Recent studies about the transcriptome-wide presence of RNA modifications have revealed their importance in many cellular functions. Nevertheless, information about RNA modifications in viral RNA is scarce, especially for negative-strand RNA viruses. Here we provide a catalog of RNA modifications including m1A, ac4C, m7G, inosine, and pseudouridine on RNA derived from an influenza A virus infected into A549 cells, as studied by RNA immunoprecipitation followed by deep-sequencing. Possible regions with RNA modifications were found in the negative-strand segments of viral genomic RNA. In addition, our analyses of previously published data revealed that the expression levels of the host factors for RNA modifications were affected by an infection with influenza A virus, and some of the host factors likely have a proviral effect. RNA modification is a novel aspect of host–virus interactions leading to the discovery of previously unrecognized viral pathogenicity mechanisms and has the potential to aid the development of novel antivirals. RNA modifications are widely distributed in the tRNA and rRNA of living organisms, including all prokaryotes, archaea, and eukaryotes. While some modifications in mRNA, such as 7-methylguanosine (m7G) in the 5’ cap-structure, were first reported in the 1970s . ExampleFlaviviridae viruses, including dengue virus, zika virus, and the hepatitis C virus, regulate RNA stability and viral replication  and erasers (ALKBH1 and ALKBH3), have been identified in previous studies . UpregulRHBDD2 gene ; readers ; and erasers (ALKBH5 and FTO) . TranscrKnockdown of an m6A writer, WTAP, decreased the viral growth, reported in two independent studies . FurtherAmong the host factors responsible for m5C modification, infection with influenza A virus downregulated NOP2 ; upregulated NSUN3 (one of six experiments), NSUN6 (one of six experiments), and NSUN7 (three of six experiments but downregulated in one experiment), and did not affect the expression levels of NSUN2, NSUN4, or NSUN5. Changes in the expression levels of TRDMT1 , another m5C writer, by viral infection yielded contradictory results: upregulation in one study and downregulation in another. Knockdown of TRDMT1 significantly decreased viral growth, suggesting its proviral role . NOP2 anIn this study, we provide a catalog of possible RNA modifications including inosine, m1A, pseudouridine, and ac4C on the RNA molecules from the influenza A virus A. We docIt should be noted that the specificity of the RIP experiment was not adequate to conclude the presence of RNA modifications. It is commonly reported and discussed that there were few overlaps in identified regions that possibly contain RNA modifications among different studies using similar methodologies, even for well-studied model organisms ,38. The Direct RNA sequencing by nanopore sequencing technology will make a significant contribution to detecting RNA modifications ,41, althStill, the present study suggested a variety of RNA modifications in RNA derived from the negative-strand RNA virus. In this study, A549 cells were infected with the influenza A virus (PR8 strain) at MOI 0.1, and intracellular RNA was extracted at a single time point, 16 h post-infection. Because A549 is a cancer cell line from the human lung, it could be subject to non-native genomic or regulatory disruptions. In the future, we should further explore the findings’ generalizability using different cells such as primary bronchial/tracheal epithelial cells, in vivo models, and clinical samples. It is possible that RNA modifications in the viral genome differ among cell types  and anatomical and physiological conditions . RNA modifications in the viral genome of viral strains other than PR8 should also be tested in the future. In our experiment, the infections were not synchronized by high MOI because we did not have prior knowledge about the dynamics of RNA modifications in the viral RNA during the viral life cycle. Future studies are expected to understand how and when those RNA modifications in the viral genome are made.We also showed how viral infection affects the host’s RNA modification factors and vice versa B. Many hInhibiting RNA modifications can be an interesting option for the treatment of viral infections. That could decrease the stability of viral RNA suppressing viral growth and/or enhance the recognition of viral RNA by innate immunity leading to the enhancement of antiviral response. Furthermore, it is intriguing to figure out how the effect of RNA modifications has influenced the evolution of the viral genome. RNA modification is a novel aspect of host–virus interactions leading to a discovery of previously unrecognized mechanisms of the viral life cycle.A549 cell lines, MDCK cell lines, and influenza A virus [A/PR8/1934 (H1N1)] were obtained from ATCC . A549 cells and MDCK cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum. Influenza A virus was propagated and titrated using MDCK cells.Influenza A virus was inoculated into subconfluent A549 cells with 0.25 µg/mL TPCK-treated trypsin at MOI 0.1 plaque-forming unit/cell. Cells were washed with PBS three times, and intracellular RNA was extracted using TRIzol  and Direct-zol  with DNase treatment at 16 h after inoculation. All experiments from viral infection to deep-sequencing were done in biological duplicates .Forty micrograms of total RNA for the stringent RIP protocol, 80 µg of total RNA for the mild RIP protocol, and 