This study was designed to investigate the efficacy and safety of immune checkpoint inhibitors (ICIs)-based therapy in proficient mismatch repair (pMMR)/non-microsatellite instability-high (non-MSI-H) metastatic colorectal cancer (mCRC).Electronic databases were screened to identify relevant trials. The primary endpoints were pooled objective response rate (ORR) and disease control rate (DCR). Stratified analysis was accomplished on ICIs-based regimens, treatment lines and RAS status.P = 0.91) and OS . It was worth noting that the addition of ICIs to anti-vascular endothelial growth factor (VEGF) agent plus chemotherapy displayed excellent efficacy in pMMR/non-MSI-H mCRC : HR = 0.9, 95% CI: 0.8–1.0, P = 0.02), especially served as first-line therapy ; and without additional treatment related adverse events (TRAEs) were observed.Totally, 1723 mCRC patients from 39 cohorts were included. The pooled ORR, DCR, 12-month overall survival (OS) rate and 6-month progression-free survival (PFS) rate of ICIs-based therapy in pMMR/non-MSI-H mCRC were 8.5% (95% CI: 4.4%-13.5%), 48.2% (95% CI: 37.8%-58.6%), 52.3% (95% CI: 46.4%-58.1%) and 32.8% (95% CI: 23.5%-42.7%) respectively. As a whole, no significantly differences were shown between ICIs-based and non-ICIs-based therapy for pMMR/non-MSI-H mCRC in terms of both PFS (HR = 1.0, 95% CI: 0.9–1.1, ICIs-based combination therapy, especially the addition of ICIs to first-line anti-VEGF agent plus chemotherapy, is promising in pMMR/non-MSI-H mCRC with good efficacy and controllable toxicity.The online version contains supplementary material available at 10.1186/s12865-023-00564-1. Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related morbidity and mortality all over the world . DespiteIt has been recorded that a lack of efficacy of the anti-PD-1 and a modest clinical benefit of the anti-PD-L1 plus the anti-CTLA-4, reserved only to patients with a tumor mutational burden (TMB) more than 28 mut/Mb on circulating tumor DNA . Based oTo overcome the limitations of individual studies and assess the overall benefit, therefore, we conducted a comprehensive survey based on a large sample size , diverse dimensions , and hazard ratio (HR)), multiple stratifications , and various evaluation indicators (incorporating objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS)) to evaluate the efficacy and safety of ICIs-based therapy in pMMR/non-MSI-H mCRC.Articles were screened through a systematic electronic literature retrieval for abstracts of relevant studies in the published literature. PubMed, Cochrane Library, and EMBASE were searched and the data were updated as of August 15th, 2022. The basic search terms were used as follows: “immunotherapy”, “immune checkpoint inhibitor”, “Pembrolizumab”, “Atezolizumab”, “Nivolumab”, “PD-1”, “Keytruda”, “Tecentriq”, “Bavencio”, “Imfinzi”, “PD-L1”, “CTLA-4”, “Ipilimumab”, “programmed cell death 1”, “programmed cell death-ligand 1”, “cytotoxic T lymphocyte-associated protein 4”, “ICI”, “Sintilimab”, “Camrelizumab”, “Tislelizumab”, “Durvalumab”, “Avelumab”, “colon cancer”, “colorectal cancer”, “rectal cancer”, “microsatellite instability-low”, “MSI-L”, “MS-S”, “MSS”, “microsatellite stable”, and “pMMR”. Full-text papers were scrutinized if abstracts did not provide substantial information. Moreover, the references of relevant articles were reviewed for additional studies. Data retrieval was accomplished in English.Initially, two investigators performed a screening of titles and abstracts respectively, then examined the full-text of articles to acquire eligible studies. For the duplicate studies based on the same study patients, only the latest or most comprehensive data were recruited.(1) Prospective or retrospective studies to evaluate the efficacy and safety of ICIs in pMMR/non-MSI-H mCRC; (2) patients pathologically confirmed as CRC; (3) the data  to evaluate the efficacy of ICIs in pMMR/non-MSI-H mCRC could be obtained or calculated from the original literature.Data extraction was implemented conforming to the PRISMA guidance Table S. All eliThe quality of included studies was assessed independently by two reviewers using the Newcastle–Ottawa Scale (NOS) for case–control and cohort studies. It encompassed three dimensions of selection, comparability, and exposure, with a full score of 9 points.P < 0.1 or I2 > 50%, remarkable heterogeneity existed. A random effect model was adopted to calculate the pooled data when heterogeneity existed, or else, a fixed effect model was employed.The primary endpoints were ORR and DCR measured by pooled rates with corresponding 95% CIs for pMMR/non-MSI-H mCRC. The secondary endpoints were pooled PFS, OS, 6-month PFS rate and 12-month OS rate. Subgroup analysis was accomplished based on various ICIs-based regimens, treatment lines and RAS status. The summary measures of prognostic parameters and adverse events (AEs) were pooled rate, ORs and HRs with 95% CIs. Funnel plots and Egger’s test were performed to evaluate publication bias. Statistical analysis was performed with R 4.0 statistical software. Survival data were obtained based on the Kaplan–Meier curves. Heterogeneity was assessed by I-square tests and Chi-square. If Initially, 421 relevant articles were scrutinized intensively. Of them, 18 were filtered for duplication, and 330 were excluded for digression after screening the titles and abstracts. Then the full text of 73 articles was thoroughly reviewed, and 38 were filtered for: they were not human research, and not in English, commentaries, case reports, review articles, or letters to the editor, and without enough data for calculation. Finally, a total of 35 articles (including 39 cohorts) incorporating 1723 patients were recruited in this study  published until August 15th, 2022 were recruited. The sample size ranged from 6 to 250. Of these studies, 4 were randomized controlled trials (RCTs), and 9 retrospective studies. Meanwhile, all of these studies involved ICIs: ICIs monotherapy, ICIs plus targeted therapy, ICIs plus chemotherapy or radiotherapy, ICIs plus both targeted therapy and chemotherapy/radiotherapy. Pooled rate and 95% CIs were used to report the ORR, DCR, 6-month PFS rate, 12-month OS rate, and AEs of ICIs in pMMR/non-MSI-H mCRC; HRs with corresponding 95% CIs were utilized to assess the PFS and OS of ICIs for pMMR/non-MSI-H mCRC. The principal traits were presented in Table P = 0.39).A total of 38 cohorts containing 1277 patients were included to investigate the ORR of ICIs-based regimens for pMMR/non-MSI-H mCRC. Overall, the pooled ORR was 8.5% (95% CI: 4.4%-13.5%) .There was no statistical difference on ORR for ICIs-based regimens in both RAS wild type (wt) and RAS mutant type (mt) pMMR/non-MSI-H mCRC .A total of 31 cohorts involving 834 patients were included to report the DCR of ICIs-based regimens for pMMR/non-MSI-H mCRC. Generally, the pooled DCR was 48.2% (95% CI: 37.8%-58.6%) .There was also no significantly difference on DCR for ICIs-based regimens in both RASwt and RASmt pMMR/non-MSI-H mCRC  .The pooled HR of OS for ICIs-based therapy versus non-ICIs-based therapy in pMMR/non-MSI-H mCRC was 1.0  Fig. a withoutAs a whole, the 12-month OS rate of ICIs-based therapy was 52.3% (95% CI: 46.4%-58.1%) (Table P = 0.91) .The pooled HR of PFS for ICIs-based therapy versus non-ICIs-based therapy in pMMR/non-MSI-H mCRC was 1.0  Fig. b without91) Fig.  and EggeP = 0.02, Fig. P = 0.60).Subgroup analysis was performed based on various ICIs-based regimens. Compared with non-ICIs-based therapy, the addition of ICIs to anti-VEGF agent plus chemotherapy brought significantly longer PFS for pMMR/non-MSI-H mCRC .Subgroup analysis was also conducted based on RAS status. There was no significantly difference on PFS for ICIs-based regimens in both RASwt and RASmt pMMR/non-MSI-H mCRC   microenvironment. Therefore, accumulating studies have been carried out recently focusing on ICIs-based combination regimens in which ICIs have been incorporated with chemotherapy, radiotherapy and anti-VEGF agent in order to transform immunologically “cold” pMMR/non-MSI-H mCRC into responsive “hot” lesions. However, the results of such studies have been inconsistent –14 and tBased on the existing studies, the pooled results of our study revealed that the addition of ICIs into anti-VEGF agent plus chemotherapy  is promising in pMMR/non-MSI-H mCRC in terms of ORR, DCR, PFS, 6-monhs PFS rate and 12-month OS rate. At the same time, it has been supported that the potential clinical efficacy of anti-VEGF agent plus ICIs combination was also founded in other tumors such as HCC and NSCLC. For HCC, compared with sorafenib monotherapy, atezolizumab plus bevacizumab IMbrave 150), and Sintilimab plus bevacizumab (ORIENT-32) were founded to significantly improve PFS and OS , 46; wit, and SinAlthough there was no difference between ICI-based therapy and non-ICI-based therapy in both OS and PFS of pMMR/non-MSI-H mCRC on the whole, the subgroup analysis revealed that the addition of ICIs to anti-VEGF agent plus chemotherapy could significantly improve PFS of pMMR/non-MSI-H mCRC; moreover, there was no directly correlation between the improvement of PFS and RAS status. At the same time, some other advantages of ICI-based therapy were still founded among these studies. The study conducted by Eng et al. found that although not superior to standard therapy, treatment with the combination of ICIs and MEK inhibitor resulted in equivalent survival without the introduction of any new AEs . BesidesWith regard to the safety, the regimens of ICIs plus chemotherapy with/without anti-VEGF agent revealed higher incidence of grade 3 or beyond AEs. Among the included studies, Bocobo et al. found that the grade 3 or beyond TRAEs only occurred in 28% patients, of which less than half (11%) were immune-related and none was associated with bevacizumab ; besidesThe best strategy and biomarkers of ICIs for pMMR/non-MSI-H mCRC remain to be established. On one hand, in order to seek the best strategy of ICIs-based therapy for pMMR/non-MSI-H mCRC, we are obliged to optimize which regimen is beneficial in combination with ICIs (with maximizing efficacy and minimizing toxicity), facilitate clinical research based on biomarkers, and explore the development of other ICIs drugs and cell-based treatment schemes ; on the This study came up with three drawbacks as follows: firstly, there were only 4 RCTs, despite containing 39 cohorts 1723 patients, for analyzing the efficacy and safety of ICIs in pMMR/ non-MSI-H mCRC; secondly, considering the limited number of studies with survival outcomes for ICIs in pMMR/non-MSI-H mCRC patients, we took the ORR and DCR as primary endpoints; finally, only studies published in English were included, which might yield language bias to some degree.ICIs-based combination therapy, especially the addition of ICIs to first-line anti-VEGF agent plus chemotherapy, is promising in pMMR/non-MSI-H mCRC with good efficacy and controllable toxicity.Additional file 1:Table S1. PRISMA ChecklistAdditional file 2:Table S2. The list of the included studies.Additional file 3:Table S3. The Newcastle-Ottawa scale for quality assessment of the studiesAdditional file 4:Figure S1. The pooled objective response rate (ORR) of immune checkpoint inhibitors (ICIs)-based therapy in RAS wild type (wt) versus RAS mutant type (mt) proficient mismatch repair (pMMR)/non-microsatellite instability-high (non-MSI-H) metastatic colorectal cancer (mCRC): (a) forest plot and (b) funnel plot; the pooled disease control rate (DCR) of ICIs-based therapy in RASwt versus RASmt pMMR/non-MSI-H mCRC: (c) forest plot and (d) funnel plot.Additional file 5:Figure S2. The funnel plot of (a) overall survival (OS) and (b) progression-free survival (PFS) for ICIs-based therapy versus non-ICIs-based therapy in pMMR/non-MSI-H mCRC; the funnel plot of PFS for (c) ICIs plus anti-VEGF agent and chemotherapy versus non-ICIs-based therapy, and (d) RAS wild type (wt) versus RAS mutant type (mt) in pMMR/non-MSI-H mCRC.


