Xenopus laevis, Ran can control branching through a canonical SAF, TPX2, which is nonessential in Drosophila melanogaster embryos and HeLa cells. Thus, how Ran regulates branching MT nucleation when TPX2 is not required remains unknown. Here, we use in vitro pulldowns and total internal reflection fluorescence microscopy to show that augmin is a Ran-regulated SAF. We demonstrate that augmin directly interacts with both importin-α and importin-β through two nuclear localization sequences on the Haus8 subunit, which overlap with the MT-binding site. Moreover, we show that Ran controls localization of augmin to MTs in both Xenopus egg extract and in vitro. Our results demonstrate that RanGTP directly regulates augmin, which establishes a new way by which Ran controls branching MT nucleation and spindle assembly both in the absence and presence of TPX2.Mitotic spindles are composed of microtubules (MTs) that must nucleate at the right place and time. Ran regulates this process by directly controlling the release of spindle assembly factors (SAFs) from nucleocytoplasmic shuttle proteins importin-αβ and subsequently forms a biochemical gradient of SAFs localized around chromosomes. The majority of spindle MTs are generated by branching MT nucleation, which has been shown to require an eight-subunit protein complex known as augmin. In  The mitotic spindle is a self-organized structure built from microtubules (MTs). Spindle MTs are made by several MT nucleation pathways that originate from various locations within the spindle , 2, 3. Mvia effector heterodimeric importins, by promoting cargo binding by importins in the cytoplasm and cargo release within the nucleus . TPX2 facilitates branching MT nucleation, whereby one MT is nucleated from a pre-existing one, enabling amplification of MTs throughout the spindle , 25. In s laevis , 28. Recs laevis . Bindings laevis . However of TPX2 , 32, andssential . Thus, tDrosophila, where subunits were shown to be required for robust spindle assembly , whereas the other end of the complex, tetramer II (T-II), recognizes and binds MTs (A). Within T-II, there are two MT-binding sites -tagged importins, as well as GST alone as a negative control, were immobilized on glutathione agarose resin and probed with recombinant X. laevis augmin complex. Because importin-α is autoinhibited by its NLS-like importin-β binding (IBB) sequence when not bound to importin-β .To determine whether augmin is a SAF, we first asked whether augmin is capable of binding importin-α and importin-β, a requirement of Ran regulation. Purified glutathione-portin-β , we usedin-αΔIBB B.A) , binding both importin-αΔIBB and importin-β with approximately equal strength. In contrast, T-III displayed no appreciable binding to either importin . Next, we analyzed the sequences of the four T-II subunits  for potential importin-binding sites .Because the augmin complex is large (∼450 kDa) and distinct functional roles are played by different subcomplexes , 38, we Fig. 1ng sites . We founng sites . Fortuitng sites , and we 1—150 was sufficient to bind to importins, we determined whether this was the sole importin-binding site on augmin by expressing and purifying Xenopus augmin lacking Haus81—150 and conducting the binding assay. Interestingly, we found that the truncated complex was still able to bind to importin-αΔIBB, although importin-β binding was abrogated (E). Based on our binding experiments with the two tetrameric subcomplexes, we suspect that this second binding site must be located somewhere within T-II, since importins do not bind T-III. However, we could not determine the location of this second importin-α binding region because of the lack of obvious predicted NLS sequences and because the entwined structure of the remaining T-II subunits prevents any other augmin fragments from folding correctly and thus retaining function . The first putative NLS is bipartite and contains residues that are highly conserved among most vertebrates, whereas the second monopartite NLS is predicted only in frogs and toads .Having identified an importin-binding region within Haus8, we sought to characterize exactly where those sites are located. Using an NLS prediction algorithm , we founFig. 2ΔIBB binding relative to wildtype (B). Moreover, mutation of NLS1 (C) had a lesser impact on importin binding than mutation of NLS2 (D). This may result either from a difference in importin-α binding affinity between the two NLS sequences, or, alternatively, may be because mutation of two residues in a bipartite NLS is less deleterious than mutation of two residues in a shorter and monopartite NLS. Nonetheless, combining mutations to both NLS sites completely abrogated importin-αΔIBB binding (E). In contrast, mutation of either or both NLS sequences had little to no measurable effect on importin-β binding (B–E). Thus, Haus8 contains two NLS regions recognized primarily by importin-α.To test whether one or both of these two putative NLS sites are required for importin binding, we mutated the NLSs by introducing double alanine mutations at either Lys-27 and Lys-28 or Lys-143 and Lys-144 , 46. We wildtype B. Moreov of NLS1 C had a l of NLS2 D. This m binding E. In con binding , B–E. Thin vitro MT-binding assays using biotinylated stabilized MT seeds attached via neutravidin to functionalized glass coverslips. GFP-labeled Haus8 NLS mutants were diluted to 100 nM, which mimics the endogenous concentration, and incubated with immobilized MT seeds. The degree of binding was assessed by total internal reflection fluorescence (TIRF) microscopy. We found that single NLS mutants decreased the binding to MT seeds, whereas the double mutant lacking both NLS sites inhibited binding altogether (F and G). Thus, the MT-binding sites in Haus8 overlap with the NLS regions, which is a common feature of other MAPs including NuMA and TPX2 .A critical role of augmin is to bind to the side of the MT lattice to recruit γ-TuRC for branching MT nucleation. We hypothesized that this function may be Ran regulated and therefore asked whether importin binding to augmin modulated its interactions with MTs. To address this, we performed d to MTs , A and BC and D). Therefore, this suggests that the secondary MT-binding site in the Haus6 subunit is insensitive to importin binding and still allows some localization to MTs. The functional consequences of the binding site in Haus6 are still under active investigation.We next tested how importins affected MT binding in the context of the full eight-subunit augmin complex to see if this behavior was reproduced. Interestingly, addition of both importin-α and importin-β significantly decreased binding of the full-length augmin to the MTs but did not inhibit it altogether , C and D1–150. We found that the N terminus of Haus8 bound strongly to a stoichiometric heterodimer of importin-α and importin-β relative to beads loaded only with GFP (A). When to the mixture was added a 10-fold excess of RanQ69L, a mutant of Ran locked in a GTP-bound conformational state, importin-αβ was lost from the resin, implying that RanQ69L triggers the release of augmin from importin-αβ (A). Furthermore, importin-αβ inhibited binding of GFP-Haus8 to MT seeds in vitro under the same conditions, which was also rescued by the addition of active RanQ69L (B and C).SAFs are not only characterized by importin inhibition but also by the ability of RanGTP to release this inhibition, thereby activating the SAF. To assess whether Ran regulates MT binding of augmin, we tested whether augmin binds the importin-αβ heterodimer, which, in contrast to monomeric importins, is the predominant form of importins taken in the cell. To test this, we pulled down importin-αβ heterodimer using beads loaded with GFP-Haus8with GFP A. When tortin-αβ A. Furthe RanQ69L , B and Cin vitro were representative of Ran regulation of augmin in a physiological environment, we next assessed the role of Ran in regulating augmin in X. laevis egg extract. To test this, we first took advantage of an antibody against Haus1 that pulls down the augmin complex without disrupting endogenous binding interactions with proteins including γ-TuRC (A), suggesting that augmin and importin-αβ interact in an endogenous setting. In addition, binding of endogenous importin-αβ was regulated by Ran, as incubation of the beads with extract spiked with RanQ69L substantially reduced the amount of importin pulled down (A). Finally, we generated branched MT structures in X. laevis egg extract and, using antibodies against Haus8 to visualize endogenous augmin, we found that augmin does not localize to MTs unless RanQ69L is present (B).To determine whether our results g γ-TuRC . Pullingg γ-TuRC A, suggesled down A. Finall present B.Figure bona fide SAF regulated by RanGTP. Augmin interacts with importin-α and importin-β and independently binds each importin through two NLS sites, which we identified. The NLS sites are located in the disordered N terminus of Haus8, which contains the primary MT-binding site, indicating that the NLS sites and MT-binding site(s) overlap. Moreover, importins inhibit binding of augmin to MTs, which can be reversed by addition of active RanGTP. Moreover, this regulatory system can be recapitulated both in vitro and in Xenopus egg extract.Here, we have shown that augmin is a While many studies have identified potential SAFs, none have previously recognized augmin as a candidate. High-throughput screens for importin clients in mammalian cells were limited to nuclear proteins ; therefoDrosophila, which might be taken to suggest that, in these systems, Ran regulates branching exclusively through augmin. However, Drosophila augmin lacks the NLS-containing disordered N terminus of its Haus8 subunit (known as Dgt4) and, in fact, previous work has suggested that, in Drosophila, augmin is not an importin client protein  supplemented with 10 μg/ml DNase I from bovine pancreas (Roche) and 1 mM phenylmethylsulfonyl fluoride. Cells were lysed using an Avestin Emulsiflex C5 and then clarified by ultracentrifugation at 105,000g at 4 °C for 30 min. Clarified Strep-GFP-Haus8 lysate was batch loaded onto 2 ml StrepTactin Superflow resin (IBA) per liter of cells, washed with 50 column volumes of lysis buffer, and eluted with five column volumes of lysis buffer supplemented with 2.5 mM desthiobiotin. GST-GFP-Haus8 constructs were purified similarly, except using glutathione Superflow agarose resin (Pierce) and eluting with lysis buffer supplemented with 10 mM reduced glutathione instead of desthiobiotin. After elution, samples were concentrated using a 30 kDa molecular weight cutoff spin concentrator (Amicon) to 1 ml and loaded onto a Superdex 200 Increase 10/300 column (Cytiva) pre-equilibrated with high-salt CSFxB -N,N,N′,N′-tetraacetic acid, 1 mM MgCl2, and 1 mM DTT). Fractions were pooled and concentrated prior to flash freezing in liquid nitrogen and storage at −80 °C. Protein concentrations were determined by Bradford concentration assay using bovine serum albumin to generate a standard curve.N-terminal Haus8 fragments, tagged either with Strep-GFP or GST-GFP, were expressed in Rosetta 2 (DE3) pLysS-competent cells (NEB) grown in modified Terrific Broth at 27 °C for 7 h, following induction at midlog with 0.5 mM isopropyl β-GST-tagged importins were previously described  and puriQ69L was purified as previously described  equilibrated in low-salt CSFxB or loaded directly onto Ni2+ agarose (Qiagen), eluted using 300 mM imidazole, then dialyzed overnight into low-salt CSFxB supplemented with 10% (w/v) d-sucrose.Heterologous expression of escribed . Full-leΔIBB and GST-importin-β pulldowns, 30 μl of magnetic glutathione agarose (Pierce) was washed three times with 300 μl Tris-buffered saline (TBS) supplemented with 0.1% (v/v) Tween-20 (TBS-T), then equilibrated into 300 μl low-salt CSFxB supplemented with 0.1% (v/v) Tween-20 (CSFxB-T). About 30 μl of freshly made binding reaction, containing either 2 μM GST or GST-importin and either 2 μM GFP-Haus8 (1–150) or 200 nM augmin complex, were added to the beads, allowed to