5 µg of total RNA for the m7G-RIP protocol were fragmented using the NEBNext Magnesium RNA Fragmentation Module  according to the manufacturer’s protocol. Incubation with magnesium ions was performed at 94 °C for 5 min, followed by RNA purification by ethanol precipitation. The peak size of fragmented RNA was ~100 bases, confirmed using Bioanalyzer .Fragmented RNA was incubated with 500 U/mL RNA 5′ pyrophosphohydrolase (NEB) and Thermopol buffer (NEB) at 37 °C for 2 h, followed by the addition of 10 mM EDTA and incubation at 65 °C for 5 min. Treated RNA was then purified using TRIzol LS (Thermo Fisher Scientific).Antibodies and their concentrations used for the RIP experiments are described in RIP experiments were performed as previously reported with a slight modification . Forty mBecause enough RNA was not retrieved for pseudouridine and ac4C using the abovementioned stringent protocol, we further modified the protocol to perform RIP under mild conditions, introducing the following changes: 80 µg of fragmented RNA was used, incubation of antibody with beads was conducted with rotating for 2 h, and incubation of RNA with antibody-conjugated beads was done with rotating at room temperature for 2 h followed by 4 °C for 16 h. The RNA-bound beads were washed with IPP buffer five times before the isolation of RNA by TRIzol.Deep-sequencing sample preparation was performed using a SMART-Seq Stranded Kit  as per the manufacturer’s protocol with a slight modification. We skipped an RNA fragmentation step for already fragmented RNA samples for RIP. Deep-sequencing of the prepared samples was conducted using the NovaSeq 6000  with a protocol of 2 × 150 base paired-end runs to produce approximately 100 Gb of data (corresponding to 660 million reads).Adapter sequences and low-quality reads in fastq files were removed using Trim Galore with default settings. Sequences of viral positive-strand segments, negative-strand segments, all human transcripts , and the mitochondrial genome  were included in a single fasta file and used as a reference for mapping. NGS reads were mapped against the reference with forward-strand only option using bowtie2 . Site-byThe criteria to identify RIP-enriched regions include at least two-fold enrichment, each antibody-specific enrichment, and enrichment in two biological duplicate experiments.Transcriptome data from virus-infected cells were retrieved from the Influenza Research Database . All expPublished data from six studies that performed siRNA knockdown screening ,29,30,31The original data of deep-sequencing are available at the DDBJ BioProject database under accession number PRJDB11331.


The biological role of RNA methylation in stem cells has attracted increasing attention. Recent studies have demonstrated that RNA methylation plays a crucial role in self-renewal, differentiation, and tumorigenicity of stem cells. In this review, we focus on the biological role of RNA methylation modifications including N6-methyladenosine, 5-methylcytosine, and uridylation in embryonic stem cells, adult stem cells, induced pluripotent stem cells, and cancer stem cells, so as to provide new insights into the potential innovative treatments of cancer or other complex diseases. In gene(U-tail) . They ar(U-tail) . Recent 6A is one of the most abundant modifications in eukaryotes, and the epitranscriptomic detection technology has been focused on m6A, making it the most in-depth modification in stem cells. However, perhaps due to the lack of effective methods for detecting other RNA modification sites, the research on m5C and U-tail in stem cells is limited. This review summarizes the molecular mechanisms of RNA methylation and the biological functions of RNA methylation in embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs), and CSCs, with a focus on m6A.Among the RNA methylation modifications, m6A, m5C, and U-tail, and their molecular mechanisms are summarized as follows.RNA methylation modifications mainly include m6A is one of the most abundant modifications in mRNA and lncRNA, which was first discovered in the 1970s [6A accounts for approximately 0.1%–0.4% of adenosines and 50% of total methylated ribonucleotides in mammalian RNA [6A modification is dynamically reversible and mainly occurs in RRACH  sequences [6A modification, including “writers,” “erasers,” and “readers.” “Writers” mediate the RNA methylation process, which is a methyltransferase complex composed of three core components: methyltransferase-like (METTL) 3, METTL14, and Wilms' tumor 1-associating protein (WTAP) [6A methyltransferase associated (VIRMA), Cb1 protooncogene-like 1 (CBLL1), KIAA1429, and HAKAI are involved in RNA methylation as auxiliary elements [6A in 18S rRNA [6A binding proteins [6A may block nonstandard A:G base pairing and affect RNA structure, it does not alter the coding ability or base pairing of adenine with uracil or thymine [6A may affect the expression level, translation efficiency, nuclear retention, splicing, and stability of RNA [6A is closely related to human obesity, cancer, and other diseases and participates in the regulation of circadian rhythm, cell meiosis, cell reprogramming, stem cell proliferation, and other biological processes [mlian RNA . The m6Aequences . Three kn (WTAP) . In addielements . METTL5 18S rRNA . “Eraser18S rRNA . YT521-Bproteins –15. Althy of RNA , 14. m6Arocesses , 17.6A, m5C is another methylated modification form widely present in RNA [5C methyltransferases