Drosophila PCID2 is a subunit of the TREX-2 mRNA nuclear export complex. Although the complex has long been studied in eukaryotes, it is still unclear how TREX-2 interacts with mRNA in multicellular organisms. Here, the interaction between Drosophila PCID2 and the ras2 RNA was studied by EMSA. We show that the C-terminal region of the WH domain of PCID2 specifically binds the 3′-noncoding region of the ras2 RNA. While the same region of PCID2 interacts with the Xmas-2 subunit of the TREX-2 complex, PCID2 interacts with RNA independently of Xmas-2. An additional RNA-binding region (M region) was identified in the N-terminal part of the PCI domain and found to bind RNA nonspecifically. Point mutations of evolutionarily conserved amino acid residues in this region completely abolish the PCID2–RNA interaction, while a deletion of the C-terminal domain only partly decreases it. Thus, the specific interaction of PCID2 with RNA requires nonspecific PCID2–RNA binding. Drosophila PCID2 protein is a component of the TREX-2 complex, which is evolutionarily conserved in eukaryotes and is responsible for mRNA nuclear export  RNA synthesis of the D was determined according to [Purified recombinant proteins corresponding to the full-size PCID2; truncated PCID2; mutated PCID2 variants; and the N, M, and C parts of PCID2 were incubated with the radiolabeled RNA fragments in 20 μL of a binding buffer, which contained 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5% Triton X-100, 5% glycerol, 1 mM EDTA, 1 mM DTT, 25x PIC, and 40x RiboLock , at 4 °C for 1 h. After binding was completed, a PAGE loading buffer was added to the samples, and the samples were applied to a native 5% polyacrylamide gel. Electrophoretic separation was carried out in a 0.5x TBE buffer at 150 V at 4 °C for 80 min. The radioactive signal was detected using a Cyclone Storage Phosphor Screen device. The signal from each lane (area) was quantitated using the ImageJ program. Direct titration reactions were plotted as bound RNA fraction versus protein concentration, and Krding to ,31.Drosophila S2 cells were maintained in Schneider’s insect medium  containing 10% fetal bovine serum  at 25 °C. Transient transfection of S2 cells was performed using the Macsfectin™ reagent according to the manufacturer’s recommendations .To extract proteins, S2 cells were lysed. Cells were centrifuged at 2000 rpm at 4 °C for 5 min and resuspended in 1 mL of 1x PBS with 25x PIC . The cells were then centrifuged once more at 2000 rpm at 4 °C for 5 min and resuspended in an LB buffer, which contained 10 mM Hepes, pH 7.0, 0.4 M NaCl, 5 mM MgCl2, 0.5% NP-40, 25x PIC, 1 mM DTT, and 0.3 μL of DNAse I, in a proportion of 1:10. The cells were incubated with the LB buffer on ice for 20 min, and the lysate was further centrifuged at 13,000 rpm at 4 °C for 15 min.Immunoprecipitation was performed as described , using eAntibodies against PCID2 , the FLAt-test was used to compare the control and treatment groups. An asterisk (*) indicates statistical significance with p-value < 0.05; (**) indicates statistical significance with p-value < 0.01.Error bars represent SD from at least three independent experiments. All data are presented as mean ± SD, and Student’s Drosophila PCID2 structure (AF ID: AF-Q9VTL1-F1) was provided by the AlphaFold Protein Structure Database , which has been created in partnership with EMBL’s European Bioinformatics Institute (EMBL-EBI) [The predicted MBL-EBI) ,34. The MBL-EBI) . The eleMBL-EBI) .Drosophila PCID2 with RNA was studied by EMSA. The ras2 (ras64B) mRNA was used in experiments. The choice was explained by our previous finding that Xmas-2 and PCID2 bound the ras2 mRNA in RNA immunoprecipitation experiments and the RNA pull-down assay [ras2 mRNA sequence was divided into five approximately equal fragments  and ras2 4_2 (right panel) fragments were used as cold competitors. A competitor was added to the reaction in 0.5- to 250-fold excess over the hot 4_2 fragment in a gradient manner. ras2 fragment 4_2. The interaction of the hot 4_2 fragment with PCID2 decreased gradually with a minimal increment in the presence of cold fragment 4_1, while cold fragment 4_2 competed with its hot counterpart and already decreased its interaction with the protein when used in a minor excess  includes the highly structurally conserved WH domain ,26. The 1–360). The full-size PCID2 and PCID21–360, each fused with the GST epitope, were tested for binding fragment 4_2 of the ras2 mRNA by EMSA  with Xmas-2 was studied in D. melanogaster S2 cells. PCID21–360 was fused with the HA epitope, Xmas-2 was fused with the FLAG epitope, and the two proteins were coexpressed in S2 cells. The results of protein coimmunoprecipitation from a cell lysate are shown in 1–360 did not interact with Xmas-2 in the experiment , which is in line with the results shown above (ras2 RNA for the M-PCID2 fragment (KD~11 nM). M-PCID2 formed several contacts with fragment 4_2 as could be seen from the migration pattern of the RNA–protein complex in gel. Thus, the N-terminal part of the PCI domain harbors an additional region responsible for RNA binding.Because the deletion of the C-terminal domain of PCID2 did not fully abolish its interaction with RNA, additional RNA-binding sites were assumed for PCID2. To identify the respective regions, we engineered the N-PCID2, M-PCID2, and C-PCID2 constructs, in which the His epitope was fused with the approximately equal (about 130 amino acids) N-terminal, middle, and C-terminal fragments of PCID2, respectively. A fragmentation scheme is shown in  by EMSA B. The C-wn above B. N-PCIDras2 RNA fragment 4_1, which does not interact with the full-size PCID2, as reported above. N-PCID2 and C-PCID2 were not found to interact with fragment 4_1  and for Arg216 in the other (PCID2R216A). PCID2R191A and PCID2R216A did not bind ras2 RNA fragment 4_2 in contrast to the wild-type PCID2 . Based on the EMSA results, a lack of the C-terminal domain partly distorts the mRNA binding, while mutations of the middle part completely abolish the mRNA binding. The finding makes it possible to conclude that the interaction of the M region with RNA is necessary for the specific RNA binding with the C-terminal region. Primary RNA binding can be assumed to occur in the M region (because its binding with RNA is highly efficient) and to allow the C-terminal domain to specifically interact with the RNA (because its binding with RNA is less efficient). The M region of PCID2 is highly conserved in evolution (up to 91% homology in higher eukaryotes). A similar role is therefore possible to assume for the respective sequences of the PCID2 homologs of other organisms.AlphaFold ,34 and A


In recent years, there has been a growing interest in the role of RNA in diseases and cancers. It is now becoming increasingly clear that non-coding RNAs, such as microRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play an active and crucial role in regulating gene expression and cellular functions. One area where the role of regulatory RNA is particularly prominent is in cancer research. Furthermore, the RNA modification field, also known as epitranscriptomics, has recently gained attention due to research into the regulation of gene expression and cellular functions. These modifications involve the addition, removal, or alteration of chemical groups on RNA molecules, and are known to impact RNA maturation, stability, and translation. Recent evidence suggests that RNA modifications also play a critical role in various diseases and cancers.International Journal of Molecular Sciences, entitled “The Emerging Role of RNA in Diseases and Cancers”, includes a total of nine contributions: one research article, one case report, and seven reviews providing new information about the role of non-coding RNAs and RNA modifications in normal and pathological conditions. This Special Issue of the Researchers have discovered that certain types of regulatory non-coding RNA, such as microRNAs and lncRNAs, can control cell growth and differentiation by regulating key signaling pathways. These RNAs can act as either oncogenes or tumor suppressors, depending on the specific context. Ciafrè et al. thoroughly reviewed the important role of lncRNAs in cancer stem cells, the different mechanisms through which they work, and how their modulation can affect tumorigenesis . On the In addition to cancer, RNA has also been implicated in a variety of other diseases, including neurological disorders, cardiovascular diseases, and viral infections. For example, mutations in RNA-binding proteins have been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Colantoni et al. investigated the effect of mutation in the RNA-binding protein FUS, which characterizes familiar forms of ALS, on the formation of circRNAs in in vitro-derived human motoneurons . The stuThe emerging role of RNA in diseases and cancers has significant implications for diagnosis, prognosis, and treatment. RNA-based biomarkers can provide important information about disease progression and treatment response, while RNA-targeted therapies, such as RNA interference (RNAi) and antisense oligonucleotides (ASOs), hold great promise for treating a wide range of diseases. However, there are also significant challenges to be overcome in translating RNA-based research into clinical practice. For example, RNA-based therapies face issues related to delivery and specificity, as well as potential off-target effects. Additionally, the development of RNA-based biomarkers requires careful validation and standardization across different platforms and patient populations. Garbo et al. reviewed the benefits and the limits of RNA therapeutics by describing the non-coding RNA molecules that are being tested in clinical trials, and by analyzing the delivery strategies for different therapeutical applications . Rincon-Lee et al. examined the role of microRNAs as biomarkers and therapeutic targets for muscle dystrophy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) .6-methyladenosine (m6A), which is the most prevalent modification found in eukaryotic mRNA. m6A has been implicated in cancer development and progression, with dysregulated m6A modification patterns associated with poor prognosis in several types of cancer. Studies have shown that m6A modification can affect mRNA stability, translation, and alternative splicing, which, in turn, can impact cell growth, proliferation, and survival. Fernandez Rodriguez et al. analyzed the role of m6A in the regulation of the global rate of mRNA translation and the selective translation of specific mRNAs in cancer [6,2′-O-dimethyladenosine (m6Am) modification by describing the methodologies for its mapping, the enzymes responsible for m6Am regulation, and their impact in gene expression regulation. Furthermore, they discussed the emerging roles of m6Am in tumorigenesis [One of the most well-studied RNA modifications is Nn cancer . Furtherigenesis .The emerging role of RNA in diseases and cancers represents a major shift in our understanding of the molecular mechanisms underlying complex diseases, such as cancer. By continuing to explore the complex roles of RNA in cellular processes, we can pave the way for the development of new diagnostic tools and therapeutic approaches that could ultimately improve outcomes for patients with a wide range of diseases.