mix, and then incubated at 4 °C for 2 h. Supernatant was then removed, and beads were washed three times with 300 μl CSFxB-T. Finally, beads were resuspended in 30 μl CSFxB-T, mixed with 10 μl 4× SDS-PAGE loading dye, and heated at 95 °C to denature and elute any bound proteins. After SDS-PAGE, gels were either stained by Coomassie to detect total protein content or transferred to nitrocellulose via iBLOT (Invitrogen) and probed for augmin using either murine α-Strep (Qiagen) or a previously published rabbit α-Xenopus Haus1  was substituted for aforementioned glutathione resin, and TBS supplemented with 0.1% (v/v) Tween-20 and CSFxB-T were supplemented with 10 mg/ml κ-casein; however, the remainder of the pulldown proceeded in the same manner.Xenopus egg extract was prepared as previously described  in the presence of 1 mM GMPCPP for 1 h at 37 °C. After 1 h, the MT seed mixture was centrifuged at 13,000g for 15 min. The supernatant was removed, and the pellet was resuspended in warm BRB80 buffer supplemented with 1 mM GMPCPP.Bovine brain tubulin was labeled following prior methods . Using Al-lysine)-PEG. Flow chambers for TIRF microscopy were prepared using parafilm and gentle heating to seal coverslips to the glass slides.About 22 mm × 22 mm cover glasses  were silanized and reacted with PEG as previously described , except Flow chambers were incubated with 5% Pluronic F-127 in water  for 5 min at room temperature and then washed with assay buffer  supplemented with 50 μg/ml κ-casein. Flow chambers were then incubated with an assay buffer containing 50 μg/ml NeutrAvidin  for 2 min on a metal block on ice and subsequently washed with BRB80. Next, flow chambers were incubated for 5 min at room temperature with Alexa568-labeled biotinylated GMPCPP-stabilized MTs diluted 1:2000 in BRB80. Unbound MTs were removed by additional BRB80 washes.To assess the binding of augmin and Haus8 constructs to MTs, augmin and Haus8 were diluted to 100 nM in CSFxB with 100 mM KCl and added to the flow chamber containing GMPCPP-stabilized MT seeds, previously attached to the coverslip surface. This was incubated for 10 min at room temperature. Unbound proteins were washed away using BRB80. For experiments with importins, importin proteins were incubated with augmin proteins on ice for 10 min prior to entering the flow chamber. All samples were imaged immediately.in vitro experiments, the objective was warmed with an objective heater at 33 °C. Images belonging to the same experiment were contrast matched. Images used for quantification of MT binding were analyzed using ImageJ. To segment MTs, the tubulin signal was thresholded via the Otsu method. MTs were isolated from the mask by setting the minimum particle area as 0.5 μm2. Average fluorescent signal per pixel was recorded for each MT with and without additional proteins. To compare augmin fluorescence intensity across experiments, the intensity was normalized with respect to the tubulin signal.TIRF microscopy was performed with a Nikon TiE microscope using a 100 × 1.49 numerical aperture objective. Andor Zyla cCMOS camera was used for acquisition, with a field of view 165.1 × 139.3 μM. Multicolor images were collected using the NIS-Elements software (Nikon). All adjustable parameters for imaging  were kept the same within experiments. For Q69L, as previously described (Branching MT nucleation reactions were carried out in 5 μl volume flow cells with Alexa568-labeled fluorescent tubulin in the presence or the absence of Ranescribed . After 1All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


EIF2AK2 are edited across all HSPC populations, which may abolish the miRNA-mediated inhibition of EIF2AK2. Elevated EIF2AK2 may thus activate the integrated stress response (ISR) pathway to initiate global translational attenuation as a protective mechanism to maintain cellular homeostasis during HSPCs’ differentiation. Besides, our findings also indicate that RNA editing plays an essential role in the coordination of lineage commitment and self-renewal of hematopoietic stem cells (HSCs). Taken together, we demonstrate the capacity of scRNA-seq data to exploit RNA editing events of cell types, and find that RNA editing may exert multiple modules of regulation in hematopoietic processes.RNA editing is a post-transcriptional modification with a cell-specific manner and important biological implications. Although single-cell RNA-seq (scRNA-seq) is an effective method for studying cellular heterogeneity, it is difficult to detect and study RNA editing events from scRNA-seq data because of the low sequencing coverage. To overcome this, we develop a computational method to systematically identify RNA editing sites of cell types from scRNA-seq data. To demonstrate its effectiveness, we apply it to scRNA-seq data of human hematopoietic stem/progenitor cells (HSPCs) with an annotated lineage differentiation relationship according to previous research and study the impacts of RNA editing on hematopoiesis. The dynamic editing patterns reveal the relevance of RNA editing on different HSPCs. For example, four microRNA (miRNA) target sites on 3ʹ UTR of  There are two canonical editing types in mammals, A-to-I and C-to-U2. Among them, A-to-I is the most common form in animal cells3. Most known RNA editing sites located in introns, and 5ʹ/3ʹ untranslated regions (UTRs), especially in the ALU repeat. A small proportion takes place in the coding sequences (CDSs), thus altering the sequence and function of their encoded proteins6. RNA editing increases the diversity of the transcriptome and expands the ways of regulation7. It has implications for various biological processes such as transcriptional stability and localization, interactions with other primary RNA processing steps such as splicing and polyadenylation, and the biogenesis and functions of small RNAs such as miRNAs and long noncoding RNAs10. Because RNA editing often occurs in the 3ʹ UTRs, in which miRNA binding sites are enriched, it may change the native binding sites or introduce new binding sites, and then affect miRNA-mediated gene silencing12. Meanwhile, as one of the important post-transcriptional modifications, RNA editing alters base composition at the transcriptional level also exhibits cell-specific features14.RNA editing alters the sequence of RNA transcripts dynamically and flexibly during cell development and in a cell type-specific manner16. They then differentiate into multipotent (produce most blood cell subsets), oligopotent (lymphoid or myeloid restricted), and unipotent hematopoietic progenitor cells (HPCs)17. Hematopoietic stem/progenitor cells (HSPCs) require the collaboration of complex pathways including regulation of the cell cycle, apoptosis, and transcription to maintain the internal homeostasis of differentiation and self-renewal24. More importantly, only HSCs own the ability of both multi-potency and self-renewal. The multi-potency of HSCs is reflected in the differentiation into different hematopoietic progenitor cells and functional blood cells. The self-renewal of HSCs is to generate HSCs themselves rather than through differentiation of other cells. The HSCs differentiate into hematopoietic progenitor cells to maintain the stability of the hematopoietic system by balancing differentiation and self-renewal25. As one of the most widespread post-transcriptional modifications, RNA editing also plays an indispensable role and is an important regulator of hematopoietic stem cell maintenance of differentiation function. For example, highly edited Azin1 in HSPCs led to an amino acid change and then altered Azin1 protein (AZI) translocation, and finally enhanced AZI binding affinity for DEAD box polypeptide 1. The edited Azin1 maintains the normal differentiation process of HSCs, whereas loss of Azin1 RNA editing makes HSCs proliferate14. MiRNAs also play a role in the stemness maintenance of hematopoietic stem cells, lineage commitment of hematopoietic progenitor cells, and function of mature effector cells29. For example, ectopic expression of miRNA-181 in B-lymphoid cells of murine bone marrow led to an increase in the proportion of B-lineage cells in vivo and in vitro31. MiR-150 also played important roles in hematopoiesis. Some studies have provided evidence that miR-150 blocks the transition from pro-B to pre-B cells during B-cell maturation33.Hematopoietic stem cells (HSCs) are defined as a group of cells with the ability to self-renew and reconstitute the hematopoietic system34, GIREMI35, RDDpred36, RED-ML37, etc. Since the development of scRNA-seq, most studies focused on gene expression changes in different cell types to explore cellular heterogeneity. Differences in RNA editing events between cell types had been identified in many species. For example in mice, neurons were typically edited at higher levels than glial cells39. In Drosophila, RNA editing had been studied in multiple neuronal populations, and each neuronal type had unique RNA editing events40. In human, it was also found that there were cell type-specific RNA editing sites and cell type-enriched RNA editing sites in glutamatergic neurons, medial ganglionic eminence-derived GABAergic neurons, and oligodendrocytes41. These results suggested that RNA editing may give rise to diverse molecular identities of different cell types. Therefore, the exploration of scRNA-seq data cannot be restricted to the expression level. Here, we developed a computational method to detect RNA editing events from scRNA-seq data, and investigated their dynamics and functions in 8 HSPC populations. By integrating aligned reads of all cells of the same cell type, we obtained the pseudo-Bulk RNA-seq of each cell type, which significantly increase the sequencing depth. Meanwhile, as scRNA-seq is strand-specific, we split the aligned reads into forward and reverse strands to improve the accuracy. Using this computational pipeline, we found RNA editing events occurred dynamically during human hematopoiesis, and some sites were co-edited in different cell types. Consistent with previous studies, most of the RNA editing events were located in 3ʹ UTRs, which may change the target sites of miRNAs and then suppress miRNA-mediated gene silencing.The rapid development of next-generation sequencing technologies has promoted in-depth research on RNA editing, and the current use of RNA-seq data for RNA editing site detection tools include Multi-Sampled MethodIt is a challenge to detect RNA editing events from scRNA-seq data because it has limited sequencing coverages. To overcome this, firstly we integrated the aligned reads of each cell of the same cell type according to the barcode tag in bam files to increase the sequencing coverages, which were merged to a ‘Bulk RNA-seq’ data called pseudo-Bulk RNA-seq data Fig. A see de” section+ cells and obtained their single-cell transcriptomes. Mobilized peripheral blood (mPB) and umbilical cord blood (CB) are two of the current sources of HSPCs. Researches show that CD34+ cells derived from CB have stronger regeneration capacities than that from mPB. These samples included 6 CB samples without in vitro cultivation, 6 CB samples with 48-h in vitro cultivation, 3 mPB samples without in vitro cultivation and 3 mPB samples with 48-h in vitro cultivation  were located in the ALU element    pathway, whose function is to balance the pressure signals that activate the cell death pathway in order to protect the cells to restore the cell balance55. PKR encoded by EIF2AK2 is one of the four stress-inducible kinases that maintain the survival-death equilibrium49. The interaction between PKR and the other three kinases  affects the phosphorylation of eIF2α56, leading to the attenuation of global translation initiation, which saves amino acid synthesis for essential cellular function, reduces the load of chaperones, and lowers the metabolic demand associated with protein synthesis57. Along with the up-regulation of EIF2AK2, We found the expression levels of EIF2AK1, EIF2AK3 and EIF2AK4  were all higher in cells expressing edited EIF2AK2 compared to those