mainly include tRNA methyltransferase 4 (TRM4), tRNA aspartic acid methyltransferase 1 (TRDMT1), and the NOL1/NOP2/SUN domain (NSUN) family [5C methylation have not been found in RNA. Although the demethylation of RNA m5C may be catalyzed by Tet, further verification is required [5C modification [In addition to mt in RNA , 19. It t in RNA –24. m5C ) family , 25, 26.required . ALYREF fication , 29.U-tail, first identified in the 1970s , can proThe discovery of RNA methyl modifiers and binding proteins and their involvement in the metabolic processing of RNA suggest that RNA methylation has crucial biological functions.6A modification is essential for the pluripotency, self-renewal, and differentiation of ESCs , which is a histone marker, and are reduced when H3K36me3 is depleted in mouse ESCs [6A methyltransferase complex, can recognize and bind to H3K36me3 and then promote the cotranscriptional deposition of m6A. In mouse ESCs, METTL14 knockdown and H3K36me3 loss significantly reduced m6A levels in transcriptome-wide and pluripotency transcripts, leading to increased cell stemness. H3K36me3 inhibits pluripotency and promotes the differentiation of mouse ESCs partially by regulating the m6A modifications of several key pluripotency genes, such as octamer-binding transcription factor 4 (OCT4), sex-determining region Y- (SRY-) box 2 (SOX2), and NANOG [6A RNA methylation and histone modification interact in the regulation of ESC pluripotency and differentiation. In addition, m6A was identified as a regulator for terminating naive pluripotency in mice to ensure timely pluripotent factor downregulation, which is necessary for proper lineage initiation and differentiation [METTL3 in preimplantation epiblasts and naive ESCs leads to near complete depletion of m6A on mRNA. However, METTL3−/− ESCs still retain their naive pluripotency. Abnormal and restricted lineage initiation subsequently occurs in the postimplantation phase and results in early embryonic lethality [6A in other stem cell developmental transitions and exploring other potential m6A functions. Sun et al. reported that extracellular regulated protein kinases- (ERK-) dependent phosphorylation of METTL3 and WTAP promotes ESC differentiation [6A-labeled pluripotent factor transcripts and traps mouse ESCs in the pluripotent state [6A methyltransferase METTL5 has recently been shown to be important for ESC pluripotency and differentiation. Ignatova et al. reported that the absence of METTL5 in mouse ESCs results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential [METTL5 causes a dramatic differentiation defect in mouse ESCs [6A methylation in ESC nuclei. ZC3H13 works together with the WTAP–VIRILIZER–HAKAI complex to promote mRNA m6A processing [ZC3H13 knockout in mouse ESCs remarkably reduced m6A levels, damaged self-renewal, and triggered differentiation [6A methyltransferases play an important role in ESC differentiation and pluripotency through different regulatory mechanisms.mrocesses . Huang euse ESCs . METTL14nd NANOG   3 and leads to polyubiquitination, which is impaired in GSK-3-knockout ESCs, resulting in increased FTO protein levels  Figure . The m6An levels . The stuYTHDF3 in ESCs resulted in the loss of pluripotency with accelerated expressions of marker genes involved in the formation of three germ layers [6A-dependent manner [The YTHDF proteins have recently been proposed to perform different cellular functions. Wang et al. reported that depletion of m layers . Liu et t manner . Lasman t manner . This su6A mRNA modification on the fate of ESCs, m6A on lncRNA also affects ESC pluripotency and differentiation. Yang et al. reported that linc1281 deletion affects the differentiation of mouse ESCs, but linc1281 was not necessary for the self-renewal of mouse ESCs [6A modification is significantly enriched in linc1281 transcripts. linc1281 containing the RRACU m6A sequence motifs could restore the differentiation capacity in linc1281-deficient mouse ESCs [6A [Similar to the role of muse ESCs . The m6Ause ESCs . MechaniESCs [6A .6A modification in different ASCs. Here, we summarize the research results of m6A modification in hematopoietic stem cells (HSCs), neural stem cells (NSCs), and bone marrow mesenchymal stem cells (BMSCs)  .6A modification plays a key role in hematopoietic development during vertebrate embryogenesis [HSCs are derived from ESCs and have high self-renewal and multiple differentiation capabilities . HSCs deogenesis .6A methyltransferase complex. METTL3 deletion promotes cell differentiation and reduces cell proliferation in human hematopoietic stem and progenitor cells (HSPCs) [METTL3 overexpression inhibits cell differentiation and promotes cell growth in vitro [METTL3 in the adult hematopoietic system resulted in HSC accumulation in bone marrow, and the blockade of HSC differentiation led to a significant reduction of reconstitution potential both in vivo and in vitro [METTL3 deletion. The authors identified m6A targets in HSCs through RNA sequencing and found notable enrichment of 2073 genes related to hematopoietic differentiation, and the m6A modification of these genes was dependent on METTL3 [MYC was identified as a major functional target of m6A in HSCs. Further validating the difference between the two studies warrants further research, and the role of m6A in HSCs and progenitor cells may require individual identification. Cheng et al. uncovered that m6A is essential for the maintenance of