Flavivirus are characterized by a complexly folded 3′ UTR with a number of RNA structural elements that are conserved across isolates of each species. The present work provides evidence of intra- and intermolecular RNA–RNA interactions involving RNA structural elements in the 3′ UTR of the West Nile virus genome. The intermolecular interactions can be visualized in vitro by the formation of molecular dimers involving the participation of at least the SLI and 3′DB elements. Certainly, the 3′ UTR of dengue virus, which lacks the SLI element, forms molecular dimers in lower quantities via a single interaction site, probably 3′DB. The functional analysis of sequence or deletion mutants revealed an inverse relationship between 3′ UTR dimerization and viral translation efficiency in cell cultures. A network of RNA–RNA interactions involving 3′ UTR structural elements might therefore exist, helping to regulate viral translation.RNA viruses rely on genomic structural elements to accomplish the functions necessary to complete the viral cycle. These elements participate in a dynamic network of RNA–RNA interactions that determine the overall folding of the RNA genome and may be responsible for the fine regulation of viral replication and translation as well as the transition between them. The genomes of members of the genus  RNA-mediated regulatory pathways have become the subject of great interest over the past two decades. The discovery of the importance of RNA–RNA interactions in the control of gene expression has allowed the deciphering of new information-coding systems across the entire phylogenetic spectrum, from viruses to humans. The small size of viral RNA genomes renders them important reservoirs of conserved structural elements that help in the maintenance of viral persistence without impeding adaptive fitness. These elements operate via the direct recruitment of cellular and viral protein factors and via the establishment of the inter- and intramolecular RNA–RNA interactions involved in complex and dynamic networks . For example, many viral and cellular RNAs form dimers in order to enlarge their conformational space, thus improving their plasticity and functional abilities . FurtheFlavivirus is the largest in the family Flaviviridae and comprises a large group of viruses commonly referred to as flaviviruses. These are small, enveloped, positive single-stranded RNA viruses. Several are human pathogens, including dengue (DENV), Zika (ZIKV), yellow fever (YFV) and West Nile virus (WNV), all of which belong to the mosquito-borne flavivirus (MBFV) group [The genus V) group ,3,4. TheEvidence exists that specific RNA elements in the 3′ UTR help regulate essential viral processes, including translation, replication and infectivity ,6,7, as Flaviviridae, has been shown to form genomic dimeric units mediated by a palindromic sequence motif (dimer linkage sequence (DLS)) [The hepatitis C virus (HCV), which also belongs to the family e (DLS))  located e (DLS)) . We havee (DLS)) . Thus, ge (DLS)) ,16.Here, it was hypothesized that, since HCV and flaviviruses show similar genetic organization and an essential information storage system based on discrete structural and functional units, they may also share common molecular mechanisms for the regulation of viral processes. Although no DLS has been identified in the flaviviral genome, RNA–RNA assembly may occur by means other than Watson–Crick and non-Watson–Crick base pairing. For example, base stacking and helix co-stacking are commonly used to establish RNA–RNA contacts in crowded RNA environments . Thus, uThis work provides evidence of the in vitro formation of molecular dimers in WNV and DENV, a process reliant on specific structural elements of the 3′ UTR RNA, and proposes the existence of a complex network of intra- and intermolecular contacts that needs to be balanced in order to achieve the control of translation. Together, the data presented support a plausible functional role for the phenomenon of genomic RNA dimerization in flavivirus infectious cycles.Flaviviridae and that it might operate as a regulatory mechanism.The 3′ UTR of HCV is known to dimerize in vitro ,20. Here2+. Different conformations were resolved by agarose gel electrophoresis under non-denaturing conditions. Up to four different conformers were detected at the highest tested concentrations of 3′WNV  were incubated in the presence of Mgof 3′WNV a, reflecof 3′WNV a. Data wof 3′WNV a.2+ and, to a lesser extent, by monovalent cations such as Na+ [2+ or Na+  technique  was usedBriefly, the RNA construct encompassing the whole 3′WNV was modified with NMIA under denaturing conditions. RNA molecules were then renatured and subjected to dimerization assays under optimal experimental conditions. The monomeric and dimeric populations were then separated by native agarose gel electrophoresis. Modified positions were detected via stoppages in the reverse transcription reaction. HMX scores were calculated from the differential reactivity profiles between the monomeric and dimeric conformers .HMX analysis showed the need for precise nucleotide conformations in essential structural elements of the 3′ UTR for the dimeric complex to form a,c. The RNAStructure software  was thenTaken together, these results support the existence of new RNA–RNA interactions that occur in the 3′ UTR of the WNV genome, and that these might be critical for preserving the functional interactome at the 3′ end of viral RNA.′ UTR, such as 3′DB, as detected by HMX. The participation of SL-I in the formation of intra- and intermolecular interactions is also suggested from the secondary structure prediction. To confirm the role of these elements in dimer formation, a collection of mutant constructs was generated by the sequential deletion of the autonomously folded structural elements within the 3′ UTR. These deletions were generated in both the 3′ end  3′UTR) was synthesized and subjected to dimerization assays using increasing RNA concentrations. Data were fitted to a non-linear curve, as described for the (+) strand. The results showed a low yield of dimer complex formation (~50%)  strand has been suggested to be critical for enhancing the synthesis of the (+) strand . The abin (~50%) .′ UTR regulate viral translation [′UTR-dependent protein synthesis mediated by the 3′ UTR, a new set of constructs was designed containing the 5′ UTR of WNV fused to the reporter gene encoding the FLuc protein  were attached to the 3′ end of the FLuc coding sequence to generate WNV-Mut_X constructs .As has been mentioned, evidence exists that specific RNA elements in the 3nslation . In an a protein a. Differnstructs a. All ofInterestingly, mutations in SL-I or CS1 induced a significant increase in FLuc synthesis in both cell types, an effect that was even stronger for WNV-Mut_CS1 in mosquito cells b. More i′DB element in WNV translation. Its deletion reduced WNV translation efficiency by up to 50%  . From aynthesis . Taken tnd 3′DB) , leading′ UTR of certain MBFVs. In WNV, this occurs at two different anchoring sites with different affinities and could overlap with the establishment of previously unreported, theoretical intramolecular interactions predicted with RNAStructure  was generated by PCR using the primers T7p3′WNV and as3′WNV-BamHI from the modified NY99-flamingo382–99 strain [′WNV oligonucleotide contained the promoter sequence of T7 RNA polymerase after the EcoRI site. The resulting amplification product was cloned into EcoRI-BamHI sites of the pUC19 vector to generate pUC3′WNV. The latter was digested using the endonuclease BamHI to generate the template for subsequent 3′WNV RNA synthesis.The plasmid pUC39 strain , a kind ′ UTRs of DENV (NC_001474.2) and YFV (KF769016.1), fused to the T7 promoter sequence, were purchased from IDT . These constructs were introduced into the plasmid pUC19, which was first linearized by BamHI using the In-Fusion® HD Cloning Kit , to generate pUC3′DENV and pUC3′YFV. These plasmids were linearized with the enzyme SalI to produce templates for transcription.The DNA fragments coding for the 3′WNV were generated by PCR amplification from the pUC3′WNV vector. For 3′ deletions, the T7p3′WNV oligonucleotide was used as a 5′ primer for all constructs, while the 3′ primers were as follows: asWNV-10950 to generate DNA T7pMut1_3′, asWNV-10904 for DNA T7pMut2_3′, asWNV-10830 for DNA T7pMut3_3′, asWNV-10740 for DNA T7pMut4_3′, and asWNV-10660 for DNA T7pMut5_3′. To generate different DNA constructs bearing 5′ deletions, the 3′ primer asWNV-BamH1 was always used, while the 5′ oligonucleotides used were as follows: T7pWNV-10500 to yield DNA T7pMut1_5′, T7pWNV-10586 for DNA T7pMut2_5′, T7pWNV-10659 for DNA T7pMut3_5′, T7pWNV-10743 for DNA T7pMut4_5′, and T7pWNV-10831 for DNA T7pMut1_5′. The resulting amplicons were used as DNA templates for RNA synthesis (see below).DNAs encoding sequential deletions in the 3′WNV was generated by PCR using the oligonucleotides rc3′ WNV and T7 GGG- asrc 3′WNV, with pUC3′WNV as a DNA template. The amplification product carried the T7 promoter sequence fused to the 5′ end of the first 625 nt of the negative strand of the WNV genome, i.e., the complementary sequence of the 3′ UTR. This DNA was used for subsequent in vitro transcription (see below).The DNA coding for (-) 3All of the above-mentioned DNAs were used as templates for the synthesis of their respective constructs by in vitro transcription using T7 RNA polymerase ver.2.0 (Takara). RNAs were purified, and their quality was analyzed as previously described .′WNV (pUC3′WNV-Flip series) or pGLWNV (pGLWNV-Flip series) [′ UTR of the NY99-flamingo382–99 WNV strain and the 3′ UTR of WNV with the indicated Flip mutations. These constructs were designed to allow fluc translation from the AUG of the 5′ UTR of WNV.DNA plasmids carrying “Flip” mutations derived from the parental pUC3 series)  vectors ′WNV was used as a template for site-directed mutagenesis to generate two constructs: Mut-SLI_GNRA, which substitutes the apical loop of SL-I for a stable GNRA tetraloop, and Mut_delSt1, which does not have the apical portion of the stem of SL-I. Mutations were introduced using the appropriate primers and employing the Phusion™ Site-Directed Mutagenesis Kit (Thermo Fisher Scientific).pUC3′WNV were synthesized by in vitro transcription from the corresponding BamH1-linearized plasmids, as described above. The RNAs derived from the pGLWNV-Flip series bearing the 5′ cap were obtained using the HighYield T7 RNA Synthesis Kit  following the manufacturer’s instructions [RNA constructs derived from pUC3ructions . The synructions . RNAs we2) [w/v) in TBM buffer  [max1)/(Kd1 + x)) + ((Bmax2)/(Kd2 + x)), where y is the percentage of dimeric conformers, Bmaxn is the maximum yield of the reaction, x is the concentration of 3′WNV RNA, and Kdn the concentration of RNA required to achieve the maximum dimer percentage.RNA dimer formation was performed essentially as previously described . Briefly2)  for 30 mM MgCl2)  at 4 °C 2+ requirements, the reaction buffer included EDTA  and increasing concentrations of Mg2+ , while for monitoring Na+ requirements, monovalent cations were added to the reaction buffer  in the range 2–200 mM .Ionic requirements were studied via dimerization assays performed as above but with appropriate reaction buffers. Thus, to test Mg260). Modified RNAs were subjected to dimerization assays as described above. Complexes were also resolved as described above. The monomeric and dimeric conformers were gel-purified and used as templates in a primer extension reaction with the appropriate fluorescently labeled oligonucleotide to detect NMIA modifications by capillary electrophoresis, as previously described [HMX analyses were performed as previously described with minor modifications . Brieflyescribed . Electroescribed . HMX scoescribed ,22.® by LifeTechnologies™, Invitrogen, Waltham, MA, USA), 2 mM L-Glutamine  and 1 mM sodium pyruvate (Sigma). Cells were grown at 37 °C in a 5% CO2 atmosphere.Vero cells, derived from African green monkey kidney cells, were cultured as previously described . Briefly2 atmosphere.Aedes albopictus larva C6/36 cells were cultured in M3 insect medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and 0.1 mM non-essential amino acids, as previously described , and mai3 cells were seeded into 24-well plates 24 h before transfection in order to reach ~80% confluence. Then, 1.5 µg of the RNA under study and 0.3 µg of cap-RLuc RNA were mixed with the transfection reagent  in Opti-Mem®  according to the manufacturer’s instructions. This mix was added to cells followed by incubation for 4 h in order to achieve maximum translational efficiency. The cells were then lysed and subjected to firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay Kit . Relative translation efficiency was calculated as the FLuc/RLuc ratio compared to that obtained for wt WNV RNA.Cells were transfected essentially as previously described ,39. Brie


Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur.An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen  Massive efforts are being expended to understand the complex interactome within living organisms. Protein–RNA interactions are an essential component and contribute to cellular regulation in manifold ways. The traditional view of an RNA-binding protein (RBP) is governed by clearly defined RNA-binding domains that the protein uses to influence the fate of specific RNAs, thereby exerting regulation. RBPs can combine several such domains to expand specificity . ProteinThe RNA–enzyme–metabolite (REM) hypothesis, an acronym introduced more than a decade ago , suggestWhile supported by a few reports in the literature, the prevalence of a REM network is currently unclear, especially in prokaryotes. The most prominent example is eukaryotic aconitase IRP1. This enzyme participates in the citric acid cycle by converting citrate to isocitrate. However, the enzymatic activity depends on an iron-sulfur cluster that is depleted under iron-starvation conditions. In the absence of the metallocofactor, the protein engages in posttranscriptional regulation and interacts with mRNAs that impact iron homeostasis ,14. AnotE. coli enzymes for specific RNA-binding properties that might hint at physiologically relevant REM functions. Our investigations were mainly based on SELEX  experiments, probing for preferential binding behavior within a library representing the transcriptome of E. coli, but also included iCLIP  and EMSA (electrophoretic mobility shift assay) studies.In recent years, high-throughput screening methods that facilitate unbiased RBP identification in bacteria have been developed and applied ,33,34. LE. coli as potential RNA-interacting proteins. The selection was based on recurring literature references to putative RNA binding of metabolic enzymes in both high-throughput screenings and targeted research. We included data for non-prokaryotic enzymes that are functionally and structurally homologous. A particular set of recently published interactome screenings in prokaryotes was given special consideration. These screenings used phase separation -ATP, gel electrophoresis, transfer, RNA elution and iCLIP2 library preparation were performed according to Buchbender et al. [Immunoprecipitation was performed in lysates using Anti-FLAG M2 Magnetic Beads  for 2 h at 4 °C, and precipitated material was washed 4 times with 1 M NaCl wash buffer  and 2 times with PNK buffer . In brief, the library was prepared by genome shearing, end-repair to create blunt-ends, 5′-phosphorylation and dA-tail addition, followed by ligation of TruSeq®-compatible adapters. The lengths of sequences comprising the DNA library were normally distributed in a range between 300 and 600 bp, as verified by automated gel electrophoresis  after synthesis. Size distribution was additionally checked by standard gel electrophoresis after each SELEX round. In order to minimize unspecific selection effects originating from the adapter sequence, truncated adapter versions were used during SELEX, and the full-length versions were restored prior to sequencing of evolved libraries. The T7 promoter sequence was added 5′ to the truncated adapter so that first nucleotides were identical in all transcripts (“GGG” plus a constant adapter sequence of 20 bases), preventing any transcriptional selection bias during SELEX. The sequences of all oligonucleotides used for SELEX are listed in The RNA library was derived from the E. coli library by MS2 phage coat protein.General SELEX functionality was verified by both amplification of aptamers from a random library by hen egg white lysozyme , and amp32P-ATP was carried out according to the manufacturer’s protocols. RNA was precipitated with 0.5 volume 0.5 M EDTA pH 8.0, 0.5 volume 7.5 M ammonium acetate, and 1.5 volumes isopropanol at −20 °C. RNA was pelletized by centrifugation and washed at least twice using 70% ethanol. RNA was resolubilized in H2O. Concentration was determined by scintillation counting, and the success of transcription reactions was intermittently verified by urea PAGE in between selection rounds.For SELEX, transcription with T7 RNA polymerase (Thermo Fisher Scientific) using α-2, and 0.5 mM DTT, pH 7.5 was used as buffer, rudimentarily resembling native E. coli conditions. For PykF, HEPES was used instead of Tris. Prior to protein addition, the RNA library was briefly heated to 95 °C and cooled, inducing unfolding and redevelopment of RNA secondary structures. For the initial 2–3 rounds of SELEX, the RNA was subjected to negative selection [The RNA library was added in tenfold molar excess to the respective target protein and incubated for 30 min. A solution of 50 mM Tris, 80 mM NaCl, 10 mM KCl, 0.8 mM MgCl®Aqua-P/C/I pH 4.5  and mixed in an Eppendorf Thermomixer 5355  for 10 min. The liquid was taken off, and filter extraction was repeated one time with half volume. The unified liquids were centrifuged for phase separation. The aqueous phase was isolated and extracted with chloroform to remove phenol traces. RNA was precipitated at −20 °C by adding 1/10 volume 3 M sodium acetate pH 6.5, 1 volume isopropanol, and 1 µL GlycoBlue™ Coprecipitant (Thermo Fisher Scientific). RNA was pelletized by centrifugation, washed with 70% ethanol, and dissolved in 70 µL H2O.After incubation, the RNA-protein solution was filtered through a 0.2 µm M24 cellulose filter slice (Cytiva Whatman™), retaining protein–RNA complexes but not free RNAs . The fil2O. Subsequently, PCR amplification using Q5 polymerase  according to the manufacturer’s protocol was used to replenish DNA, providing enough starting material for the subsequent SELEX cycle. PCR amplification was verified by agarose gel electrophoresis.Reverse transcription was carried out with SuperScript IV reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s protocol. The resulting DNA was precipitated at −20 °C by adding 1/10 volume 3 M sodium acetate, 1 volume isopropanol, and 1 µL GlycoBlue™ Coprecipitant. DNA was pelletized by centrifugation and dissolved in 70 µL HAll DNA libraries were sequenced on an Illumina MiSeq device. Libraries from iCLIP were sequenced using an Illumina 50 cycles MiSeq Reagent Kit v2. Libraries from SELEX were sequenced using an Illumina 500 cycles MiSeq Reagent Nano Kit v2.Escherichia coli K-12 substrain MG1655 with standard parameter settings.For SELEX, in the first step of read processing, adapters were removed using Cutadapt version 2.8  with theE. coli K-12 substrain MG1655 instead of STAR-mapper.For iCLIP, read processing was conducted according to a published protocol . To accoMapping data (.bam files) and crosslinking-sites (.bed files) were examined in the most current version of the integrative genomics viewer IGV .E. coli. This comprised identification of areas with significantly above-average read density, inspection of peak shapes, and comparison between evolved libraries to ensure specificity of peaks . Only deduplicated reads were considered for iCLIP reads. For SELEX libraries, mapped reads were evaluated, and the deduplicated dataset was inspected to validate peak calls. Peak calling by PureCLIP [Peak calling was mainly achieved by thorough manual inspection of mapping data, applicable to the moderate genome size of PureCLIP  was consPureCLIP .32P-labeled with T4 polynucleotide kinase (New England Biolabs) according to the manufacturer’s protocol and tested for interactions with respective target proteins in native polyacrylamide gel electrophoresis. Components for native gels were 40% acrylamide/bisacrylamide 18:1 , 2.5% v/v glycerol, H2O, and 1× TB or 0.5× TBE buffer, polymerized by TEMED and APS. For development of secondary RNA structures, all RNAs were heated in assay buffer (see below) for 1 min to 95 °C and subsequently cooled by placing them into a metal rack at room temperature. Pre-incubation of RBP candidates and RNAs started by mixing protein and RNA and proceeded for 30 min at 37 °C under the same buffer conditions used for incubation of the respective RBP candidate and RNA library during genomic SELEX: 50 mM Tris, 80 mM NaCl, 10 mM KCl, 0.8 mM MgCl2, 0.5 mM DTT, pH 7.5. For PykF, HEPES was used instead of Tris. In the case of competition assays, target RNA and competitor RNA were treated separately to allow secondary structure formation and were added to the protein concurrently. Samples to be subjected to electrophoresis were supplemented with 10% glycerol to facilitate sinking into gel pockets. For visualization of 32P-labeled RNAs, gels were wrapped into plastic foil and exposed to a PhosphoImager screen for at least 2 h. The phosphoscreen was visualized on a Cyclone Storage Phosphor System . Quantification of bands was performed with the OptiQuant 3.0 Software supplied by Canberra Packard. Data of EMSA titrations were fitted in SigmaPlot 14 using the common hyperbolic binding equation to determine a Kd-value.RNA probes were d values.MST assays for quinone oxidoreductase were conducted on a Monolith NT.115Pico device . The Pico RED detector (excitation wavelength 600–650 nm) was used to measure fluorescent Cy5-labeled RNA. Prior to titration experiments, base fluorescence intensities of free RNA and RNA–protein complex solutions were measured to ensure homogeneity of the signal. The buffer used during RNA identification in SELEX was used as assay buffer, supplemented with 0.05% Tween-20 to avoid adsorption of biomolecules to the capillaries. A sigmoidal equation as provided in the NanoTemper evaluation software was fitted to the titration data to obtain KFor prediction of RNA secondary structures, the most recent version of RNAfold  was rout