expressing unedited EIF2AK2 , N4BP2L2 may be involved in the positive regulation of HSC proliferation and negative regulation of HSC differentiation  using cell annotation information to obtain pseudo-Bulk RNA-seq of different cell types; (ii) optimizing the marking duplicates method to increase the sequencing depth; and (iii) using strand information to detect RNA editing sites. Compared with other studies, the distribution of RNA editing sites across different genomic elements, and the proportion of RNA editing sites within ALU elements were consistent with previous research reportsEIF2AK2 with the shared RNA editing sites in all HSPC cell populations may have an impact on maintaining self-homeostasis during HSPCs differentiation through ISR pathway. Though a study has found that ISR activation in HSCs/MPPs is more active than in hematopoietic progenitor cells49, our findings may indicate that the ISR pathway not only safeguards HSCs/MPPs but also affects all HSPCs with high differentiation potential. This implies that HSPCs with higher differentiation potential may be more sensitive to external stimulus stress. They perform specific regulation of their own cellular homeostasis through RNA editing to maintain the integrity of the hematopoietic system. Meanwhile, we found lymphoid-specific and myeloid-specific RNA editing sites may involve in lineage commitment and HSPCs differentiation. According to a previous study, RNA editing may influence lineage commitment during hematopoiesis. They found the frequency of RNA editing alters at the branch point of HSPCs differentiation14. Our results indicate that not only changes in editing frequency, but also specific RNA editing sites occur in different lineages during differentiated development. This may suggest that RNA editing sites occur at genes critical to lineage commitment. Besides shared sites, we found specific editing sites in HSCs may have functions on differentiation and self-renew. The capacity to self-renew and to differentiate into other hematopoietic cells are important features of HSCs15. This also suggests that RNA editing may play an important role in maintaining the balance of self-renewal and differentiation of HSCs. However, those results still require more experiments to verify the role of RNA editing for HSPCs.We then explore the shared RNA editing sites and specific RNA editing sites among different HSPC populations and found that RNA editing is dynamic between different cell types during HSPCs differentiation and self-renewal, and has specific editing events for different cell types. However, our study did not further resolve the underlying mechanisms conferring to this dynamic change. It seems that RNA editing may serve as a molecular marker for different cell types and have implications for the performance of specific functions of different cell types. Since ADAR enzymes catalyze RNA editing events, there may be some differences in the expression of ADAR enzymes in different cell types, all of which need to be investigated in detail by additional work. Moreover, our study found that More broadly, our approach validates the feasibility and usability of RNA editing event studies using high-throughput scRNA-seq. And we showed the overall differences RNA editing of the HSPCs and the possible function. In summary, these efforts confirm the great potential and value of scRNA-seq for the study of biological process mechanisms. Though future studies will be required to confirm and clarify the role of RNA editing.https://db.cngb.org/cnsa/) of CNGBdb with accession code CNP0000978. Both the reference and query dataset were CD34+ cells obtained from human CB and mPB, and the method of cell cultivation, scRNA-seq libraries and pre-processing were as previously described42. The raw sequencing data of query dataset generated in this study was deposited in the CNGB Sequence Archive  of China National GeneBank DataBase (CNGBdb) with accession code CNP0003367.The reference dataset  was downloaded from CNSA . All methods were performed in accordance with relevant quidelines and regulations.+ cell selection. Downstream experiments were conducted on CD34+ cells after sorting.Human CB and mPB samples were obtained from healthy donors with informed consent. We got mononuclear cells (MNCs) using centrifugation on Lymphoprep medium. CD34 Microbead kits and LS columns using MACS magnet technology (Miltenyi) were used for MNC enrichment for CD34+ cells were applied to cell culture in vitro or to single-cell RNA-seq (scRNA-seq). CD34+ cells were resuspended in SCGM medium (Cellgenix) using the following recombinant hematopoietic cytokines: recombinant human stem cell factor (rhSCF) 100 ng/ml, recombinant human thrombopoietin (rhTPO) 100 ng/ml, recombinant human fms-related tyrosine kinase-3 ligand (rhFlt3-L) 100 ng/ml and cultured in 24-well tissue culture plates at 37 °C in an atmosphere of 5% CO2 for 48 h (Thermo Fisher). The DNBelab C4 platform was used to perform scRNA-seq. Single-cell suspensions were used for droplet generation, demulsification, microbead collection, reverse transcription, and cDNA amplification to generate barcode libraries. The manufacturer’s protocol was used to construct indexed libraries. QubitTM ssDNA Assay Kit  was used to quantify the sequencing libraries. DNA nanoballs (DNBs) are loaded into a pattern nanoarray and sequenced at ultra-high throughput with the following read lengths used by the DIPSEQ T1 sequencer: 30 bp for read 1, inclusive of 10 bp cell barcode 1, 10 bp cell barcode 2 and 10 bp unique molecular identifier (UMI), 100 bp of transcript sequence for read 2 and 10 bp for sample index.The fresh CD34+ cells, we used “Mapping and annotating query datasets” method of Seurat package (v. 4.1.0) in R (v.4.0.5)67. This approach allows the comparison of the similarity of cells in the reference and target datasets, resulting in cell annotation of the target dataset with unknown cell types. The reference data set was obtained from CNGBdb with accession code CNP000097842. Then the query dataset was mapped to the reference data for annotating cell types. First, we imported the final cell-gene expression matrix of the query dataset into the Seurat package to create a Seurat objects. Cells with fewer than 200 detected genes and for which the total mitochondrial gene expression exceeded 5% were removed. Besides, we used the IQR Method of Outlier Detection to remove cells with outlier gene number. Genes expressed in fewer than three cells were also removed.To identify HSPC populations in CD34Downstream analyses were also performed using Seurat package (v. 4.1.0). Normalizing SeuratObject used the NormalizeData function and the ScaleData function. The following functions were run together when mapping the query dataset to the reference dataset: FindIntegrationAnchors, TransferData, IntegrateEmbeddings, ProjectUMAP. The FindAllMarkers function was then used to find the cell type-specific genes. Differential expression analysis was performed based on the Wilcoxon Rank Sum test. Finally, the merge function of Seurat was used to merge the query dataset and reference dataset. All methods use default parameters unless otherwise specified.The AverageExpression function of Seurat was used to calculate the average expression of 8 HSPC cell types. And then we used hclust function to perform the hierarchical clustering.https://github.com/Genki-YAN/Cell2Editing).To get pseudo-Bulk RNA-seq of 8 cell types, we got the barcode and sample information for each cell of the 8 cell types from SeuratObject. Based on the CB (Cell Barcode) tag in the bam file we can determine the cell from which the reads originated, so we can use the cell barcode of each cell type to obtain aligned reads for each cell barcode. And then, we integrated multiple cells aligned reads of each cell type to obtain a pseudo-Bulk RNA-seq for each cell type. The code of this method is available on Github (https://github.com/Genki-YAN/Cell2Editing).Considering the difference between scRNA-seq data and Bulk RNA-seq data, we designed the procedure of marking duplicates: taking the aligned position, UMI barcode, and cell barcode of reads into account for the definition of duplicates. Only the reads from the same cell with the same unique molecular identifier (UMI) were considered as PCR duplicates. And we retained the read with the highest base quality. The code of this process is available on Github  in bam file using Samtoolshttps://broadinstitute.github.io/picard/; Broad Institute) method. We counted and compared the non-duplicates reads in the bam files processed by the two methods. The tool locates and marks duplicates in BAM or SAM files, where repeat reads are defined as originating from a single DNA fragment. Duplication may occur during sample preparation, such as library construction using PCR. If the 5’ position, strand and base alignment are the same, Picard compared base alignment quality to mark duplicate reads and available reads.We compared the number of available reads detected by the improved method with the Picard 37 with the default parameters to detect the RNA editing sites in the pseudo-Bulk RNA-seq using the GRCh38 genome and GRCh38 SNP database (https://ftp.ncbi.nih.gov/snp/organisms/human_9606_b151_GRCh38p7/VCF/All_20180418.vcf.gz), and removed RNA editing sites with coverage less than 30× to facilitate subsequent analysis. When we investigated cell type-specific RNA editing sites, we used the mpileup function of Samtools for investigating site sequencing coverage, keeping sites with coverage greater than 30× with the following commands: samtools mpileup -B -f -s - output-QNAME -min-MQ 20 -min-BQ 20 -excl-flags DUP.After the above pre-process of data, we used the RED-ML (https://annovar.openbioinformatics.org/en/latest/)69 table_annovar.pl with the reference genome of GRCh38. We used the following commands: -remove -protocol refGene, phastConsElements20way, wgRna, cytoBand -operation g,r,r,r -nastring . -csvout -polish. Based on the strand of the aligned reads, we only retain the RNA editing sites with genes transcribed from the same strand.RNA editing sites were annotated utilizing ANNOVAR 73.To identify the RNA editing sites that were co-edited in different cell types, the co-editing modules of filtered RNA editing sites in 8 cell types were analyzed using WGCNA package (v. 1.70.3)EIF2AK2, we removed cells that did not express EIF2AK2.Since RED-ML is a method designed for RNA editing sites by Bulk RNA-seq, and there is no existing method to detect RNA editing sites in individual cells, we used the mpileup function of Samtools to find the aligned reads with RNA editing sites. The cells with at least 1 read supporting RNA editing events were defined as edited cells. Samtools mpileup was performed using the following parameters: samtools mpileup -B -f -s -output-QNAME -min-MQ 20 -min-BQ 20 -excl-flags DUP. To calculate the proportion of cells with edited 73.The expression of genes with RNA editing sites in cells was quantified using ScaleData function of Seurat. To explore the function of editing sites with different editing patterns, we performed GO enrichment analysis using clusterProfiler package (v.4.0.5)https://www.targetscan.org/vert_80/)50 was used to predict miRNAs that may target and bind to candidate genes. Then we compared the binding region with the RNA editing sites to determine whether RNA editing sites are located in the miRNA binding region. The reference file of UTR sequence was downloaded from TargetScanHuman website. The version of RNA sequence used was GRCh38.TargetScanHuman (https://github.com/aet21/SCENT)60 with the default parameters. The P-value was calculated using T-Test.To demonstrate that RNA editing may be correlated with differentiation potency, we first removed cells that do not express genes with the RNA editing, and then we calculated the differentiation potency of cells using SCENT (Supplementary Figure S1.Supplementary Legends.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Tables.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.