HSC identity and symmetric commitment, with normal asymmetric commitment upon METTL3 depletion [METTL3 resulted in defective fetal hematopoietic progenitor proliferation, lineage commitment, and maturation, with accumulation of immature HSPCs and resultant hematopoietic failure [6A modification in the fate of HSPCs during vertebrate embryogenesis. The authors found that m6A peaks are obviously enriched in RRACH motifs in zebrafish, and this result is consistent with studies in mammals [METTL3-deficient embryos, delayed YTHDF2-mediated mRNA decay of the arterial endothelial genes notch1a and rhoca resulted in a significant decrease of m6A levels and blockade of HSPCs [METTL3-deficient embryos blocked the endothelial-to-hematopoietic transition, the production of the earliest HSPCs was inhibited [METTL3 is a key component of the m (HSPCs) . By contin vitro . Notablyin vitro . Howevern METTL3 . In addiepletion . Gao et  failure . Zhang e mammals . In METTof HSPCs   and inhibited the proliferation of aNSCs without affecting their homogeneity [METTL3 deficiency not only inhibits the development of neurons but also makes the differentiation of aNSCs more inclined toward glial lineage; the morphological maturation of new neurons in the adult brain is also affected [6A modification regulates histone methyltransferase Ezh2 expression at the translational level. Ezh2 overexpression could alter neuronal development and neurogenesis defects caused by METTL3 deficiency [METTL3 is increased in NSCs [6A methyltransferase METTL14 is required for NSC proliferation and maintains NSCs in an undifferentiated state. METTL14 knockout decreases NSC proliferation and promotes premature NSC differentiation, which suggests that m6A is necessary for NSC self-renewal [mogenesis . Loss ofogeneity . METTL3 affected . METTL3-ficiency . In addi in NSCs . Another-renewal .6A demethylase FTO is key in neurodevelopment and neurogenesis. FTO is strongly expressed in neurons and aNSCs and dynamically expressed during postnatal neurodevelopment [FTO deletion results in reduced brain size and body weight. The lack of FTO can inhibit the proliferation and neuronal differentiation of aNSCs in vivo, leading to learning and memory impairment in mice [Similarly, melopment . FTO del in mice . Cao et  in mice   and the spatiotemporal generation of neurons and other cell types [−/−YTHDF2 embryos in both in vivo and in vitro experiments [−/−YTHDF2 neurons cannot produce normal functional neurites, and expression of genes rich in neural development pathways is remarkably interfered [6A modified transcripts are caused by delayed degradation of mRNAs in −/−YTHDF2 NSPCs, which may result in neurogenesis defects [The deletion of the mll types . NSPC preriments . YTHDF2−terfered . Increas defects .6A modification all participate in the development of the nervous system and have crucial effects on neurogenesis.Taken together, the writers, erasers, and readers of m6A modification is involved in the development and differentiation of BMSCs. The expression of m6A methyltransferase METTL3 is significantly upregulated in the adipogenic process of porcine BMSCs (pBMSCs) [METTL3 in pBMSCs can promote adipose formation and mediate Janus kinase 1 (JAK1) expression in an m6A–YTHDF2-dependent manner [METTL3 deficiency reduces the m6A levels of JAK1, thus enhancing the stability of YTHDF2-dependent JAK1 mRNA. JAK1 affects adipogenesis by regulating STAT5 expression and activity. STAT5 can directly bind to the CCAAT/enhancer binding protein (C/EBP) β promoter to regulate its activity and mediate JAK1-regulated adipogenic gene expression and thus affect adipogenesis [6A modification in regulating BMSC differentiation into adipocytes and may provide crucial reference values in stem cell regenerative medicine and obesity treatment.BMSCs have multidirectional differentiation potential, can support hematopoiesis, promote HSC implantation, participate in key biological processes such as tissue regeneration and immune privilege, and have broad application prospects in stem cell therapy and regenerative medicine . The m6A(pBMSCs) . A lack t manner . METTL3 ogenesis   [METTL3 by short interfering RNA (siRNA) decreased m6A methylation levels and inhibited osteogenic differentiation of BMSCs and reduced bone mass [METTL3 overexpression in BMSCs can prevent osteoporosis caused by estrogen deficiency in mice [6A downstream pathway in BMSCs [METTL3 conditional knockout reduces the translation efficiency of PTH1R in BMSCs and interferes with PTH-induced osteogenic and adipogenic effects in vivo [6A in bone health and disease as well as new evidence for the regulation of stem cell differentiation by m6A.Moreover, ntiation . METTL3 ed BMSCs . Loss ofof BMSCs . Yan et  (RUNX2) . Silenceone mass . In addi in mice . In termin BMSCs . METTL3  in vivo  , and c-Myc, into differentiated somatic cells and reprogramming them. iPSCs have broad application prospects in the treatment of human diseases and organ transplantation . Becauseipotency   interact with the METTL3–METTL14–WTAP complex in an Activin/Nodal signal-dependent manner in both hESCs and human iPSCs (hiPSCs)  Figures. The intl signal . The mec6A reader proteins YTHDF2 and YTHDF3 are required for reprogramming somatic cells into iPSCs [YTHDF2 is highly expressed in iPSCs and downregulated during neural differentiation [YTHDF2 in iPSCs leads to stabilization of a group of m6A-modified transcripts associated with neural