Toxoplasma gondii is limited. In this study, RNA modifications in three T. gondii strains  with distinct virulence abilities were determined by liquid chromatography-tandem mass spectrometry. We compared the levels of modifications of four nucleotides in tRNA and sncRNA, characterized RNA modification patterns of different T. gondii strains, and determined the diversity of RNA modifications. We detected and quantified 22 modified nucleosides in both tRNA and sncRNA. Significant differences in the diversity of the modified nucleosides were found between the three T. gondii strains. RNA modifications were correlated with the expression of many T. gondii virulence proteins. Some of the identified modifications  play a role in mediating the host-parasite interaction. These results provide novel insight into the global modifications in tRNA and sncRNA, and the diversity of RNA modifications between T. gondii strains with different virulence backgrounds.Many RNA modifications have been detected in rRNA, tRNA and small noncoding RNA (sncRNA) as well as in low-abundance RNA species such mRNA. Although RNA modifications play roles in many cellular and biological processes in various domains of life, knowledge about the diversity and role of RNA modifications in IMPORTANCE Although RNA modifications play roles in many cellular and developmental processes in various domains of life, knowledge about the patterns and functions of RNA modifications in T. gondii is limited. Here, a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to study global RNA modifications in T. gondii strains of distinct virulence backgrounds. We quantified 22 modified nucleosides in both tRNA and sncRNA. Significant T. gondii strain-specific differences in RNA modifications were detected. More tRNA modifications correlated with T. gondii virulence proteins than sncRNA modifications. RNA modifications were significantly correlated with virulence proteins. Our data provide the first comprehensive profiling of the modifications tRNA and sncRNA in T. gondii, expanding the diversity of RNA modifications in this parasite and suggesting new regulators for modulating its virulence. Toxoplasma gondii is a widespread protozoan parasite that infects most warm-blooded animals and roughly one-third of the world’s human population  are involved in many biological and pathological processes and play various roles across different domains of life –12. RNA –asmodium , Leishmadonovani , Trypanoa brucei , and Echranulosa , has attranulosa  and mediranulosa .N6-modification methyladenosine (m6A), is the most abundant modification in mRNA and long intergenic ncRNAs (lincRNAs) as well as found in primary miRNA and rRNA , type II , and type III , whose virulence varies significantly in mice  approach to analyze the spectrum of RNA modifications in T. gondii type I (RH strain), II (PRU strain), and III (VEG strain) using LC-MS/MS-based approach , only 18 types of tRNA and sncRNA modifications were consistently quantified, and thus, shown in the results.We determined the patterns of modifications in tRNA (~80 nt fragments) and sncRNA (17 to 50 nt fragments) between approach . Our anaapproach . HoweverT. gondii strains , uridine (U), cytidine (C), and guanosine (G) was calculated according to the levels of RNA modification. The result showed that tRNA exhibited more modifications than sncRNA, where 1–3% of each nucleotide was modified in tRNA, while < 1% of each nucleotide was modified in sncRNA, except A nucleotide (>2%) in the three  strains  and B. An sncRNA . Interes strains  and D, iT. gondii strains  and two sncRNA modifications (m1A and m5C) were expressed at significantly different amounts between RH, PRU, and VEG strains (3U) were uniquely detected between RH and PRU, whereas two (Ψ and m3C), one (Gm), and four  differentially abundant sncRNA modifications were detected between RH versus PRU, RH versus VEG, and PRU versus VEG, respectively. PCoA plots showed a strain-specific RNA modification patterns in tRNA and sncRNA, which clearly separated T. gondii strains into three distinct clusters  , which plays a role in the differentiation of T. gondii from acute to chronic, suggesting its important role in mediating the host-pathogen interaction  , rhoptris (MICs) , 32. MIClication . Additioeraction .T. gondii virulence, we investigated the correlation between the parasite virulence proteins and RNA modifications. We used LC-MS/MS approach and next-generation sequencing to quantify the expression of RNA modifications (Table S1) and genes encoding virulence-related proteins ; most of those proteins had positive correlation with I, Am, Im, and m227G. We also analyzed the correlation of sncRNA modifications with T. gondii virulence proteins and found moderate correlations between sncRNA modification patterns and T. gondii virulence factors  was significantly higher than that of the less virulent strain PRU (genotype II) and avirulent strain VEG (genotype III).Our analysis revealed a significant diversity of tRNA and sncRNA modification signatures between T. gondii. Alterations of tRNA modifications can affect codon decoding fidelity . A previous study in mice detected a correlation between the level of sperm tsRNA mcRNA m2G . The intcRNA m2G . WhetherT. gondii is defined by three main clonal lineages which differ significantly in virulence capacity mediated by the expression of effector proteins secreted by the specific organelles MICs, ROPs, and GRAs , PRU strain (type II), and VEG strain (type III), were provided by Dr. Jin-Lei Wang, Lanzhou Veterinary Research Institute. The growth of the tachyzoite stage of these T. gondii strains in monolayers of human foreskin fibroblasts (HFFs) was performed as previously described  for 1 h in 1× TBE buffer . The gel was stained with SYBR GOLD , and based on the position of the RNA maker , RNA fragments of ~80 nt and 17 to 50 nt were extracted from the gel as previously described  was added to a 1.5 mL microcentrifuge tube containing 1 × 10escribed .2 and 5 mg/mL BSA), 1 IU benzonase , 0.2 IU alkaline phosphatase , and 0.05 IU phosphodiesterase I  at 37°C for 3 h. Then, the enzymes in digestion mixture were removed using a Nanosep 3K spin filter . Before transferring the digested RNA samples, the spin filter was rinsed twice with 400 μL Watsons distilled water at 14,000 × g for 15 min at room temperature. The processed RNAs were stored at −20°C for LC-MS/MS analysis. The LC-MS/MS-based RNA modification detection was performed using waters ACQUITY-UPLC I-class carrying Xevo-TQ-S mass spectrometry system . The RNA libraries were prepared from 1 μg total RNA of each T. gondii sample using the NEBNext Ultra RNA Library Preparation Kit  in accordance with the manufacturer’s recommendations. Each library was sequenced on the Illumina HiSeq 2500 . Adapter sequences and low-quality reads were removed using Trimomatic (v.0.39) (T. gondii reference genome (ftp://ftp.ensemblgenomes.org/pub/release-31/protists//fasta/toxoplasma_gondii/dna/) using ersion 2.0 with default parameters (We used next-generation sequencing to obtain the transcriptomes of genes associated with virulence properties in (v.0.39) . The filrameters . The HTSt test and one-way ANOVA with multiple comparisons. Data are presented as the means ± standard error of means (SEM) of three independent replicates. P values < 0.05 were considered statistically significant. Linear correlation analysis was performed to study the correlation between RNA modifications or between RNA modifications and RNA modification enzymes. The transcriptomic data of selected genes encoding virulence-related proteins were used to explore the correlation between RNA modifications and virulence-related proteins. Hierarchical clustering heatmaps and correlation matrices were obtained using the R package corrplot and correlations were calculated based on the mean values of the measured modifications using the “Spearman” method. Correlation analysis was used to assess the degree of covariance among the various RNA modifications for each parasite strain, with correlation coefficients calculated using GraphPad Prism 8. Principal coordinates analysis (PCoA) of RNA modification levels was performed using the R package scatterplot3d. The PCoA and chord plots were generated using the R packages scatterplot3d and circlize, respectively.Statistical analyses were performed using GraphPad Prism 8 . Differences in the levels of ribonucleosides in different strains were examined by Student's T. gondii have been submitted to NCBI under the accession number PRJNA932999.RNA-seq reads for the


However, relatively few proteins have been confirmed to bind directly to RNA containing these modifications. Furthermore, for many of these protein readers, the exact binding mechanisms as well as the exclusivity for recognition of modified RNA species remain elusive, leading to questions regarding their roles within different cellular processes. In the case of the YT-521B homology (YTH) family of proteins, both experimental and in silico techniques have been leveraged to provide valuable biophysical insights into the mechanisms of m6A recognition at atomic resolution. To date, the YTH family is one of the best characterized classes of readers. Here, we review current knowledge about epitranscriptome recognition of the YTH domain proteins from previously published experimental and computational studies. We additionally outline knowledge gaps for proteins beyond the well-studied human YTH domains and the current in silico techniques and resources that can enable investigation of protein interactions with modified RNA outside of the YTH-m6A context.To date, over 150 chemical modifications to the four canonical RNA bases have been discovered, known collectively as the epitranscriptome. Many of these modifications have been implicated in a variety of cellular processes and disease states. Additional work has been done to identify proteins known as “readers” that selectively interact with RNAs that contain specific chemical modifications. Protein interactomes with N6-methyladenosine (m  For instance, the m6A modification has been shown to affect processes like alternative splicing of pre-mRNAs Since the initial discovery of chemical modifications to RNA in the form of pseudouridine in 1957 6A mark has led to a more generalized model for dynamic “writing” or “erasing” of modifications to RNA  domain proteins 1A), N3-methylcytosine (m3C), and m6A 6A, m1A, N5-methylcytosine (m5C), and 8-oxo-7,8-dihydroguanosine (8-oxoG) 6A, but we also outline current gaps in knowledge around the binding mechanisms for non-YTH readers of m6A and readers for other RNA modifications. Finally, we discuss the challenges and promise of conducting in silico biophysical studies of protein interactions with modified RNA and highlight current work in characterizing these interactions.Characterization of the enzymes that deposit or remove the ms to RNA . In the 22.1i.e., through protein-protein interactions or possibly RNA structure-specific interactions) to the modified RNA. These proteins are then isolated from whole cell lysate through affinity pulldown of the RNA 6A-containing RNA bait versus an unmodified control. This was the first study to identify the YTHDF2 and YTHDF3 proteins as m6A interactors. In the case of YTHDF2 and YTHDF3, both proteins contain a YTH domain that is widely conserved across eukaryotes [38]. After the discovery of YTHDF2 and YTHDF3 as putative m6A readers, these proteins as well as the three others found in humans that contain the YTH domain  were investigated for their recognition of m6A-containing transcripts in vitroE. coli, purified, and subjected to in vitro binding experiments (via the dissociation constant (KD). It is difficult to directly compare the reported KD values in some cases because of the effects of different buffer compositions, incubation temperatures, and incubation times on these values 6A or the unmodified adenine. Regardless of RNA length, the YTH domains showed micromolar to sub-micromolar affinities and selectivity for m6A with respect to adenine. Specifically, these proteins showed either no binding to unmodified RNA D value to unmodified RNA for those studies that did show binding 6A-containing RNA, relative to unmodified RNA. Another common observation regarding m6A binding can be found in the effects of RNA sequence and length on KD. The YTH family of proteins has been shown to recognize predominantly a RRACH sequence motif, where R represents a purine base (A or G), A is modified to m6A, and H represents either A, C, or U 6A modification (“G-1”), show the preference of YTHDC1 for RGAC D from 0.3 μM to 2.0 μM for YTHDC1 6A-containing RNA sequences bound at