Inula japonica flowers and showed that it inhibits the coronavirus 3 chymotrypsin-like (3CL) protease as well as viral replication. We showed that eupatin treatment inhibits SARS-CoV-2 3CL-protease, and computational modeling demonstrated that it interacts with key residues of 3CL-protease. Further, the treatment decreased the number of plaques formed by human coronavirus OC43 (HCoV-OC43) infection and decreased viral protein and RNA levels in the media. These results indicate that eupatin inhibits coronavirus replication.The coronavirus disease 2019 (COVID-19) pandemic has caused more than six million deaths worldwide since 2019. Although vaccines are available, novel variants of coronavirus are expected to appear continuously, and there is a need for a more effective remedy for coronavirus disease. In this report, we isolated eupatin from  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19) pandemic, resulting in more than 6 million deaths worldwide . Among t6-C3-C6 carbon skeleton consisting of two benzene rings (rings A and B) linked with a three-carbon heterocyclic ring (ring C) −. The characteristic UV absorption maxima (255 and 345 nm) of this compound suggested the presence of a flavonol skeleton  and ABX aromatic system protons at δH 7.58 , 7.56 , and 7.10   were assigned to the methoxy groups at C-6, C-7, and C-4′, respectively. The 13C-NMR spectrum showed that this compound contained 18 carbons, of which 15 carbon signals were assigned to the 3-O-substituted flavonol structure, and three carbon signals  were assigned to three methoxy groups substituted in the flavonol structure  . The sigtructure . Finallytructure A.50 value of the compound was 34.9 ± 1.7 μM  genes were used to evaluate the level of coronavirus RNA. Similar to protein expression, eupatin treatment did not affect the level of coronavirus RNA in infected cells. However, eupatin treatment effectively decreased the level of coronavirus RNA  in the conditioned media C. These I. britannica is one of the most commonly used plant species in traditional Chinese medicine for treating digestive disorders, bronchitis, and various types of inflammation −. 1H-NMR : δH 7.58 , 7.56 , 7.10 , 6.85 , 3.90, 3.86, 3.80 . 13C-NMR : δC 150.2 (C-2), 137.9 (C-3), 178.2 (C-4), 148.9 (C-5), 129.7 (C-6), 155.4 (C-7), 90.9 (C-8), 154.6 (C-9), 105.6 (C-10), 122.5 (C-1′), 115.1 (C-2′), 145.7 (C-3′), 146.3 (C-4′), 111.9 (C-5′), 120.3 (C-6′), 59.7 , 56.3 , 55.7 .Eupatin: Yellow amorphous powder. UV (MeOH) λThe assay was performed as described previously . A totalRD cells were maintained in Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum  and 1% antibiotic-antimycotic solution . RD cells were infected with HCoV-OC43 at the indicated dilution of virus-infected cell media, and the infected cells were maintained in minimum essential medium (MEM) containing 2% FBS and 1% penicillin-streptomycin (Welgene). The cell viability was assessed using EzCytox cell viability assay kits . RD cells were seeded in the wells of a 24-well plate and incubated with the indicated concentrations of eupatin for 24 h. RD cells were obtained from the Korean Cell Line Bank . The plaque formation assay was performed as previously described .Quantitative RT-PCR was used to measure the level of HCoV-OC43 RNA in infected cell media . For quaWestern blotting was used to measure the level of coronavirus protein in cells and media. For western blotting, the cells and media were harvested and resuspended in cell lysis buffer  containing a protease inhibitor cocktail . Equal amounts of proteins were subjected to western blotting, and virus proteins were detected with a 1:5000 dilution of primary anti-HCoV-OC43 antibody . Images were acquired using a Chemi-Doc XRS+ .t-test using Excel software . p < 0.05 was considered significant.Results are shown as mean ± standard error values. Differences between groups were evaluated with a 2-tailed Student’s


The new water-bridged U–U mismatch resulted in high base-pair opening and single-sided cross-strand stacking interactions, which in turn dominate the CUG RNA structure. Furthermore, we performed molecular dynamics simulations that complemented the structural findings and proposed that the first and third U–U mismatches are interchangeable conformations, while the central water-bridged U–U mismatch represents an intermediate state that modulates the RNA duplex conformation. Collectively, the new structural features provided in this work are important for understanding the recognition of U–U mismatches in CUG repeats by external ligands such as proteins or small molecules.Secondary structures formed by expanded CUG RNA are involved in the pathobiology of myotonic dystrophy type 1. Understanding the molecular basis of toxic RNA structures can provide insights into the mechanism of disease pathogenesis and accelerate the drug discovery process. Here, we report the crystal structure of CUG repeat RNA containing three U–U mismatches between C–G and G–C base pairs. The CUG RNA crystallizes as an A-form duplex, with the first and third U–U mismatches adopting a water-mediated asymmetric mirror isoform geometry. We found for the first time that a symmetric, water-bridged U-H The polymorphic nature of RNA is important for its diverse functions. RNA can undergo various motions and transitions, and presence of such heterogeneous conformations has important implications for ligand binding, signaling, or catalysis , 2, 3. TThe structural properties of CUG repeat RNA have been studied with a range of experimental and theoretical methods, including NMR, X-ray crystallography, single-molecule techniques, and molecular dynamics (MD) simulation , 15, 16.To provide the key information of water mediation within pathogenic RNA structures, we solved an RNA crystal structure containing three continuous CUG repeats and identified three different states of U–U mismatches, MM1, MM2, and MMT. The first and third mismatches exhibit a mirror isoform, asymmetric, single hydrogen bonded geometry in which one of the uridines is tilted into the minor groove (MM1 and MM2 states). At the central repeat, we discover a new type of U–U mismatch displaying a planar symmetry with two uridines separated about 6 Å apart (MMT state). The MMT state contains a central water molecule bridging the two uridines, resulting in a symmetric duplex structure. The detailed structural analysis revealed how a specific U–U mismatch conformation can modulate the CUG-repeat RNA features. We also used all-atom MD simulation in explicit solvent to understand the exchange between the different conformations of U–U mismatch in the CUG RNA structures. Our analysis indicates that MM1 and MM2 are the predominant structural forms, while MMT is an intermediate conformation. The presence of U–U mismatches is shown to disrupt adjacent G–C base pair stacking interactions in a manner that depends on the mismatch conformation. These results provide new structural insights into the U–U mismatches in DM1-related CUG repeat RNA, which are unique features for recognition by CUG-binding proteins or small molecule ligands.P3221. The crystal diffracted X-rays at a resolution of 1.8 Å. A clear electron density map resolved for all RNA heavy atoms in the final refinement indicates no disorder in the structure (A). The biological assembly formed an intermolecular duplex in the crystal, while the asymmetric unit contains only a single RNA strand, indicating a 2-fold crystallographic symmetry. This RNA is denoted as M3 duplex in the following discussion. We also solved another crystal structure containing two U–U mismatches and a A–U Watson–Crick pair in the center at a resolution of 1.58 Å that similarly shows a duplex in the asymmetric unit and is referred to as M2 duplex (B). The five U–U mismatches from two crystal structures showed a high-quality fit of the electron density map for all atoms and water molecules mediating these mismatches . The two RNA duplexes adopt an A-form as based on the C3′-endo conformation of the sugar pucker and average glycosyl torsion angles (χ) of about −160° for all residues (B). The averaged helical twist (31.6°) and rise (2.7 Å) in both M2 and M3 are close to those in A-form structures. The two RNA duplexes exhibit end-to-end stacking to generate infinite pseudocontinuous CUG helices, which resemble the stem structure of stem-loops known to form in long CUG repeats (C). The sum of roll angles at the central base pair steps of the M3 and M2 duplexes is 13° and 17°, respectively, indicating a sharp bending of the RNA toward the major groove (D). Interestingly, the helical twist at the three U–U mismatch sites in the M3 duplex has larger values (average = 26.3 ± 1.0°) compared with those in M2 (average = 21.1 ± 0.5°). Local differences in translational parameters are also observed in both duplexes. The upper half of both RNA duplexes shows positive values for base pair buckle, while the lower half shows negative buckle values. The C–G and G–C base pairs adjacent to the U–U mismatches in the M3 RNA showed excessively high buckle values (up to ± 11.6°) compared with the M2 duplex  (The oligonucleotides in these duplexes are numbered from U1 to A13 in one strand and U14 to A26 in the other complementary strand in the 5'→3′ direction A. The twresidues B. The av repeats . For com repeats C. The sur groove D. Intereand -7°) . In contA). In the M2 crystal, the λ-angle showed more variations with lower values of 26.8 ± 4.5° and 56.7 ± 1.5°, respectively, indicating that these U–U mismatches are more inclined toward the minor groove compared with those in the M3 structure . The average C1′–C1′ interstrand distance measured between these U–U mismatches is 10.8 ± 0.1 Å, which is higher than that of the standard A-helix (10.4 Å).The two crystal structures presented here have five U–U mismatches stacked between C–G and G–C Watson–Crick base pairs. To get insight into the polymorphic nature of U–U mismatch conformations in these sequences, we compared the geometries of these mismatches. The majority of the mismatches are inclined toward the minor groove and shifted away from the helical axis, and three mismatch types as MM1, MM2, and MMT are identified . The incFig. 