the 2 interior base pairs comprising G•U wobbles, which are thus particularly susceptible to melting. An alternative UCCU thermometer is present in the 5′-UTR of the cyanobacterial se pairs . HSP17 cse pairs .The ROSE element consists of 2–4 SLs . OriginacIII gene of the bacteriophage λ, exists in 2 conformations at equilibrium, like the bacterial RNA thermometers. However, based on in vitro structure probing with DMS  report whole-transcriptome restructuring during heat. In yeast, Chang and colleagues  extractePHYTOCHROME INTERACTING FACTOR 4 (PIF4) was a milestone in understanding temperature signaling mechanisms in plants. PIF4 upregulates the expression of several key auxin biosynthesis genes at high temperatures, including TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS  and SMALL AUXIN UP RNA (SAUR) family genes that are known from T-DNA knockout studies to be required for plant development and heat-stimulated hypocotyl elongation  also mediates thermomorphogenesis, as knockout of either PIF4 or PIF7 impairs heat-stimulated hypocotyl and petiole elongation. Interestingly, a recent study revealed that the enhanced translation of PIF7under warm temperatures is mediated by temperature-responsive structural changes in the 5′-UTR of the PIF7 mRNA , suggesting a conserved temperature-responsive translational regulatory machinery at the RNA structural level that allows plants to acclimate to temperature changes  operon. Research from the Yanofsky lab on a series of deletion mutants of trp demonstrated that a structure in the 5′-leader sequence which they dubbed the terminator, consisting of a very stable G-C-rich hairpin followed by a series of Us that make unstable base pairs with templating dAs -induced transcription termination  gene of mination . Breakermination  demonstr2+ ions interacting with the pyrophosphate moiety of TPP  have also been documented. In addition, as noted by Breaker and colleagues . As elegantly revealed by PCR-based and Northern blot analyses of THIC transcript composition and length  is a precursor for the formation of TPP, an important cofactor for enzymes in the TCA cycle and pentose phosphate pathway. Thiamine levels rise under abiotic stresses associated with ROS production , and exogenous thiamine application reduces H pathway . Thiamin pathway , and in  pathway . HoweverxLyG)4, where L is a loop nucleotide and x ≥ 2 and y is typically ≤7. For instance, the sequence 5′-GGAGGAGGAGG can fold into a 2-tier parallel-stranded quadruplex, in which G1, G4, G7, and G10 form 1 quartet, and G2, G5, G8, and G11 form the second quartet, which stacks on the first one. Biological sequences can also possess more complex but still contiguous motifs containing irregular repeats of Gs and Ls. Additionally, GQs can form from noncontiguous stretches of Gs, especially as elucidated in structures of dye-binding aptamers 4, or by the association of up to 4 different strands in which case (xLyG)n can form with n < 4, making this interaction plausible in the concentrated environment of a phase-separated liquid droplet (Tandem repeats of Gs can lead to a structure known as a G-quadruplex (GQ), which involves pairing of 4 different Gs in a planar array called a “G-quartet” . These saptamers , and pro droplet .In RNA, the GQ is often found in the 5′-UTR  where it+ levels, are enriched in GQ sequences. This seminal bioinformatic study suggested that GQ sequences could regulate the expression of key proteins during stress response. In the last decade, additional studies have provided further support for GQ formation in plants.Perhaps the first study that suggested GQ formation in plants was a bioinformatic analysis from our labs   has beenHIRD11 mRNA (At1G54410) encodes a KS-type dehydrin and contains a G2 (with 2 G-quartets) GQ in its 3′-UTR, as recently identified from an in vivo SHALiPE-Seq structure profiling study (hird11 GQ mutants exhibit shorter roots relative to wild type (HIRD11 transcript abundance and root morphology through a mutagenesis study of GQ (mutGQ), in which the 4 G residues were substituted by A-residues, which exclude the possibility of intramolecular GQ folding. Higher HIRD11 transcript abundance and longer roots were observed in the mutGQ hird11 transgenic plants than in wtGQ hird11 plants, suggesting an important role of GQ in HIRD11 posttranscriptional regulation and root development.In addition, several studies have functionally implicated GQs in root