lower levels of affinity to YTHDF1 and YTHDC1, which could be linked to the importance of regions flanking m6A for stabilizing the protein-RNA complex 6A, the key regions responsible for this selectivity needed to be determined. To elucidate the protein features responsible for the selective recognition of m6A containing transcripts, crystal structures were resolved in tandem with binding characterization efforts for all human YTH domains Initial identification of proteins that recognize modified RNA transcripts has been accomplished through large scale RNA affinity chromatography studies eriments . These b2.26A-containing RNA (referred to as bound or “holo” structures) and in the absence of RNA (referred to as unbound or “apo” structures)  and bound (holo) crystal structures of YTHDF2, that contains Tyr418, Trp432, Trp486, and Trp491 CU pentanucleotide 6A-bound (holo) forms 6A recognition. This was validated for YTHDC1 and YTHDF1 through mutagenesis of the corresponding tryptophan residues and subsequent in vitro binding assays Studies have investigated human YTH domains in complex with muctures) . This ind Trp491  A [40], 6A recognition, the m6A-bound and unbound states show noteworthy differences. When the apo crystal structure of YTHDF2 was first resolved, the same study also resolved a holo crystal structure of YTHDF2 in complex with an m6A mononucleotide for comparison 6A. This loop contains Trp486 and Trp491, which accounts for two out of the four residues in the aromatic cage. Trp486 forms the “base” of the aromatic cage in both the apo and holo crystal structures of YTHDF2. The aromatic rings of Trp491, however, “flip” from the apo structure to become parallel to the aromatic rings of Trp432 in the holo structure 6A recognition 6A versus unbound structures. These residues include Trp491 in YTHDF2, as well as Trp470 and Tyr397 in YTHDF1  simulations, alchemical transformations, and metadynamics 6A to examine in detail the overall contribution of the methyl group to YTH domain binding 6A to adenosine. The steps along this unphysical transformation were analyzed using thermodynamic integration (TI) 6A containing RNA and its unmodified counterpart 6A)CU oligomer adopts a more favorable conformational state for association with the YTH domain in solvent relative to the GGACU unmodified RNA. When examining attributes of the binding pocket that could lead to m6A specificity, multiple microsecond-scale simulations of the apo YTH domain showed that conformational shifts of Met438 and Trp428 lead to metastable states that rearrange the structure of the aromatic binding pocket. In addition to the structural rearrangements, Thr379 was shown to be key for maintaining indirect interactions with m6A via a conserved water molecule. Follow-up crystallography experiments with a Thr379Val mutant to disrupt hydrogen bonding with the particular conserved water (referred to as “water 1”) resulted in a 140-fold decrease in affinity for a GG(m6A)CU RNA oligomer as measured by differential scanning fluorimetry Rattus norvegicus YTH-G(m6A) bound form of this protein 6A was shown to expel invading water molecules that occupied the m6A binding pocket in the apo simulations.To further investigate the interactions between the YTH domain and m6A were resolved in the crystal structures of m6A-bound human YTH domains. In YTHDF1, a water molecule mediates interactions between the N7 of m6A and sidechains of Trp411 and Asp507  was identified as a promising candidate for further design due to its IC50 of 39 μM and its ligand efficiency of 0.6 kcal mol-1 nHA-1. Additionally, this finding further underlines the importance of interactions with polar residues in the binding pocket of YTHDC1 and motivates additional development of a small molecule inhibitor for YTHDC1.The regulation of m6A-YTHDF2 binding activity. It is important to note that due to the structural and sequence similarity of YTHDF2 to YTHDF1 and YTHDF3, these small molecules might act as general inhibitors for all of these proteins 6A nucleobase and uracil analogs; as well as pyrazolopyrimidine, triazine, and pyrimidine derivatives. These molecules were analyzed for their inhibitory effects with HTRF, and their interactions with the YTH domain from YTHDF2 were determined using X-ray crystallography. One of these molecules, 6-cyclopropyluracil (referred to as compound 11) represents a promising candidate for future drug development with an IC50 of 174 μM and a ligand efficiency of 0.47 kcal mol-1 nHA-1, owing its potency to the interaction of the cyclopropyl group with the aromatic cage of YTHDF2 in vitro binding assays and through immunoprecipitations from PC-3 prostate cancer cells treated with a non-lethal concentration of ebselen for 24 h. Ebselen was found to bind covalently with Cys412 through selenium sulfide bonds or reversibly with the m6A binding pocket depending on the reducing or oxidizing nature of the binding pocket environment as determined by X-ray crystallography The YTHDF2 and YTHDF1 proteins have also been selected as targets for small molecule drug design. In addition to crystallizing the first structure of the YTH domain of YTHDF2 in complex with a pentanucleotide (PDB: 7z26) 33.16A-interacting proteins have also uncovered readers outside of the YTH family such as the insulin-like growth factor 2 binding proteins (IG2BPs) 6A-containing transcripts. The IGF2 binding proteins (IGF2BP1/IGF2BP2/IGF2BP3) have been found to bind m6A-containing transcripts with a 3-to-4-fold higher affinity relative to unmethylated transcripts 6A methylation in both single-stranded and structured, hairpin RNA. These proteins contain two RNA recognition motif (RRM) and four K-homology (KH) RNA binding domains. The KH3 and KH4 domains of these proteins were shown to be key for binding a single-stranded m6A oligomer through RNA pulldown experiments followed by Western blotting to visualize the protein-RNA complex 6A binding pocket found in the YTH domain; however, the specifics of the structural similarities (or differences) between the m6A binding pockets of these proteins remains unclear. Furthermore, these proteins are currently being investigated as druggable targets in the context of colorectal cancer proliferation 6A could elucidate the impact of a small molecule inhibitor on the IGF2BP-m6A interaction.RNA chromatography studies that identify m6A base, a subclass of proteins recognizes m6A in a more indirect, structurally dependent manner 6A reader that is thought to bind to methylated transcripts via an indirect mechanism involving RNA structure 6A modification, creating a structure distinct from its unmodified counterpart, which is preferably recognized for binding by the HNRPNC reader protein. One such example of this “m6A switch” behavior within the local RNA structure is found in the human metastasis-associated lung adenocarcinoma transcript (MALAT1) 6A methylation motif in a dsRNA region. Upon methylation of the adenine in this motif, the base pairing of adenine to uracil in the dsRNA region within the hairpin loop is disrupted, leading to a partial opening of the stem loop. The now single-stranded region of the partially opened stem loop previously bound to the GGACU motif becomes accessible to proteins like HNRNPC, which has been well characterized for its binding to MALAT1 in an m6A-dependent manner 6A methylation event 6A “reading” as it appears not to recognize the m6A modification through direct binding, but rather recognizes unmodified regions of RNA that are made accessible for binding through structural rearrangements dependent on the presence of m6A. While other HNRNPs have been identified as m6A interacting proteins 6A 6A modified RNA when compared with relative to unmodified for a 5-mer- 8-mer and 10-mer, respectively 6A is still an eluding problem. Thus, additional studies are needed to explore the nature of the molecular recognition mechanism.In addition to readers like the YTH proteins and the IGFBPs that bind directly to the modified m6A have only been recently investigated in terms of their mechanisms of binding to methylated transcripts. Excitingly, however, these investigations have not only identified a possibly novel mechanism for direct recognition of m6A, but also a potentially indirect, structurally dependent mechanism that might extend to other m6A reader proteins (besides HNRNPC). Further work is needed to characterize the dynamics of these binding events as well as to uncover alternative binding surfaces for m6A beyond the YTH aromatic cage.Overall, non-YTH readers for m3.26A modified RNAs, similar mass spectrometry techniques have been used to identify proteins interacting with m1A 5C 1A with a 34-mer RNA probe designed to carry a portion of the SOX18 gene known to be modified in vivo1A readers. Follow-up in vitro binding characterization was conducted with the YTH domain proteins via EMSA 1A at a lower affinity relative to the same oligomer containing m6A. Despite this lower affinity, these proteins were selective for both m1A and m6A relative to an unmodified RNA oligomer containing adenine  led to an abrogation of m1A binding activity, suggesting that m1A modified RNA may be recognized through a similar mechanism to m6A modified RNA; however, the other proteins identified in the m1A interactome from this study were not investigated further. The large difference in affinity of these YTH proteins for m1A and m6A modified RNA was later investigated using follow-up EMSAs and microscale thermophoresis (MST) experiments D values with m1A containing oligomers for both YTHDF1 and YTHDF2, as well as a 3-to-5-fold decrease in affinity for an equivalent unmodified oligomer  can serve as a stepping stone to study and identify the protein-RNA interface, and subsequently build an initial interaction model between protein and RNA e.g., of the monomer models with respect to the experimentally resolved complex) and then evaluated through scoring functions measuring structural similarities between the monomer models, as well as the complex template components An “appropriate” protein structure , as well as with respect to the protein 4.3E. coli, which has been previously investigated in human cells for its selective recognition of 8-oxoG in vitro alongside m5C, a modification screened out at the explicit solvent phase Simulations have been widely employed to study protein-RNA interactions 56A, with the in silico investigations focusing on the YTH family of protein readers 6A modified RNA and the possible redundancy in binding mechanisms for the YTHDF proteins. However, the YTH domain family represents only a single model of recognition for m6A, and the binding mechanisms for non-YTH domain readers such as the IGF2BPs 6A modification 1A, m5C, and 8-oxoG. The in silico investigation of the YTH family of proteins represents a proof-of-concept for the power of atomistic MD simulations for mechanistic understanding of epitranscriptome reader proteins. The development of both protein structure prediction tools, molecular docking, and molecular mechanics parametrization of more than 100 different modified RNA species The budding field of epitranscriptomics offers new and exciting opportunities for investigation of novel protein-RNA interactions. Large-scale studies to identify proteins that interact with a handful of RNA modifications have been conducted Lucas G. Miller: Conceptualization, Writing – original draft preparation, Reviewing & editing. Madeline Demny: Methodology, Writing – original draft preparation, Reviewing & editing, Visualization. Phanourios Tamamis: Conceptualization, Writing – reviewing & editing, Supervision, Funding acquisition. Lydia M. Contreras: Conceptualization, Writing – reviewing & editing, Supervision, Funding acquisition.The authors declare no conflicts of interest.