2A). Moreover, the base pair opening is about 35°, resulting in a large distance between the carbonyl oxygen and the N3 amino group of the two uridine residues, so there is no direct hydrogen bonding (B). In contrast, the U7–A20 formed two hydrogen bonds with a lower base pair opening of about 6°. Based on the analysis of nine CUG repeats within a single crystal structure, Tamjar et al.. The values for the λ-angle are about 72° for both uridine bases within the mismatch. These λ-angle are normally observed for Watson-Crick base pairs (left and right λ-angles around 54°) of the A-form helix. Therefore, upon replacing the central U7-U20 mismatch with U7-A20 Watson-Crick base pair, the λ angles are about 58° and 59.9°, respectively. The U7-U20 mismatch shows a shear and buckle value of zero, compared to −0.2 Å and 0.4 Å in the U7-A20 base pair, respectively A. Moreov bonding B. In conar et al. proposedA). On the other hand, the two O4 carbonyl atoms of the U4–U23 and U10–U17 mismatches form hydrogen bonds with the common water at 3.2 to 3.3 Å in the major groove (A). In the M2 duplex, the hydrogen bonds of U4–U23 and U10–U17 mismatches with the water molecules are 2.5 Å and 2.8 Å in the minor groove and 3.1 Å and 3.5 Å in the major groove, respectively (B). In the M3 structure, the U7–U20 mismatch pair showed a unique planar water-bridged interaction. The symmetrically stretched U–U form hydrogen bonds between two carbonyl oxygen atoms O2, each 3.0 Å apart. Another water bridges the interactions between two N3 amino atoms of two uridine residues with a short intermolecular distance of 2.9 Å. We named the current base pairing as a water-bridged U-H2O-U mismatch pairing. The current crystal structure is the first to capture such unique geometry in U–U mismatches, highlighting the importance of water molecules in mediating nucleic acid structural motifs.The presence of water molecules in the minor and major grooves forms hydrogen-bonding interactions with the U–U mismatches. In both the crystal structures presented here, each U–U mismatch is solvated by two water molecules, one in the minor groove and the other in the major groove. In the U4–U23 and U10–U17 mismatches of the M3 duplex, the O2 oxygen and the N3 amino nitrogen form hydrogen bonds with a common water in the minor groove and the distance to water oxygen is 2.7 Å A. On ther groove A. In theectively B. In theA). The profile of joint probability density of d and d was evaluated based on the 1-μs trajectory data . It can be observed that MM1 and MM2 are the two major forms of U–U mismatch, and MMT has a minor population. Furthermore, the time series of d and d indicate that transition between MM1 and MM2 occurs frequently during the dynamical simulation, and MMT is an intermediate state between the two dominant structural forms . Similar to the crystal structure, we also observed three distinct types of solvation structures, whb1, whb2, and whbt, during the simulations. Interestingly, the probability of finding the whb1, whb2, or whbt solvation state is shown to depend on the particular U–U mismatch state  . For example, whb1 is mostly found in the MM1 state while whb2 is specific to MM2. For MMT, in particular, the identified solvation structure tends to be whbt. Such dynamics and solvation structures of the U–U mismatch state are illustrated in Thus, the crystal structures of CUG-repeat RNA exhibit three distinct MM1, MM2, and MMT forms of U–U mismatches bridged specifically by water molecules. In order to further quantify the distribution of these three states, we performed 1-μs MD simulations starting from the M2 and M3 duplexes in explicit solvent model. The results of the central U–U mismatch in M3 duplex, U7–U20, are presented in Fig. 3A). In contrast, when the central U–U mismatch is replaced with A–U Watson–Crick base pair, the lower base-pair opening retained the base pair within a helical axis and formed continuous stacking interactions on both sides of the A–U pair, similar to a standard A-form duplex .Stacking interactions play an important role in maintaining nucleic acid structures , 21. DepFig. 4Consistent with the structural findings, the MD simulation results also exhibit the dependence on the different structural form of U–U mismatch and cross-strand stacking interactions. For example, the U7→G21 cross-strand stacking (where the → notation indicates the stacking interaction) appears to go with U7–U20 being in the MM1 state . On the via an induced fit mechanism. Since no complex structure of MBNL1 bound to CUG repeats is available, most studies largely depend on RNA features in understanding the binding mechanism of MBNL. Thus, the availability of new crystal structures could shed light on this important pathogenic interaction.Aberrant expansion of trinucleotide repeats is a major cause of many neurodegenerative diseases , 23. Of et al. suggests that GC element recognition follows a chain-reversal loop trajectory . To formet al. have shown that a mercury-mediated T–T mismatch is capable of causing a structural switch from a nonhelical form to the B-form of DNA (A) (B) . Although we do not observe associated cations in the current crystal structure, the distance between two stretched uridines offers the possibility that the ordered water molecule could be replaced by metal ions. Such structural features could therefore provide a basis for rational design of metal-conjugated RNA nanomaterials containing U–U mismatches.Apart from the biological roles of novel U–U mismatches in CUG repeats, the naturally occurring nucleobases including thymine or cytosine selectively form stable metal-mediated base pairs. For example, Kondo m of DNA A 39). A. Aet al. DNA (A) B 40). T. Tet al.ed pairs C. AlthouIn summary, we have presented here new structural motifs of CUG repeat RNA duplexes with a water-bridged symmetric U–U mismatch geometry. Our results showed that the chemical symmetry of the central base pair is observed in U–U mismatches, suggesting that such U–U mismatch could exist as transient conformation in CUG repeat sequence context. More detailed studies would be required to confirm the biological consequences of symmetric U–U mismatch geometry. The new structural features presented here help to expand the current repertoire of U–U mismatches in CUG context. The structural understanding from this study would also help in the development of new U–U mismatch-selective chemical probes for targeting CUG repeats in DM1.2O. Oligonucleotides were dissolved in 0.1% (v/v) diethyl pyrocarbonate-treated ddH2O by heating at 95 °C for 5 min followed by slow cooling (−0.5 °C/min) to room temperature to allow annealing of duplexes. The absorbance of the oligonucleotide concentrations was determined using JASCO V-630 ultraviolet-visible spectrophotometer  with a quartz cuvette (1 cm path length) at a wavelength of 260 nm. The quantitative concentration of the oligonucleotides was calculated through Beer's law with the approximate values of extinction coefficients for each oligonucleotide  diethyl pyrocarbonate-treated ddHcleotide .For the crystallization, HPLC-purified oligonucleotides were denatured by heating to 95 °C for 5 min and then stored on ice for annealing. To grow M3 RNA crystals, 0.2 mM RNA solution was mixed with mother liquid containing 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (pH 6.5), 3.5 mM spermine tetrahydrochloride, 10% (v/v) 2-methyl-2,4-pentanediol in 1: 1 ratio, and equilibrated with 300 μl mother liquid. Crystals suitable for X-ray crystallography experiments were obtained by the sitting-drop vapor diffusion method. After 1 to 2 weeks, translucent long rod-shaped crystals were obtained at 4 °C. To obtain M2 RNA crystals, 0.2 mM RNA oligonucleotides were mixed in solution containing 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (pH 6.5), 0.5 mM spermine tetrahydrochloride, 10% (v/v) 2-methyl-2,4-pentanediol in a 1: 1 ratio and equilibrated with 300 μl mother liquid. Bright and transparent broad rod-shaped crystals grew after about 1 week at 4 °C.P3221 and P32, respectively. The crystallographic model of a typical A-form RNA duplex was created using Discovery Studio 2020 client software (version 20.1.0.19295) and used as template for initial phase determination of the duplexes. Structural refinement using phenix.refine in PHENIX (version 1.18.2-3874) (2Fo–Fc electron density maps were generated using the fast Fourier transform in CCP4i and PyMOL (version 2.3.2), which was used to draw graphical representations of the refined structures.X-ray diffraction data were acquired using a fixed-exit double crystal monochromator with images collected on a Rayonix MX300HS CCD area detector and a Rayonix MX300HE CCD area detector, respectively, at the Biological Crystallography Facility of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. Data collection wavelength of 1 Å was used for a single crystal cooled to 100K with a stream of nitrogen. Diffraction data reduction, processing, integration, and scaling were performed using the HKL-2000 package. PHENIX (version 1.18.2-3874) was used to determine the phases for M3 and M2 RNA duplexes using molecular replacement (Phaser MR) in space groups .2-3874)  and WinC.2-3874)  were perA standard A-form RNA structure with the same sequence as CUG RNA duplex was created using Discovery Studio 2020 client software (version 20.1.0.19295) for structural comparison. The RNA structural parameters, including helix, torsion, local base pair parameters, and local base pair step parameters were analyzed using the Curves plus web server program . Values m) analysis, RNA oligonucleotides were prepared in the same buffer (20 mM Mops [pH 6.5] and 1 mM spermine). Circular dichroism (medg) curves were determined by increasing the temperature from 4 to 95 °C at a rate of 1 °C/min and recording every 1 min at 268 nm.Circular dichroism (CD) spectral analysis was performed at 25 °C on a ChirascanTM V100 CD spectrophotometer (version 4.8.3.313) with Pro-Data software suite (version 4.8.3.0) in quartz cuvettes with path length of 1 mm. RNA duplex, 10 μM, was mixed in a buffer containing 20 mM Mops (pH 6.5) and 1 mM spermine tetrahydrochloride. The samples were annealed in the buffer from 95 to 4 °C and then stored overnight. The determination of the ellipticity was carried out in the range of 400 to 200 nm, at a sampling rate [time per point (s)]. The ellipticity of the circular dichroism was superimposed and normalized by curve fitting using Pro-Data Software Suite core (version 4.8.3.0). To perform melting temperature (T+ and Cl− ions were added to achieve charge neutrality and ionic strength of 0.15 M. For the system of M2 duplex, there are 43 Na+ and 19 Cl−, and there are 44 Na+ and 20 Cl− in the system of M3 duplex. The resulting M2 system has 20,898 atoms and the M3 system contains 21,326 atoms. The cut-off radius for van der Waals interactions and real-space particle-mesh Ewald terms of electrostatics (via LINCS (The crystal structures of M2 and M3 duplexes were used as the initial structures to perform MD simulations. Both systems were solvated in dodecahedron boxes of explicit water molecules with at least 10 Å between any nucleic atom and box edges. Naostatics  was 12 Åia LINCS . After iia LINCS . All theia LINCS  and the ia LINCS . A snapsia LINCS , 51.7Y2B and 7Y2P.The atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank with accession codes This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Nanomedicine 2.0 refers to the next generation of nanotechnology-based medical therapies and diagnostic tools. This field focuses on the development of more sophisticated and precise nanoparticles (NPs) for targeted drug delivery, imaging, and sensing. It has been established that the nuclear delivery of NP-loaded drugs can increase their therapeutic efficacy. To effectively direct the NPs to the nucleus, the attachment of nuclear localization signals (NLSs) to NPs has been employed in many applications. In this review, we will provide an overview of the structure of nuclear pore complexes (NPCs) and the classic nuclear import mechanism. Additionally, we will explore various nanoparticles, including their synthesis, functionalization, drug loading and release mechanisms, nuclear targeting strategies, and potential applications. Finally, we will highlight the challenges associated with developing nucleus-targeted nanoparticle-based drug delivery systems (NDDSs) and provide insights into the future of NDDSs. Nanoparticle-based drug delivery systems (NDDSs) have revolutionized the medical field by providing targeted and efficient drug delivery while minimizing side effects . NuclearNucleus-targeted nanoparticles (NPs) are designed and engineered to selectively accumulate in the nuclei of cancer cells, where they deliver drugs, genes, or other therapeutic drugs directly ,9. In orThis review will first provide a brief introduction to the structure of NPCs and the mechanisms of classic and transactivator of transcription (TAT) nuclear import. We will then focus on various nanoparticles, covering topics such as their synthesis, functionalization, drug loading and release mechanisms, nuclear targeting strategies, and potential applications. Finally, we will discuss the challenges associated with nucleus-targeted NDDSs and provide perspectives on the future of NDDSs.The nuclear pore complex (NPC) is a large structure that exhibits octagonal rotational symmetry. In vertebrates, the NPC has a diameter of approximately 120 nm and a length of about 200 nm along the transport axis ,11,12. TAs is shown in The best-understood type of facilitated diffusion is the classical nuclear import pathway. Proteins destined for transport into the nucleus