development and abiotic stress response. Roots serve a variety of functions, including water and nutrient uptake, nutrient storage, and plant anchorage. Several recent studies have implicated GQs in the regulation of genes involved in root development. For example, ng study . hird11 ild type . The GQ SUPPRESSOR OF MAX2 1-LIKE4/5 (SMXL4/5) mRNAs  and its RNA targets, the 5′-UTRs of the 5) mRNAs  (Cho et 5) mRNAs . JUL1 prild type . Notably as well .SHORT ROOT (SHR) is a GRAS-type  transcription factor that is a pivotal player in root development. Overexpression of SHR with a 35S constitutive promoter results in extra cell layers in the root cortex, which show evidence of mis-specification as vascular tissue (SHR RNA accumulates in cytosolic structures reminiscent of phase-separated compartments. SHR contains a GQ that can promote RNA-driven liquid–liquid phase separation (LLPS) in vitro   ; stress-Stress granules and P-bodies exemplify LLPS, wherein specific RNA-binding proteins could direct their RNA partners to the condensate, or vice versa. Sequestration in stress granules can render RNA translationally inactive while protecting it from degradation . LLPS W and aggrRegarding the versatile functions of GQs in RNA stability and protein translation, and the possibility of their regulation by helicases, it is worth mentioning that the first plant-type GQ helicases have been identified recently by a GQ-binding protein pull-down mass spectrometric study . The strRNA structure in the living cell is influenced by myriad factors: the distinct environments of the nucleus, cytosol, and RNA-containing organelles; a panoply of RNA-binding proteins that can directly (e.g. RNA helicases) or indirectly (e.g. by binding and sequestering transcript regions) alter structure; and the possibility of co-transcriptional folding. This complexity dictates that test tube simulations of in vivo conditions will fall short of capturing the true in vivo structures of all but the most rock-like of RNAs. While in silico modeling, particularly of the most thermodynamically stable structure of an RNA sequence, is quite advanced, RNAs in vivo can become trapped in alternative structures, never reaching the thermodynamic minimum . This isIn vitro methods of probing the structures of individual RNA species include NMR, crystallography, and cryo-EM. These low throughput methods are complemented by methods in which the structure of an RNA species is read out on gels or via capillary electrophoresis, using either end-labeling of the RNA or RT. These approaches are based on structurally mediated sensitivity to (i) spontaneous degradation (in-line probing); (ii) nucleases that specifically target single-stranded or double-stranded regions; or (iii) chemical probes of RNA structure. With the exception of hydroxyl radical footprinting of cell cultures, in which accessible regions of the RNA are oxidized in vivo and the resultant cleavage patterns then read out , only thPhe upon progressive denaturation.Chemicals that covalently modify RNA were described as early as the 1960s (reviewed in ). The firplJ operon f complex. These DMS results, combined with in vitro structure probing with nucleases, suggested the presence of 2 hairpins that promoted translation, the upstream of which also was required for RNA stability. The 5′-UTR of rbcL, which encodes the LSU of Rubisco, similarly has a 5′-upstream hairpin, identified by DMS probing and structure prediction  that were assessed, did not exhibit these light-induced changes in structure; however, similar structure changes were also observed in other plastid genes that, like psbA, have weak SD sequences  modification of 3′-UTRs in Arabidopsis  plants, nor was protein binding correlated with m6A modification. The salt-induced m6A modifications were correlated with increased RNA stability and, thereby, proposed to enhance salt-stress-responsive protein translation. The authors report that they did not observe the inverse relationship between change in RNA structure and change in RNA abundance seen in salt stress by 6A modified transcripts reported by 6A modification, similar effects of salinity on the composition of the transcriptome were observed in the 2 studies  for everything else. This division reflects the relatively recent recognition of the regulatory function of noncoding RNAs (ncRNAs). There are many categories of ncRNA such as the aforementioned tRNA, rRNA, snRNA, ribozymes, riboswitches, and