Understanding how ligands bind to ribonucleic acids (RNA)is importantfor understanding RNA recognition in biological processes and drugdevelopment. Here, we have studied neomycin B binding to neomycin-sensingriboswitch aptamer constructs by native top-down mass spectrometry(MS) using electrospray ionization (ESI) and collisionally activateddissociation (CAD). Our MS data for a 27 nt aptamer construct revealthe binding site and ligand interactions, in excellent agreement withthe structure derived from nuclear magnetic resonance (NMR) studies.Strikingly, for an extended 40 nt aptamer construct, which representsthe sequence with the highest regulatory factor for riboswitch function,we identified two binding motifs for neomycin B binding, one correspondingto the bulge-loop motif of the 27 nt construct and the other one inthe minor groove of the lower stem, which according to the MS dataare equally populated. By replacing a noncanonical with a canonicalbase pair in the lower stem of the 40 nt aptamer, we can reduce bindingto the minor groove motif from ∼50 to ∼30%. Conversely,the introduction of a CUG/CUG motif in the lower stem shifts the bindingequilibrium in favor of minor groove binding. The MS data reveal site-specificand stoichiometry-resolved information on aminoglycoside binding toRNA that is not directly accessible by other methods and underscorethe role of noncanonical base pairs in RNA recognition by aminoglycosides. Previous studies found that high-affinitybinding is not necessarily highly specific, and low-affinity bindingis not necessarily unspecific. For example, RNA aptamers selectedfor higher-affinity binding did not show higher specificity for theguanosine triphosphate ligand.6 Likewise,neomycin B binds to the HIV-1 rev response element (RRE) stem II RNAwith ∼100-fold higher affinity than kanamycin A, but the specificityof binding to RRE stem II RNA is higher for kanamycin A than for neomycinB.8 Tor and coworkers have even suggested that the inversecorrelation between binding affinity and specificity observed in aminoglycosideand aminoglycoside derivative binding to RRE stem II RNA may representa general principle of small molecule-RNA binding.7 Apparently, ligand binding to RNA is far more intricatethan what a simple model would predict and could involve more thanone binding motif.13To better understand biological processesthat involve ribonucleicacids (RNA) and to advance the development of potential therapeuticsthat target RNA,17 The interactions of aminoglycosides with RNA arelargely electrostatic in nature due to the positive charge of theaminoglycosides and the negative charge of the RNA at neutral andnear-neutral pH but can be highly specific when the binding interfaceincludes networks of directional intermolecular interactions suchas hydrogen bonds.20 Numerous studies have investigated RNA-aminoglycosideinteractions using a variety of methods, including nuclear magneticresonance (NMR) spectroscopy,28 crystallography,31 and enzymatic or chemical probing approaches.34 The majority of NMR and crystal structures of RNA-aminoglycosidecomplexes show aminoglycoside binding to various motifs formed bynucleotides in and next to internal loops or bulged regions,15 and noncanonical base pairs were suggested tobe important for recognition by the aminoglycoside ligand.35 However, since aminoglycosides are conformationallyflexible and can adopt to RNA structures, which themselves can behighly dynamic,36 it is challenging topredict binding affinity and specificity.37 To learn more about RNA-aminoglycoside interactions, Disney andcoworkers developed a two-dimensional combinatorial screening methodfor the identification of high-affinity, specific interactions betweenaminoglycosides and model RNAs with various hairpin and internal loopmotifs.42 More recently, Hargrove and coworkers used data from aminoglycosidebinding to model RNAs with different secondary structure motifs and principal component analysis to develop an unbiased approach forthe classification of RNA structures.44Aminoglycosides can bind to many different types of RNA, includingribosomal RNA, viral RNA, transfer RNA, and riboswitches, which iswhy they have been described as excellent model ligands for the studyof RNA recognition.61 For example, in separate native ESI MS studies, Loo et al., Hofstadleret al., and Fabris and Hagan observed RNA-aminoglycoside complexeswith higher stoichiometry, which they interpreted as either unspecificbinding or low-affinity binding to sites other than those of the 1:1complexes.62 Inspired by our recent studies of RNA-peptide complexes,64 we wondered if we could use native ESI MS in combination with low-energycollisionally activated dissociation (CAD)66 to contribute to a better understanding of how small molecules interactwith RNA, especially with respect to specific versus unspecific bindingand multiple binding motifs. A major advantage of native ESI combinedwith CAD is that this approach can be used to characterize wild-typeRNA-ligand complexes even when their conformational dynamics complicatethe interpretation of NMR data67 or interfere with RNAcrystallization.70 As a model system for our studies, we chose 40 ntaptamer constructs based on the sequence of a synthetic neomycin-sensingriboswitch, which showed high ligand specificity for neomycin B anddose-dependent regulation of gene expression.72 Moreover, we have studied a 27 nt aptamer construct of the neomycin-sensingriboswitch for whose complex with paromomycin, an NMR structure, isavailable.73A method that can complement the above techniquesand does notrequire isotope labeling, crystallization, enzymes, or chemical reagentsis native mass spectrometry (MS). Since the late 1990s, RNA-proteinand RNA-drug complexes have been studied by native electrospray ionization(ESI) MS, which can directly reveal the identity of RNA binding partnersand the stoichiometry of the complexes present in solution.1, 77 with increasing pH, which in turn decreases the number of electrostaticinteractions essential for aminoglycoside binding to the RNA.79 The KD values calculated from the fractionsof RNA complexes in the spectra from ESI at room temperature (21 °C)and assuming a single binding site62 (model1 in the Supporting Information) were 29± 5 nM at pH ∼6, 0.14 ± 0.05 μM at pH ∼7,0.7 ± 0.2 μM at pH 7.5, and 6 ± 1 μM at pH ∼9 by Wöhnert and coworkers.73 Data from ITC experiments at 21 °C with solutions closely resemblingthose for ESI (with the RNA concentration increased to 8–10μM for reasons of sensitivity) were not suitable for the determinationof KD values even though imino protonspectra from nuclear magnetic resonance (NMR) clearly showed thatthe RNA was folded . We thenconducted ITC experiments at pH 7.5 and 21 °C using a buffertypical for ITC of RNA.80 The ITC dataindicate two binding sites for RNA 1  with KD values (2 ±2 nM and 0.6 ± 0.2 μM) that differ by ∼2.4 ordersof magnitude, meaning that in 1:1 mixtures of RNA 1 withneomycin B, the latter is almost exclusively bound to the high-affinitysite. Fitting the ITC data with a single-site model instead resultsin a KD value (0.7 ± 0.2 μM)that matches the KD value from ESI atthe same pH . Higher concentrations of the aminoglycoside ligand were requiredto observe similar fractions of 1:1 complexes of RNA 1 and paromomycin by native ESI MS. For a solution with RNA 1 (1 μM) and paromomycin (10 μM) at pH ∼6and room temperature , the ESIdata showed 67 ± 6% RNA complexes, which indicates a KD of 4.6 ± 1.4 μM, in good agreementwith the KD of 5.13 ± 0.26 μMfrom ITC measurements (at pH 6.8 and 37 °C).73 We conclude that although native ESI MS62 and ITC83 are conceptually different,ESI MS spectra can be used for the calculation of KD values that agree well with those from ITC, providedthat a single-site model adequately describes the binding equilibrium.For systems with more than one binding site, the ESI MS data can atleast provide an estimate of the overall binding affinity. However,without site-specific binding information, it is generally difficultto distinguish between single and multiple binding sites.9 To further investigate the neomycin-sensing riboswitchaptamer and to address this issue by determining the sites of neomycinB and paromomycin binding, we used our recently developed MS approachwith ion isolation in the quadrupole followed by collisionally activateddissociation (CAD) in the collision cell of a Fourier transform ioncyclotron resonance (FT-ICR) instrument.65at pH ∼91D, all o1 + neomycin B - 9H)9–, at m/z ∼1016 produced c and y fragmentsfrom phosphodiester backbone bond cleavage85 with and without neomycin B attached and only ∼1% free RNAfrom neomycin B dissociation (relative to undissociated complexes),which shows that the electrostatic interactions between neomycin Band RNA 1 are sufficiently strong for binding site mappingby CAD.65c fragmentswith neomycin B attached (white circles) and the fraction of complementary y fragments without neomycin B attached (whitetriangles) plotted against the RNA cleavage site. The below data interpretationfor the identification of ligand binding regions of RNA64 is conceptually the same as for nucleobase modifications86 and considers site-specific fractions of c and y fragments with and withoutneomycin B attached. No neomycin B was found on fragments c1–c3, and no complementary fragments y26–y24 without neomycin B were observed, indicating that neomycin B doesnot bind to G1-C3. The fraction of c fragments with neomycin B attached sigmoidally increased from site3 to site 10, along with a corresponding increase in the fractionof complementary y fragments withoutneomycin B, both of which indicate binding of neomycin