contain amino-acid-targeting sequences called nuclear localization signals (NLSs), which are recognized by importin α in the cytoplasm, linking it to importin β (importins are nuclear transport receptors responsible for importing cargo into nucleus). Importin β then mediates the interaction of the trimeric complex with the nuclear pore as it translocates into the nucleus. Once the import complex reaches the nucleus, it is dissociated by Ran-GTP. The binding of Ran-GTP to importin β causes a conformational change that results in the release of the importin α-cargo complex. The importin β will return the cytoplasma by itself and importin α will be exported by exportin CAS (exportins are nuclear transport receptors responsible for exporting cargo out of nucleus) ,22. The The classical nuclear import pathway offers distinct advantages compared to free diffusion. Firstly, it enables efficient and unidirectional transport, preventing the escape of cargo containing nuclear localization signals (NLSs) from the nucleus and promoting their accumulation within the nucleus. This unidirectional transport ensures that NLSs cargo remains confined to the nucleus, enhancing its functional effects. Secondly, the classical nuclear import pathway permits the entry of larger cargo molecules, as it can accommodate cargo with a diameter of up to 40 nm, whereas free diffusion is limited to smaller molecules with a diameter of around 10 nm. This ability allows for the nuclear import of larger macromolecules and facilitates their involvement in crucial nuclear processes.132), polyoma large T antigen (VSRKRPRP196), hepatitis D virus antigen (EGAPPAKRAR75), murine p53 (PPQPKKKPLDGE322), NF-κB p50 (QRKRQK372), NF-κB p65 (EEKRKR286), and human c-myc (PAAKRVKLD328/RQRRNELKRSF374) usually contain a single cluster of four or five basic residues. In contrast, bipartite NLSs, such as those found in Xenopus nucleoplasmin (KRPAATKKAGQAKKKKLD171), rat glucocorticoid receptor (YRKCLQAGMNLEARKTKKKIKGIQQATA524), and RCC1 (MSPKRIAKRRSPPADAIPKSKKVKVSHR28), have two clusters of basic residues. The first cluster is located in the usual position, but a second basic cluster can be found approximately 9–12 residues downstream of the first one [The cNLSs are widely considered as the prototype and most extensively studied form of NLSs. They were the initial NLSs to be discovered and characterized, leading to the identification of numerous proteins that utilize the classical import pathway. As a result, a wealth of examples have demonstrated the utilization of the cNLSs for nuclear import. The cNLSs typically consist of either one cluster (monopartite) or two clusters (bipartite) of basic residues. Monopartite NLSs, such as SV40 large-T antigen  repetitions, which collectively form a domain responsible for binding to the NLS. Structural studies have revealed the presence of two distinct NLS binding sites within the central domain. The first binding site, situated between the ARM motifs 1–4, directly interacts with the amino acids of monopartite NLSs as well as the longer amino acid sequence of bipartite NLSs. On the other hand, the second binding site, located in the ARM motifs 7–8, specifically binds to the shorter amino acid sequence found in bipartite NLSs. Structural analysis has demonstrated that mutations in the amino acids of the first NLS binding site severely impede the interaction between importin α and both types of cNLSs, and amino acid substitutions in the second NLS binding site decrease the affinity between importin α and bipartite NLSs, but do not affect the interaction with monopartite NLSs .Experimental findings have revealed a direct correlation between the binding affinity of a classical nuclear localization signal (cNLS) for importin α and the nuclear accumulation and import rate of the corresponding cNLS cargo. A successful cNLS exhibits a binding constant of approximately 10 nM for importin α. Alternatively, a weak binding between importin α and the cNLS results in inefficient import of the cargo, while a tight binding hinders the effective release of the cNLS cargo from importin α within the nucleus. Computer simulations have further demonstrated that the import and accumulation of cNLS cargo in the nucleus are influenced by both the affinity of the cNLS cargo for importin α and the concentration of the importin α receptor .The recognition of the cNLS by importin α is only the initial step in the formation of functional import complexes. The interaction between importin α and importin β is essential for an efficient transport process. This interaction is facilitated by the importin beta binding (IBB) domain located in the N-terminal region of importin α. Experimental evidence strongly supports a model in which proteins containing a cNLS have a higher affinity for importin α when it is associated with importin β, compared with binding to free importin β alone. Import complexes traverse the nuclear pore through a mechanism that involves the interaction between importin β and the FG domains of nucleoporins . Further48 GRKKRRQRRRAPQN 61), derived from human immunodeficiency virus-1 (HIV-1), has demonstrated remarkable capabilities in facilitating the delivery of various cargoes into cells and the nucleus. These cargoes range from nanoparticles, peptides, and nucleic acids to proteins. The TAT peptide contains overlapping regions responsible for cellular localization, including a classical nuclear localization signals (cNLSs) (48GRKKRR), and a cell-penetrating peptide (CPP) signal (48GRKKRRQRRRAPQN). These distinct regions within TAT play crucial roles in efficiently transporting cargoes across cellular barriers and facilitating their entry into the nucleus [The transactivator of transcription (TAT) peptide  within its sequence, the mechanism by which the TAT peptide is imported into the nucleus is still a topic of debate. Some evidence suggests that TAT does not interact with importin α but instead directly binds to importin β. In vitro nuclear import assays indicate that TAT’s nuclear entry is facilitated by its direct binding to importin β . In a stThe nuclear import pathway of TAT peptide is illustrated in There are various types of nanoparticles that have been utilized as drug delivery systems , each wiMetal nanoparticles are small particles made of various metals, such as gold, silver, platinum, iron, and copper, at the nanoscale level ,42,43. TGold nanoparticles (GNPs) have shown great promise as effective carriers for drug delivery. Their negatively charged surfaces make them easily functionalize with various biomolecules such as drugs, genes, and targeting ligands, allowing them to transport multiple therapeutic agents to targeted sites. Additionally, GNPs offer unique physical and chemical properties that allow for controlled drug release through external stimuli such as light or internal stimuli such as pH or glutathione. GNPs are biocompatible, non-toxic, and safe for use in biological systems. They can be synthesized easily and produced in a variety of shapes and sizes, ranging from 1 nm to over 100 nm, including spherical, rod-like, cage-like, and other forms. The distinctive surface effects, ultra-small size, macroscopic quantum tunneling effect, and presence of surface plasmon resonance (SPR) bands make GNPs ideal for various biomedical applications, such as biosensing, molecular imaging, and drug delivery systems .Spherical gold nanoparticles (GNPs) ranging from 1 to 100 nm are a versatile choice for drug delivery applications. They can be synthesized using various methods, including the conventional synthesis method, which involves reducing aqueous chloroauric acid with sodium citrate. In this process, citrate acts as a stabilizer and reducing agent, and the size of the resulting GNPs can be adjusted by altering the stoichiometric ratio between chloroauric acid and sodium citrate. Precise control of the synthesis process is crucial, since the size and shape of GNPs significantly impact their drug delivery efficiency. Alternative synthesis methods also exist for producing spherical GNPs, which can be selected based on specific application requirements.The functionalization of gold nanoparticles (GNPs) with various biomolecules, such as DNA, peptides, and antibodies, is essential for their biomedical applications. These biomolecules can be attached to the surface of GNPs through either noncovalent interactions, such as electrostatic interactions, hydrophobic entrapment, and van der Waals forces, or covalent modifications, which involve immediate chemical attachment, linker molecules, or click chemistry . NoncovaKang et al. conducted a study to investigate the interactions and effects of gold nanoparticles (GNPs) in living cells . To accoIn a study conducted by Peng et al., a simple method was employed to prepare cationic gold and silver nanoparticles functionalized with TAT peptides for gene delivery to epidermal stem cells  Figure . Positiv2, which is significantly lower than the maximum permissible exposure of skin. Unlike conventional PTT, this low level of irradiation did not cause cell necrosis. Tumors treated with GNRs-NLS under this ultralow NIR irradiation exhibited significant regressions. This report demonstrated that intranuclear PTT using GNRs-NLS greatly mitigated side effects and improved biosafety, making it a promising approach for cancer therapy.Photothermal therapy (PTT) is a promising technique that utilizes light-converting agents to induce heat and “burn” cancer cells. In a recent study by Pan et al. , a stratPolymeric micelles are small colloidal particles with a size typically ranging from 10 to 100 nm . These mPolymeric micelles possess various advantages that make them ideal for drug delivery in cancer treatment. Firstly, they can improve the solubility of hydrophobic or poorly water-soluble drugs, thereby enhancing their bioavailability. Secondly, their small size allows for extended circulation in the blood by evading the mononuclear phagocytic system, while also being large enough to avoid fast renal clearance. The high structural stability of polymeric micelles allows for drug retention and stability upon dilution in the body. Furthermore, they can be targeted to specific tissues by modifying their surface with various ligands using different surface chemistries. Additional benefits include reduced side effects, easy and reproducible scale-up, ability to slow down opsonization, and longer circulation times when hydrophilic moieties such as PEG are incorporated to provide an effective steric barrier ,51,52.The active targeting of nanoparticles (NPs) has emerged as a strategy to enhance the therapeutic potential of NP-based drugs by facilitating their delivery to specific cellular locations. This is achieved by attaching ligands that specifically recognize the target cells onto the surface of NPs, such as micelles. The ligands can selectively bind to receptors or proteins that are overexpressed on the surface of the target cells, promoting the accumulation of NPs in these cells. Different types of ligands, including peptides, antibodies, aptamers, and small molecules, can be used depending on the target cells and the desired therapeutic effect. Overall, the active targeting of NPs has shown promise in preclinical and clinical studies as a way to improve the delivery and efficacy of drug therapies.Yu et al.  develope111In) and the HER2-specific antibodies trastuzumab  and NLS peptide (CGYGPKKKRKVGG) to directly target the nucleus of HER2-overexpressing breast cancer cells. These micelles, called 111In/NLS2-TmAb-Fab-BCMs, were investigated in mice with subcutaneous BT-474 and MDA-MB-231 xenografts expressing high and low levels of HER2, respectively, for their pharmacokinetics, biodistribution, tumor uptake, and intratumoral distribution. The TmAb-Fab fragments on the micelle surface facilitated the binding and internalization of BCMs by HER2-positive breast cancer cells, and NLS conferred