RNA thermometers within UTRs. These particular ncRNAs are highly structured, and many of their structures have been solved by crystallography or NMR. There are many other classes of ncRNAs as well, which have been revealed by next-generation sequencing and bioinformatics, as well as a growing awareness that such RNAs exist and should be looked for. A major class of ncRNAs are small RNAs, typically 21–24 nt, which are generally not structured in their mature form. As previously mentioned, they are covered by other articles in this Focus Issue, so we do not discuss them in this review. Another class of ncRNAs are the lncRNAs, which have received increased attention over the last decade . These u2 triplexes or as R-loops   R-loops . Methods R-loops . In 1 expression . R-loopsn  . It appeudoknots .AUXIN-REGULATED PROMOTER LOOP) lncRNA recognizes multiple sites in trans via short segments leading to the formation of R-loops. This interaction has a number of effects , with over 20,000 in Arabidopsis alone . As desc effects  includinCOOLAIR, involved in flowering time regulation, and PHO1.2, involved in phosphate nutrition, have definitively shown a role for lncRNA structure in controlling the abundance of the corresponding sense mRNA. These 2 lncRNAs are discussed in detail below.Targeted analysis of 2 Arabidopsis natural antisense lncRNAs, FLOWERING LOCUS C (FLC) represses flowering initiation and is centrally involved in vernalization-dependent flowering time regulation  in COOLAIR among natural Arabidopsis accessions resulted in varied isoforms and distinct secondary structures that were found to be involved in tuning flowing time. For example, the haplotype of the Var2-6 accession results in a shorter H4 helix with a larger internal loop that likely decreases the stability of COOLAIR; this allele was less inhibitory of FLC transcription when introgressed into Col-0, resulting in later flowering  and were associated with reduced FLC abundance and shortened time to flower under warm conditions -type Pi transporter (PHO1.2) loads Pi into the xylem for distribution via the vascular system, playing an important role in maintaining Pi homeostasis and grain production (PHO1.2 gene (cis-NATPHO1.2) in Arabidopsis has been shown to have a unique regulatory function in enhancing the translation of PHO1.2  is an essential macronutrient in both plants and animals, as it is an essential building block for nucleic acids, phospholipids, various cofactors, and ATP, the energy currency of the cell. In plants, Pi modulates starch synthesis through regulating the activity of ADP-glucose pyrophosphorylase. The rice oduction . Notablyf PHO1.2 . Pi star1.2 mRNA . The addis model . AlthougAccompanying the publication of the first draft human genome sequence  was a seAmong the possible functional consequences of an SNP is a change in RNA structure. The term “riboSNitch” was coined by Alain Laederach and colleagues to designate such structure-changing SNPs . ExperimThe first transcriptome-wide experimental assessment of human riboSNitches was provided by Howard Chang and colleagues, who analyzed the ex vivo structuromes of a mother/father/child trio using nuclease susceptibility . They foWith the proliferation of genome sequences, computational methods have been developed for riboSNitch prediction  and a daCOOLAIR, where natural variant sequences were shown to differentially affect levels of the sense transcript, FLC which, as discussed above, encodes a key regulator of flowering time  and COTTON-GOLGI-RELATED3 (CGR3). Particularly for ZR3, the alternative structure, with its larger loop and shorter stem  allele being associated with continental regions with greater variation in temperature  RNA and SARS-CoV-2 (As an additional level of complexity that is layered on dynamic cellular conditions, many RNAs can adopt multiple structures, and many of these can co-exist simultaneously in vivo as structural ensembles, as recently revealed for variants of the plant lncRNA RS-CoV-2 . EmerginRS-CoV-2 , also hoRS-CoV-2 .At the level of quaternary structure, intermolecular RNA–RNA interactions can also influence, and be influenced by, structure. That RNA–RNA interactions can promote condensate formation is well established , yet theFinally, in an RNA-centric view of modern biology, the efficacious delivery of mRNA vaccines against SARS-CoV2 via lipid nanoparticles stands as an astounding scientific success story  Dolgin . Notably