development, loss of pluripotency, and induction of neural-specific gene expression [The mto iPSCs . YTHDF2 ntiation . Depletipression   cells. In addition, METTL3 loss in human myeloid leukemia cell lines can lead to cell differentiation and apoptosis and hinder the progress of leukemia in recipient mice in vivo. m6A can promote the translation of c-Myc, Bcl-2, and PTEN mRNAs in the human AML MOLM-13 cell line [METTL3 is a crucial gene for AML cell growth [METTL3 leads to cell-cycle arrest, leukemia cell differentiation, and leukemia establishment failure. METTL3 is related to chromatin and is located at the transcriptional initiation sites of active genes. Moreover, most of these genes have the CAATT-box binding protein CEBPZ at the transcription initiation site, which is necessary for METTL3 to be recruited into chromatin [6A modification in the coding region of relevant mRNA transcripts and improves its translation efficiency by mitigating ribosome stagnation [Compared with healthy HSPCs or other types of cancer cells, mell line . Barbierl growth . In immuhromatin . Promoteagnation . These r6A methyltransferase, is strongly expressed in AML cells carrying t(11q23), t, or t and is reduced during myeloid differentiation [MYB and MYC, through m6A modification.METTL14, another mntiation . METTL14ntiation . METTL146A-dependent mechanisms [The demethylase ALKBH5 plays critical roles in leukemic cell transformation, AML development and maintenance, and LSC/LIC self-renewal through posttranscriptional regulation of critical targets via mchanisms . Shen etchanisms . Wang etchanisms   and demonstrated the carcinogenic effects of METTL3 in promoting stemness and metastasis of CRC. Colorectal cancer stem-like cells are a cancer cell type with self-renewal and multiple differentiation potential and have strong tumorigenicity and metastasis abilities. METTL3 inhibition could enhance chemotherapy response and reduce CSCs in CRC [6A–IGF2BP2-dependent regulatory mechanism in CRC cells and then promotes CRC stemness and metastasis [One study revealed the basic functions of ms in CRC . METTL3 tastasis   self-renewal and tumorigenesis. Visvanathan et al. reported that METTL3-mediated m6A modification plays a key role in GSC maintenance and glioma cell dedifferentiation [METTL3 expression is elevated in GSC and attenuated during differentiation. The METTL3-dependent GSC maintenance is mediated by SOX2 mRNA stabilization, and human antigen R (HuR) recruitment to m6A-modified sites is required for SOX2 mRNA stabilization [METTL3-silenced GSCs exhibited increased sensitivity to γ-irradiation and decreased DNA repair. Exogenous overexpression of 3′UTR-less SOX2 exhibited potent DNA repair in METTL3-silenced GSCs [6A regulome in METTL3-silenced GSCs showed global disruption in tumorigenic pathways that are indispensable for GSC maintenance and glioma progression [METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs [METTL3 or METTL14 deletion can significantly enhance GSC growth, self-renewal, and tumor progression [METTL3 overexpression or FTO suppression can inhibit GSC growth and self-renewal. In addition, FTO inhibition can suppress tumor progression and prolong the lifespan of GSC-transplanted mice [6A modification participates in regulating the expression of genes with crucial biological functions in GSCs, which may be a target for GBM treatment.The mntiation . METTL3 lization  , whereas ALKBH5 deletion can inhibit GSC proliferation and tumorigenesis [The levels of migenesis . Kowalskigenesis . These digenesis .Yarmishyn et al. demonstrated that YTHDF1 is involved in Musashi-1-mediated GBM tumorigenesis processes such as cell proliferation and migration and also regulates the stem-like properties of GBM cells . YTHDF2 6A RNA methylation and decrease the levels of NANOG and KLF4 under hypoxic conditions [α and ALKBH5 were consistent in human breast cancer biopsies analyzed. Knockout of ALKBH5 in MDA-MB-231 breast cancer cells remarkably reduced breast-to-lung metastasis in immunodeficient mice [6A demethylase ALKBH5 and m6A methyltransferase inhibitor ZNF217 have important effects on BCSC phenotype and breast cancer metastasis. Zhu et al. reported that hypoxia-induced lncRNA KB-1980E6.3 is involved in the self-renewal and stemness maintenance of BCSCs by recruiting IGF2BP1 to regulate c-Myc mRNA stability. The lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis may potentially be a therapeutic target for breast cancer [The breast cancer stem cells (BCSCs) are the crucial factors for the occurrence, growth, metastasis, and recurrence of breast cancer . BCSC phnditions . The expent mice   RNA m classes . This stpiration . Althougaffected . ESC difaffected . Thus, tfunction .5C RNA methylation is involved in the regulation of NSC differentiation and motility. The m5C methyltransferase NSUN2 is expressed in neuroepithelial stem and progenitor cells during early human brain development, and its expression gradually decreases during neural differentiation [NSUN2−/− brains [NSUN2 depletion inhibits the migration of neural cells to chemical attractant fibroblast growth factor 2, leading to impaired neural differentiation of neuroepithelial stem cells [5C RNA methylation has crucial effects on neural development, and its role in other stem cells requires further exploration and research.mntiation . Deletio− brains . NSUN2 dem cells  (Table 1U-tail is closely related to stem cell differentiation and cancer . Zcchc11DIS3L2 is a 3′–5′ exonuclease responsible for the decay of uridylated let-7 precursor in mouse ESCs , 100. PiRNA methylation, as a vital posttranscriptional mechanism of gene regulation, is crucial to physiological and pathological processes. As high-throughput sequencing technology is rapidly developing, the understanding of the biological functions of RNA methylation is deepening.6A in stem cells is limited and may require further exploration and research as well as more advanced molecular biology techniques to make breakthroughs.Numerous studies demonstrated a close correlation between RNA methylation and stem cells. RNA methylation plays a key role in regulating stem cell maintenance, differentiation, reprogramming, and controlling in mammalian developmental stages. The regulatory factors of RNA methylation are involved in regulating stem cell fate and function. These findings have opened new directions for the clinical treatment of various diseases, including cancer. The aberrant expression of one or more RNA methylation regulatory factors may be used as a diagnostic or prognostic biomarker. Further in-depth analysis of RNA methylation may aid in developing inhibitors targeting writers, erasers, or readers to further explore the potential mechanisms for controlling gene expression in stem cells in physiology and pathology. However, many obstacles remain that must be overcome when treating diseases by targeting RNA methylation. In addition, although RNA methylation involves many patterns, research on RNA methylation patterns in addition to m


Highly pathogenic avian influenza viruses (HPAIVs) evolve from low pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes. This evolution is characterized by the acquisition of a multi-basic cleavage site (MBCS) motif in the hemagglutinin (HA) that leads to an extended viral tropism and severe disease in poultry. One key unanswered question is whether the risk of transition to HPAIVs is similar for all LPAIVs H5 or H7 strains, or whether specific determinants in the HA sequence of some H5 or H7 LPAIV strains correlate with a higher risk of transition to HPAIVs. Here, we determined if specific features of the conserved RNA stem-loop located at the HA cleavage site-encoding region could be detected along the LPAIV to HPAIV evolutionary pathway. Analysis of the thermodynamic stability of the predicted RNA structures showed no specific patterns common to HA sequences leading to HPAIVs and distinct from those remaining LPAIVs. However, RNA structure clustering analysis revealed that most of the American lineage ancestors leading to H7 emergences via recombination shared the same viral RNA (vRNA) structure topology at the HA1/HA2 boundary region. Our study thus identified predicted secondary RNA structures present in the HA of H7 viruses, which could promote genetic recombination and acquisition of a multibasic cleavage site motif (MBCS). More precisely, HA nucleotide sequences were downloaded, translated, and then subjected to alignments based on the amino acid sequence. Finally, codons of each sequence from the original nucleotide sequences were retrieved and replaced the corresponding amino-acid residues of the protein alignment. Referring to H5 and H7 HPAIV emergence mechanisms, specific HA sequences underwent nucleotide insertions and recombination with other genes, making the HA1/HA2 region alignment difficult. The consideration of indels remains challenging in phylogeny and is absent in commonly used DNA models, which potentially constitute a source of bias for tree inference. Thus, cDNA alignments of HA sequences were manually edited to remove nucleotide insertions corresponding to codon duplication and recombination (H7 HPAIV) events at the HA-CS encoding region. The minimal consensus motifs of H5 and H7 LPAIV-HA-CS motif, ‘QRETR|GLF’ and ‘XPKGR|GLF’, respectively, have been preserved during the alignment editing. HA sequences were annotated as HPAIV or LPAIV-HA depending on the HA-CS motif on non-edited alignment. HPAIV/LPAIV-HA assignment was verified from the literature .2.1.2The phylogenetic analysis was conducted with the IQ-Tree-2.0.6 software using a maximum-likelihood (ML) approach .The best DNA models were estimated and selected via Akaike Information Criterion . HA5 and2.1.3Molecular clock analysis using the least square dating (LSD2) method was performed . The col2.1.4Every internal node of the HA5 and HA7 time-scaled trees was estimated by the empirical Bayesian method. Only HA-estimated sequences of internal nodes that meet the following criteria were retained for RNA structure predictions: (1) every nucleotide position around the HA-CS encoding region of the estimated HA sequence had to have a posterior probability superior to 0.80, and (2) the estimated HA sequence did not harbour a MBCS motif in its amino-acid sequence.2.22.2.1The aim of the study was to analyse the predicted RNA structures encompassing the conserved HA-CS encoding region of all H5/H7 LPAIV and HA sequences from other subtypes. During genome replication, the polymerase may be locked between the pairing of entering and exiting RNA strands that constitute the cSL, which can partially re-hybridize . Moreove2.2.2We used an analytic pipeline to address the question of how structural features of the RNA secondary stem-loop evolve along the evolutionary pathway of HA sequences. Most of the codes were realized with the R-software 4.0.2  and Pyth2.2.3k=1.A