B to U4–U10(binding region I). After a plateau at sites 11–16, which indicatesno binding of neomycin B to C11-A16, the fraction of c fragments with neomycin B and y fragments without neomycin B further increased to 100% atsite 22, which indicates binding of neomycin B to A17-C22 (bindingregion II). Finally, the 100% values for c fragments with neomycin B and y fragmentswithout neomycin B at sites 23–26 are consistent with no bindingof neomycin B to C23–C27. The fraction of c fragments with neomycin B and the complementary y fragments without neomycin B differ by up to ∼15%in the plateau region, which can be attributed to a higher stabilityof c and y fragments with neomycin B attached against secondary backbone cleavagecompared to fragments without a ligand.64 CAD of the1:1 complexes of the neomycin-sensing riboswitch with paromomycin,(RNA 1 + paromomycin - 9H)9–, alsoat m/z ∼1016 and using thesame energy, produced very similar data  has been published.73 Neomycin B differs from paromomycin only by the presenceof an aminium group instead of a hydroxyl group at carbon 6′of ring I 9–, from ESI of solutions at pH ∼6,∼7, and ∼9 produced very similar data , which suggests that the pH of the solution onlyaffects the fraction of RNA complexes , eventhough their fraction decreased from 84% at pH ∼6 to 12% atpH ∼9  comparedto those from solutions without ammonium bicarbonate, but the datafrom CAD of the 1:1 complexes showed no significant differences . Further, varying the energy usedfor CAD of the (RNA 1 + neomycin B - 9H)9– ions from ESI of solutions at pH ∼7 between 99 and 108 eVhad no significant effect on the percentage of c and y fragments with and withoutneomycin B, respectively , butCAD at lower energies resulted in signal-to-noise ratios for the lessabundant fragments that were too small for reliable relative quantificationof fragments with and without neomycin B attached.CAD of the 1:1 complexes of the 27 nt RNA lar data S4, whichlar data S4, whichlar data S4, whichlar data S4, whichc and y fragments85 but must also have dissociated some of the noncovalentbonds between the aminoglycoside ligand and the RNA, leaving it boundto either the c or the complementary y fragment. The plateaus for c fragments with and y fragments without an aminoglycoside ligand in 1 + neomycin B - 9H)9– and (RNA 1 + paromomycin - 9H)9– ions,the interactions of paromomycin and neomycin B with binding regionI are somewhat more stable than those with binding region II. Datafrom CAD of (RNA 1 + neomycin B - 10H)10– ions were similar to those of the (RNA 1 + neomycinB - 9H)9– ions  but more scattered because competitive dissociation into a and w fragments,which is common in CAD of RNA ions with a higher net negative charge,92 resulted in lower signal-to-noise ratios for c and y fragments.Because theaminoglycoside ligand interacts with both binding regionsI and II of the riboswitch 2C, vibra1 + neomycin B - 9H)9– ions, the plateau values were not affected by solution pH  or the energy used for CAD , but the plateau values increasedto ∼80% for the complexes with a net charge of 8– and7– . This observationis consistent with an increased number of protons that can be mobilizedduring CAD85 in the complexes with a netcharge of 8– and 7– and preferential proton transferto negatively charged sites94 in binding region II, which substantiallyweakens the interactions between binding region II and the aminoglycosideligand  shows that dissociation by CAD is not governed by simpleCoulombic interactions as the distances between the phosphorus atomsof the RNA phosphodiester moieties and the nearest nitrogen atom ofparomomycin in the NMR structure would predict an overall higher stabilityof aminoglycoside interactions with region II compared with regionI  agree well with the NMRdata 13– ions, with a similar number of net charges per nucleotide(0.325 charges/nt) to the (RNA 1 + neomycin B - 9H)9– ions (0.333 charges/nt)  2A, indic and III 4A. The ld III in ∼50% of the complexes and to the binding pocket formed bythe loop and the bulge of RNA 2  in the other ∼50% of the complexes. This resultis in line with ITC data  thatindicate two binding sites for RNA 2 with KD values of comparable magnitude (0.4 ± 0.6 and 3.1± 1.5 μM). Replacing the noncanonical base pair G4•U32of RNA 2 by the canonical base pair U4-A32 . These findings are consistent with the proposed role of noncanonicalbase pairs in RNA recognition and binding of aminoglycoside ligands35 and show that CAD MS can be used to detect changes in the locationof ligand binding sites as well as for the determination of relativepopulations of different binding motifs in a given RNA-ligand complex.Finally, to get an idea about how the different binding motifs ofRNAs 2–4 affect the overall bindingaffinity for neomycin B, we performed competitive ESI MS binding experimentsusing RNA 1 as a reference (method 2 in the Supporting Information). The dissociation constantsfor the complexes of neomycin B with RNAs 2–4 were the same within error limits and did not reflect thesubtle differences in the occupation of binding motifs A and B derivedfrom CAD data  offer two sequence-distantstrand regions for specific binding of neomycin B, referred to hereas binding regions I and II. The CAD data for the 27 nt constructdesigned (and optimized) for NMR studies (RNA 1) showthat neomycin B simultaneously utilizes both regions I and II, whichform a single binding pocket for neomycin B (binding motif A), inexcellent agreement with NMR studies.73 Importantly, in the functionally more relevant 40 nt aptamer constructs(RNA 2–4), we identified two additionalbinding regions 0 and III (binding motif B). The proportion of complexeswith neomycin B binding to motifs A and B and thus the correspondingbinding specificities strongly depend on the presence and nature ofnoncanonical base pairs. For the RNA with the sequence correspondingto the highest regulatory factor for riboswitch function (RNA 2), we find that binding motifs A and B are equally occupied.Although the biological meaning and relevance of the second bindingmotif is unclear at this time, one can speculate that the additionalbinding motif fine-tunes riboswitch regulatory function. For instance,simultaneous occupation of both motifs may impact the repressor/antirepressorstem fold equilibrium, which is responsible for the accessibilityof the AUG start codon (nucleotides 38–40) in translationalcontrol of riboswitches.With regard to binding specificity, ournative top-down MS datareveal that all neomycin-sensing riboswitch aptamer constructs studied(RNAs 2) in the lowerstem with the canonical base pair U4-A32 (RNA 3), whichresulted in decreased binding to motif B. Further, the introductionof a different noncanonical base pair, U4•U32, as part of aCUG/CUG motif in the lower stem (RNA 4), not only increasesthe number of nucleotides in the lower stem that interact with neomycinB but also increases binding to motif B. Our data thus show that neomycinB preferentially binds to regions of the riboswitch aptamer constructsthat contain noncanonical base pairs35 and unpairednucleobases, which also supports the hypothesis of Wöhnertet al. that “ligand specificity of the neomycin riboswitchis encoded at the level of structural dynamics”.73 In future studies, we will extend our nativetop-down mass spectrometry approach to other RNA-aminoglycoside complexesand also investigate new classes of ligands with the ultimate goalto establish general principles of RNA recognition to guide the developmentof drugs that target RNA.We further stress that a major strengthof our FT-ICR MS approachis the experimental ease to investigate sequence mutations. The approachis complementary to NMR spectroscopy and crystallographic approachesand fills a method gap for the determination of middle-to-low affinityRNA small molecule binding sites with the advantage of very littlesample amount requirements, no need for stable isotope labeling, andno need for labor-intensive and tedious RNA construct optimization.Any mutation or sequence variation can be easily integrated in theoriginal experimental protocol as demonstrated here by replacing thenoncanonical base pair G4•U32 . RNAs 2–4 were prepared by solid-phase synthesisand purified by HPLC. H2O was purified to 18 MΩ·cmat room temperature using a Milli-Q system , andCH3OH  was HPLC-grade. RNA was desalted bydiluting ∼100 μL of RNA solution in H2O with∼400 μL of aqueous ammonium acetate (100 mM) solutionfollowed by concentration to ∼100 μL using centrifugalconcentrators ; theconcentration–dilution process was repeated 10 times and followedby 6 cycles of concentration and dilution with H2O. TheRNA concentration was determined by UV absorption at 260 nm usingan Implen nanophotometer . ESI spectra were obtainedby operation of the linear quadrupole in the transmission mode. ForCAD, ions of interest were isolated in the quadrupole and dissociatedin the collision cell using the laboratory frame collision energy92 indicated in the figure legends. For ESI andCAD, 25–100 and 500 scans were added for each spectrum, respectively.Data reduction utilized the SNAP2 algorithm  or FASTMS, software programmed in our group by Michael Palasser,34 as well as manual inspection of the spectra.NMR (at 25 °C) and ITC (at 21 °C) experiments were carriedout using a 600 MHz Avance II+ NMR spectrometer equipped with a ProdigyTCI probe (Bruker) and a PEAQ-ITC instrument , respectively.MS experiments were performedat room temperature (21 °C)on a 7 T Fourier transform ion cyclotron resonance (FT-ICR) instrument equipped with an ESI source, a linearquadrupole for ion isolation, a collision cell for CAD, and an ICRcell for ion detection. RNA