nuclear localization capability. Targeting BCMs resulted in a 5-fold increase in tumor uptake in HER2-overexpressing BT-474 tumors, as well as a greater level of cellular uptake and nuclear localization compared to non-targeted formulations. Overall, this study presents a promising platform for the effective delivery of chemo- and/or radiotherapy in vivo, providing a potential strategy for the treatment of HER2-overexpressing breast cancer [Hoang et al.  designedt cancer .Wang et al. developed a highly efficient nucleus-targeted co-delivery vector capable of delivering p53 genes and doxorubicin (DOX) directly into the nucleus of cancer cells  Figure . The vecDendrimers, which are highly branched, nanoscale macromolecules with diameters ranging from 1 to 20 nm, have shown great potential as drug delivery candidates . DendrimDendrimers can be synthesized using two main methods: convergent ,60 and dPolyamidoamine (PAMAM) dendrimers have demonstrated superior biocompatibility and a larger nucleic acid loading capacity compared to branched polyethylenimine (PEI). The spheroidal shape, nanoscale size, and cationic surface of PAMAM dendrimers also aid in the cellular uptake of the nucleic acid complexes .Lee et al.  developeIt is widely acknowledged that the hydrophobic drugs can be accommodated in the interior hydrophobic cavities of dendrimers. Wu et al.  developeIn one study, Wang et al. introduced a novel delivery carrier called TAT-SS-PAMAM-D3, which was designed to specifically target the nucleus while offering the ability to adjust its size  Figure . The surDespite much progress, the clinical translation of dendrimer-based drug delivery systems has been limited due to their biocompatibility and toxicity which are closely linked to the size and surface chemistry of the dendrimer. For example, PAMAM dendrimers of generation 6 and above have high costs and severe toxicity , therefoAmong various nanocarriers, mesoporous silica nanoparticles (MSNs) have exhibited exceptional characteristics and versatility, consequently finding extensive applications in the field of nanomedicine delivery in recent years. Owing to their large surface area and tunable pore sizes, MSNs enable highly efficient drug loading . The higPan et al. pioneered the development of a cell-nucleus-targeted delivery system by conjugating TAT peptide with mesoporous silica nanoparticles (MSNs)  Figure . This inThe TAT peptide, characterized by a highly cationic motif, has been widely utilized for the cellular uptake and intracellular delivery of various cargoes. Nonetheless, it is important to highlight that this peptide lacks specificity for tumor cells, as it can bind to the cell membranes of both cancerous and normal cells. Hence, to ensure the precise delivery of nanoparticles to tumor cells, it is critical to block TAT peptides prior to the release of TAT-labeled nanoparticles into the cell cytosol. To block the TAT peptide, Zhao et al. developed a novel multifunctional nano-drug delivery system based on the reversal of peptide charge  Figure . This na3O4@mSiO2, exhibited a diameter of 91 nm (the diameter of the Fe3O4@mSiO2 nanoparticle is 50 nm and the pore diameter is 2.8 nm). Tirapazamine (TPZ) is an anticancer drug that demonstrates remarkable selectivity in targeting hypoxic cancer stem cells (CSCs). TPZ exerts its effects within the nucleus by interacting with reductases, leading to the generation of a transient oxidizing radical. The targeted delivery of drugs to the nucleus resulted in the induction of apoptosis in CSCs through a combination of thermotherapy and hypoxia-activated chemotherapy. In vivo experiments demonstrated that the nucleus-targeted nano delivery system effectively suppressed tumor growth with minimal side effects. Molecular mechanism studies revealed that the system achieved CSC elimination by inhibiting the hypoxia signaling pathway. This innovative nucleus-targeted nano delivery system holds great promise for the development of efficient platforms for CSC-targeted cancer therapy.Li et al. conducted a study to develop a novel nucleus-targeted system using mesoporous silica nanoparticles (MSN)  Figure . The maiLiposomes are a class of spherical structures  composed of one or more lipid bilayers that encapsulate various drugs within their aqueous and lipid layers ,85. Theiw/w) liposome. This delivery system was successfully transfected into SKnSH mammalian neuroblastoma cells, resulting in a three-fold increase in luciferase expression compared to cationic liposome controls. By investigating the factors that impede the rate of transgene expression, it is possible to uncover new strategies for improving the efficiency and effectiveness of non-viral gene therapy methods [In one study, researchers developed a delivery vehicle composed of a synthetic NLS peptide derived from the SV40 virus, a luciferase-encoding PGL3 plasmid, and a cationic lipid DOTAP-: DOPE -labeled DNAhsp65 (referred to as NLS/DNA/cationic liposome) for the treatment of tuberculosis . The tarIn addition to gene therapy, liposomes have been utilized for the delivery of anticancer drugs to the nucleus. In one particular study, a nuclear-targeted delivery system was developed to overcome drug resistance in breast cancer (MCF-7/Adr) by leveraging nucleolin’s active transport to the nucleus and its affinity with aptamers. The drug doxorubicin hydrochloride (Dox·HCl) was inserted into the aptamer AS1411 (Ap-Dox) and encapsulated within the aqueous interior of liposomes (Lip (Ap-Dox)). In vitro investigations demonstrated that upon diffusion of Lip (Ap-Dox) into MCF-7/Adr cells, the Ap-Dox complex strongly bound to nucleolin, which contains a bipartite nuclear localization signal (NLS: KRKKEMAKQKAAPEAKKQKV), leading to its eventual entry into the cell nuclei. By utilizing this drug delivery system, Dox·HCl efficiently accumulated in the nuclei, effectively inducing cancer cell death .Wang et al. engineered a liposome formulation that incorporated a monoclonal antibody called TuBB-9, designed to selectively recognize a biologically active form of the protein Ki-67, which predominantly localizes in the nucleus . To enhaIn one study, Torchilin et al. demonstrated the effectiveness of liposomes modified with TAT peptide (referred to as TATp-liposomes) in facilitating rapid and efficient translocation into the cell cytoplasm, followed by migration into the perinuclear zone . In vitrA summary of nanoparticles and their applications in nuclear drug delivery is depicted in In the context of therapeutic gene delivery, non-viral systems are expected to play a significant role. Although they have traditionally exhibited lower efficiency in gene expression compared to viral delivery systems, non-viral systems offer several advantages such as simplified production, reduced toxicity, and no risk of infection. However, a limitation of many non-viral systems is the lack of a mechanism for efficient gene transport into the nucleus. To overcome this challenge, the incorporation of nuclear localization signal (NLS) peptides can combine the enhanced expression capabilities of viral delivery systems with the safety and ease of preparation associated with non-viral delivery systems. By integrating NLS peptides, non-viral systems can improve the targeting and transport of therapeutic genes to the nucleus, thereby enhancing their overall effectiveness as gene delivery vehicles.Classical nuclear localization signals (cNLSs) rely on importin α and importin β to facilitate the import of cargo into the nucleus. This process is unidirectional and enables the accumulation of cargo in the nucleus. Moreover, cNLSs offer specific nuclear targeting capabilities for delivering cargo. However, the maximum size of nanoparticles that can traverse the nuclear pore complex (NPC) using cNLSs is approximately 40 nm in diameter. Depending on the physical properties of the cargo, larger cargo (>40 nm diameter) can potentially be transported through the NPC with the aid of flexible shapes during transport. However, for rigid-core nanoparticles such as metal nanoparticles or mesoporous silica nanoparticles (MSNs), which do not change shape during transportation, cNLSs cannot facilitate their transport if the core diameter exceeds 40 nm.In contrast to cNLSs, the TAT peptide can facilitate the import of nanoparticles with larger and rigid cores (>40 nm in diameter) into the nucleus. The exact mechanism behind this phenomenon is still not fully understood. Therefore, it is important to investigate how these rigid and large nanoparticles traverse the nuclear envelope after being labeled with TAT. One possible explanation is that in live cells, TAT-labeled nanoparticles indirectly bind to nuclear components during cell division, allowing the large cargo to remain inside the nucleus. To test this hypothesis, the use of digitonin-permeabilized cells is essential, because cell entry is not a concern and the intact nuclear envelope prevents cargoes from binding to nuclear components. The other explanation is that the TAT peptide can penetrate the nuclear envelope and allow large cargo to enter the nucleus and bypass the nuclear pore complexes (NPCs) . CompareThe efficiency of importing NLS-labeled nanoparticles depends on the density of NLS labeling ,47,96, h


In this review, focusing on the mechanisms and available quantitative information, we discuss first how the METTL3/14 m6A writer complex is specifically targeted to RNA assisted by protein–RNA and other interactions to enable site-specific and co-transcriptional RNA modification and, once introduced, how the m6A modification affects RNA folding and protein–RNA interactions.Protein–RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein–RNA assemblies rely on spatially and temporally coordinated interactions between specific proteins and RNAs to achieve a functional form. RNA folding and structure are often critical for successful protein binding and protein–RNA complex formation. RNA modifications change the chemical nature of a given RNA and often alter its folding kinetics. Both these alterations can affect how and if proteins or other RNAs can interact with the modified RNA and assemble into complexes. N Protein–RNA complex assemblies are at the core of many essential cellular machineries, such as the spliceosome and the ribosome. In recent years, protein–RNA complexes have gained importance in more cellular processes, and studying them has become increasingly relevant to understanding the regulation of many cellular processes.Similarly, since the discovery of the first modified nucleotide (pseudouridine) in yeast tRNA , the lisNew and advancing technologies enable the discovery and characterization of currently around 170 chemically distinct RNA modifications on highly abundant RNAs such as ribosomal RNA (rRNA) and on rare and short-lived species including mRNAs, a subset of long noncoding RNAs (lncRNAs) and miRNAs , 2022. T6-methyladenosine (m6A) is among the best-studied internal RNA modifications found mainly on RNA polymerase II (Pol II) transcripts, including mRNAs, lncRNA, and primary microRNAs (pri-miRNA)  . Within p codons . m6A is nslation .6A modifications happens mainly co-transcriptionally by functionally distinct methyltransferases, also termed m6A writers, which each display different substrate specificities  or alpha-ketoglutarate–dependent dioxygenase alkB homolog 5 (ALKBH5) (The mversible . Therefo(ALKBH5) .6A RNA modification requires the formation of protein–RNA complexes for its deposition and removal, and it itself modulates the formation of functionally distinct RNPs on modified RNAs by either enabling or preventing the binding of a defined set of protein interactors or other RNAs.The m6A, are modulated through the dynamic assembly of RNP complexes during the “writing” process and how the modification affects RNA structure, RNP assembly, and function   . METTL3 ex (MAC)  (Wang etex (MAC) . Posttraex (MAC) .6A homeostasis, recent