complementary approach was performed to evaluate structural differences or similarities in the predicted RNA structures encompassing the conserved HA-CS encoding region. We performed a clustering of RNA structures from all LPAIV-HA sequences uploaded from the NIAID IRD and from the predicted ancestors (both on the vRNA and cRNA) by RNAclust perl-script . In brie3.3.1To test if the ability of H5 and H7 AIV to acquire MBCS correlated with specific differences in the cSL structure, we performed an analysis of the thermodynamic stability of the predicted cSL structure focusing on a region of eighty nucleotides. The thermodynamic stability of the predicted MFE structures was calculated and expressed in dG0 value : higher dG0 values indicated lower thermodynamic stabilities. As vRNA and cRNA can exhibit different RNA structures, we performed the analysis on both strands. All available HA-encoding sequences from avian origin were downloaded from NIAID IRD. Groups were designed in terms of the HA subtype. H17 and H18 HA CDS sequences (from bat origin) were also included in the analysis. On the eighty-nucleotide window, the thermodynamic stability of the predicted MFE structure was variable within and between HA subtypes . The pre3.2Previous work  and the As a first step towards this objective, we generated phylogenetic trees with 1,116 and 1,928 HA sequences for the H5 and H7 subtypes, respectively, corresponding to all HA sequences from H5/H7 LPAIV and known HPAIV emergences (detailed in x=0) towards the root of the phylogenetic tree   –6. As thIndividual evolutionary pathways in the H5 ‘LPAIV-only’ group showed significant variability in their cSL structure stability . No appaIn the case of H7 sequences, evolutionary pathways that did not lead to any known emergence event also showed high variability in terms of MFE stability values . We did 3.3RNA molecules with similar thermodynamic stabilities can have different secondary conformations. Thus, to determine if specific RNA structure topologies could be identified among HA sequences evolving towards HPAIV, we analysed RNA structures using the RNA structure clustering software RNAclust. The analysis was performed on all LPAIV-HA sequences and the HA sequences estimated at each internal node of the time-trees, including the HPAIV progenitors. RNA structure clusters that contain ancestors specifically leading towards HPAIV emergence events were highlighted by a blue rectangle on the computed cluster trees . Two HPAIn the case of HA5 sequences, we identified eight different clusters containing HPAIV ancestors on the vRNA and eight clusters containing HPAIV ancestors on the cRNA . All theIn the case of HA7 sequences, we identified ten different clusters containing HPAIV ancestors on the vRNA and twelve clusters containing HPAIV ancestors on the cRNA . AncestoThis analysis thus revealed that most of the American H7 ancestors (four over five events of recombination belonging to the American lineage) leading towards HPAIV emergence events via recombination shared similar cSL RNA structure topologies.4.Previous studies suggested a role of the cSL structure in acquiring a MBCS and thus in the evolution towards HPAIVs in the case of H5/H7 AIV . HoweverAnalysis of the cSL thermodynamic stability was insufficient to explain why only specific H5 and H7 LPAIVs could acquire the MBCS pathogenic motif. No specific feature in the cSL was detected for the HPAIV emergence events via nucleotide insertions or substitutions. Interestingly, we observed a specific clustering of vRNA structures in HA1/HA2 encoding region from most of the American H7 HPAIV ancestors via recombination events. We therefore hypothesize that a putative evolutionary convergence mechanism involving the HA1/HA2 vRNA topology contributes to H7 HPAIV emergence via recombination . ComparaInfluenza virus evolution is driven by several viral and environmental factors, making these biological processes complex. Therefore, LPAIV-to-HPAIV evolution might result from multiple interacting and cooperating factors, involving other known pathogenicity factors . MoreoveThe biological relevance of the shared RNA structure identified amongst H7 HPAIV ancestors via recombination must be further explored to establish a functional link between RNA structure and evolution via recombination. At least, these predicted structures constitute a new marker of HA sequences that evolved towards HPAIV via recombination, which may be used to generate a new classification of viral sequences. Moreover, predicted RNA structures in the HA1/HA2 boundary region must be compared with RNA structures determined chemically in the context of the NP and the polymerase complex . Other sAs the cSL’s biological role is still under study, it may have other roles beyond evolution. Other RNA structures located at the internal region of other vRNA (M and NS segments) have been linked to genome packaging . Moreove4.1Based on the analysis of the cSL across HA subtypes and along the distinct evolutionary pathways, we conclude that the thermodynamic stability of the cSL cannot be used to explain why specific H5/H7 HA sequences can acquire the MBCS pathogenic motif.Using RNA clustering analyses, we observed that most of the American H7 emergence events via recombination (four over five) shared the same cSL structure, suggesting a possible role of RNA topology in polymerase jump and template switching.veab093_SuppClick here for additional data file.