findings show that METTL3 competes with E3 ubiquitin ligase STUB1 for binding to METTL14 and enhances METTL14 stability by preventing its degradation , within which the central adenosine base gets methylated  Csepany. In vitrBesides modifying RNAs, the METTL3/METTL14 enzyme has also been reported to be active on single-stranded DNA in vitro, which has been linked to DNA repair at UV- or X-ray-induced double-stranded DNA lesions in vivo .6A marks, directly recruits translation initiation factors CBP80/20 and eIF4E, and facilitates recruitment of eIF3 , an epigenetic mark for active transcriptional elongation, and with active histone marks H3K27ac and H3K4me3  Barbier. METTL3,r YTHDC1   to these sites via a direct interaction. KDM6B can then remove H3K27me3 marks and promote transcriptional activation of the affected genes  Dou et .6A modification of chromatin-associated regulatory RNAs (carRNAs) by METTL3 leads to transcriptional repression of the affected genes. Unmodified carRNAs bind and recruit proteins, such as CBP/EP300 and YY1, to promote open chromatin and activate transcription. Once methylated, the m6A-marked carRNAs are bound by nuclear m6A reader protein YTHDC1 and then targeted for degradation via the nuclear exosome targeting complex, depleting carRNA-associated transcriptional activators from the affected sites  splicing factor Wilm’s tumor 1-associated protein (WTAP) is frequently found to be associated with the me enzyme  d recruits the METTL3/METTL14 complex to actively transcribing genes via direct interactions with the transcription elongation factor and histone chaperone Spt6 . In this6A modification on 3′UTRs and near stop codons   Yue et . VIRMA ip codons  (Yue et p codons . Anotherp codons .6A modification by facilitating the m6A RNP assembly (The composite interaction interface created by WTAP and VIRMA recruits Zinc-finger CCCH-type containing 13 (ZC3H13) protein and E3 ubiquitin ligase HAKAI to the core enzyme  Su et a. ZC3H13 assembly   and are considered integral players in regulating m6A deposition.Because of their frequently observed association and regulatory functions in complex with the METTL3/METTL14 enzyme, the adapter proteins WTAP, VIRMA, HAKAI, and ZC3H13 are referred to as regulatory subunit m6A core enzyme to specific DRACH motifs on lncRNA Xist  and its paralog RBM15B and recruits it to the METTL3/METTL14 core enzyme . RBM15/1RNA Xist   on the pre-mRNA  by ∼fourfold to ninefold for an RNA–RNA or RNA–DNA duplex containing an m6A-U/T base pair. In contrast, the rate constant for duplex dissociation (koff) changes only by 0.7–1.7fold  by altering processes that are directly dependent on base-pairing or (2) by altering the RNA structure to enable or prevent protein association.6A modification in U6 snRNA, the 5′SS is less conserved than in lower eukaryotes, especially at the +4 position. Two studies, in A. thaliana and S. pombe, report that in the presence of the m6A in the U6 snRNA, 5′SSs with an A in position 4 are favored as the modification slightly stabilizes the m6A-A base pair by 0.7 kcal/mol as compared with an A–A base pair. A 5′SS with a U at position 4, on the other hand, gets destabilized by 0.5–1.7 kcal/mol and preferentially base-pairs with the loop 1 region of U5 snRNA instead of the U6 snRNA  of the U6 snRNA is specifically modified by methyltransferase METTL16 . A43 is U6 snRNA  Fig 3B)6A modifi6A-modified nucleotide influences efficient base-pairing between the codon on the mRNA and the anticodon on the cognate tRNA leading to only near-cognate codon-anticodon interactions, thereby slowing down proper tRNA accommodation. Under high-accuracy conditions (1.3 mM MgCl2), this results in a reduction of the Michaelis–Menten parameter (kcat/Km)pep for peptide bond formation at m6AAA codons by a factor of 18 as compared with AAA codons    (Qiu et s (RLRs) .6A modification leads to subtle changes in the RNA folding kinetics that result in small changes in the local RNA structure. Those changes carefully fine-tune cellular processes sensitive to base-pairing such as splicing and translation, and can affect the immunogenicity of endogenous or pathogenetic RNAs.Overall, the m6A modification are also associated with RNA structural rearrangements that facilitate protein–RNA interactions.The altered base-pairing characteristics induced by the mThis is exemplified by the interaction of splicing factors heterogeneous nuclear proteins C and G (hnRNP-C and hnRNP-G) with pre-mRNAs and lncRNAs , 2017.6A-dependent manner subsequently leads to the generation of hnRNP-C-dependent splicing variations , via its intrinsically disordered or low-complexity domain. Strikingly, the hnRNP-G binding motif strongly overlaps with the m to bind . The binsplicing   binds AGG and UAG motifs via its two RNA-recognition motif domains through a similar mechanism as hnRNP-G. hnRNPA2B1 binds these motifs in a subset of pri-miRNAs and interacts with Dgcr8, a component of the microprocessor complex, thereby promoting miRNA processing .6A-induced base-pairing changes has been termed an “m6A switch.”The release of specific single-stranded recognition sequences by m6A methylation by the METTL3/METTL14 complex. An m6A modification within the Box D motif interferes with formation of a structurally critical trans Hoogsteen-sugar A-G base-pair, disrupting the kink-turn fold. The kink-turn fold is required for binding of the Box C/D-associated proteins. Therefore, the Box C/D snoRNP cannot be assembled in the presence of an m6A modification in the Box D motif . Box C/D snoRNAs are the core component of the Box C/D snoRNP, which installs 2′-O-methylation marks in rRNA during ribosome biogenesis. For correct assembly of this multicomponent RNP, the Box C and D consensus motifs on the snoRNA have to fold into a characteristic kink-turn conformation to allow the binding of the protein core components. The Box D motif contains a GAC motif which is potent for m D motif .6A modification also affects the availability of single-stranded RNA-binding interfaces. Therefore, the modification can fine-tune the binding of specific RNA-binding proteins and affect cellular processes such as splicing or miRNA processing.In summary, the subtly altered base-pairing kinetics resulting from the presence of an m6A modifications can influence the protein interactome of a given modified RNA either by (1) serving as an “m6A-switch” as described in the previous section, (2) by providing m6A-specific binding pockets CU motif (6A modifications via their K homology (KH) domain, though how exactly the binding specificity is achieved is still unclear. In contrast to YTHDF proteins, IGF2BP proteins promote the stability of their mRNA targets in the cytosol and promote their translation.Insulin-like growth factor 2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) are a family of distinct mCU motif . IGF2BPs6A reader proteins is FMR1 and its paralogs FXR1 and FXR2. These proteins have been known to bind RNAs with the consensus motifs ACUG/U or U/A/GGGA, which show strong overlap with the METTL3/METTL14 DRACH consensus sequence  is a recently discovered meraction .6A modification. Protein–RNA interactions that are negatively affected by the m6A modification include human Pumilio 2 (hPUM2), stress granule proteins G3BP1, G3BP2, RBM42, USP10, CAPRIN1, and RNA-binding proteins LIN28A, and EWSR1  results in an increase of the KD from 0.14 to 0.34 nM with one m6A and 5.3 nM with three m6A bases , N1-methyladenosine (m1A), 3-meThymine (3-meT), and 3-meUracil (3meU)-modified nucleotides  to demethylate RNA. FTO localizes mainly in the nucleus and nuclear speckles but is also present in the cytosol . ALKBH5,leotides  (Jia et leotides .6A and m6Am modifications on mRNA and snRNA and m1A modifications on tRNA (6A sites nearby to promote demethylation (The main substrates for FTO are internal m on tRNA . The inf on tRNA . A large on tRNA . The splhylation . SFPQ-di6A-modified nucleotides. Furthermore, it shows a sequence preference toward m6ACU motifs, which notably overlaps with the known METTL3/METTL14 DRACH motif (6A methyltransferase METTL3/METTL14, it also localizes to actively transcribing genes. A recent article showed that ALKBH5 associates with the newly identified m6A reader RBM33, which activates the demethylase by suppressing SUMOylation and regulates the demethylation target selection (ALKBH5 seems to act only on internal mCH motif . ALKBH5 election . ALKBH5 election .6A demethylases. Recent years have unveiled the structural and catalytic basis for the demethylation, and important binding partners have been identified. Nevertheless, target specificity, interaction modes, specific functions, and potential functional redundancies are poorly understood. It is also unclear if specific RNA molecules can get methylated and demethylated multiple times. Future research in these directions will shed more light on the importance of reversing RNA modifications in the coming years.So far, only little is known about FTO and ALKBH5 in their function as m6A modifications, from their deposition until their removal, are functionally coupled to RNA folding and protein–RNA complex formation.Protein–RNA assemblies play an integral part in many cellular processes, and the coordinated installment of RNA modifications can modulate their assembly. In this review, we aimed to summarize how m6A core enzyme METTL3/METTL14 to sites of active transcription. This process is mainly mediated by direct protein–protein interactions between the core enzyme and defined histone marks or the C-terminus of Pol-II. The close spatial proximity between the enzymatic machinery and the nascent RNA allows for co-transcriptional installation of the modification (The process of the modification itself is a highly coordinated chain of events that relies on protein–RNA and protein–protein complex assembly. It starts with the site-specific and direct recruitment of the mfication . The corfication . Interesfication , 2013. Tfication . For sitfication .6A changes the base-pairing kinetics, by weakening Watson–Crick m6A-U base-pairs and stabilizing Hoogsteen–Hoogsteen/Watson–Crick m6A-A base pairs (6A readers and bind RNA preferentially in the presence of the modification.After installation, mse pairs . This alse pairs . Further6A reader YTHDC1 and the METTL3/METTL14 core enzyme localize to chromatin, and can thereby cooperate to maintain a repressive chromatin state at murine retroviral elements (Writers and readers can also cooperate. For example, both the melements .6A modifications allow for regulated competition between inhibitors and facilitators, as is the case for m6A “anti-reader” LIN28A and m6A reader hnRNPA2B1 (Different writers can also compete with each other. For example, during pri-miRNA processing, the mnRNPA2B1 .6A, are regulated by the coordinated assembly of protein–RNA assemblies and play an integral role in modulating the assembly of diverse sets of protein–RNA complexes and thereby affect their downstream functions. Although more and more roles are being proposed for m6A in different pathways and the interactome is constantly expanding, only a few and often very specific cases describe the mechanistic details of m6A-mediated regulation in protein–RNA assemblies.Through the examples illustrated in this review, it becomes clear that RNA modifications, here specifically m6A target-site selection, how the modification and the process of modification affect RNA folding, and temporal information on the coordinated action of m6A writers, readers, and erasers remain to be answered. Also, little is still known about the mechanistic details of how m6A modification is functionally coupled to cellular processes such as transcription and the effect of RNA modification on co-transcriptional splicing (We still do not have high-resolution structural information on the RNA bound to MAC or MAC/MACOM. Furthermore, questions such as msplicing . Quantitsplicing  will be