Elevated level of oxidized RNA was detected in vulnerable neurons in Alzheimer patients. Subsequently, several diseases and pathological conditions were reported to be associated with RNA oxidation. In addition to several oxidized derivatives, cross-linking and unique strand breaks are generated by RNA oxidation. With a premise that dysfunctional RNA mediated by oxidation is the pathogenetic molecular mechanism, intensive investigations have revealed the mechanism for translation errors, including premature termination, which gives rise to aberrant polypeptides. To this end, we and others revealed that mRNA oxidation could compromise its translational activity and fidelity. Under certain conditions, oxidized RNA can also induce several signaling pathways, to mediate inflammatory response and induce apoptosis. In this review, we focus on the oxidative modification of RNA and its resulting effect on protein synthesis as well as cell signaling. In addition, we will also discuss the potential roles of enzymatic oxidative modification of RNA in mediating cellular effects. See (via its oxidative stress biomarker provides a useful method for diagnosing age related diseases (see review (see review (Reactive oxygen species (ROS) and free radicals play major roles in normal biological functions. However, they can also react rapidly with biomacromolecules, such as nucleic acids, proteins, or lipids, and alter their biological functions. Therefore, they have been implicated in a wide range of age-related neurodegenerative diseases and aging . Howevee review , 8-oxo-Ge review , mRNA . Howevee.g. 8-oxo-G as well as 8-oxo-dG could exhibit an antioxidant function (via oxidative modification and the apurinic/apyrimidinic endoribonuclease1 (APE1) (c (cyt c) could form a cross-linked complex with oxidized RNA, and facilitate its dissociation from mitochondria to the cytosol, a process required to initiate the mitochondria-mediated apoptosis. Our findings imply that oxidative modification of RNA facilitates cellular apoptosis via a protective signal in response to oxidative stress  . We havee stress . In addie stress . Pokeweee stress . As illu2O2 are ubiquitously generated from the electron transport chain , H2O2 is converted to hydroxyl radical, which is a highly reactive ROS . Therefe.g.  .Abasic site is a sugar moiety formed after a nucleobase is cleaved off. It is estimated that the abasic sites in genomic DNA are the most abundantly generated oxidative modification. It yielded approximately 10,000 to 50,000 sites mediated by site-specific spontaneous reaction , enzymatith H2O2 . As showith H2O2  and charith H2O2  and prefith H2O2 . In ordeith H2O2 . While t 8-oxo-G , the oxiIn vitro oxidoreduction of yeast RNA in the presence of NADH and K3Fe(CN)6 gave rise to the hydroxylated pyrimidines 5-OH-C and 5-OH-U in addition to 8-oxo-G and 8-oxo-A , and then becoming sulfinic acid (-SO2H), and then sulfonic acid (-SO3H), which is desulfrated to generate 4-pyrimidinone ribofuranoside (H2U) (2O2 treatment (c (cyt c) mediated peroxidation  is oxidized de (H2U)  and uridreatment  under phreatment  and cytoxidation . Conversxidation .i.e. . For pareatment . Proteomnd cyt c . Cyt c c of H2O2  and exhientified . Thus, ain vitro reactions between hydroxyl radicals and RNA strands lead to strand scission, in which nucleobase radicals are primarily generated, followed by H-abstraction from ribose rings, and resulting in a strand break -EDTA. The cleavage site and chemical structure of the terminal overhangs were identified by their molecular weights. In addition to 3′-PO4, 2′,3′-cyclic PO4 and 3′-phoshoglycolate were the main 3′-end structures, while 5′-PO4 and 5′-OH were the main 5′-end structures. Based on previous DNA studies, it was deduced that there are three different modes of cleavage reactions, hydrolysis, H-abstraction, and 2′-OH mediated transesterification: 3′-PO4 was generated by the hydrolytic process; 3′-phosphoglycolate formation occurred by oxidative 4′-H abstraction in the ribose moiety, and base 2-propenal was generated as a byproduct. In addition, 2′,3′-cyclicPO4 could be generated due to 2′-OH mediated transesterification , but also a small amount of Met (5%). Even though the translation was not efficient, the AP site was found to be decoded in certain nucleotides but not a single nucleotide. Conversely, HEK293T eukaryotic cells did not translate the modified codon 6 and NADH. The redox potentials (E7) for 5-OH-C, 5-OH-U, 8-oxo-A and 8-oxo-G were found to be 0.42, 0.44, 0.72, and 0.38 V, respectively. Considering the redox potentials of K3Fe(CN)6 (0.42 V) and NADH (−0.35 V), it is reasonable that 5-OH-C, 5-OH-U, and 8-oxo-G but not 8-oxo-A could catalyze the oxidoreduction between the two compounds . Chung’ectively . Additioectively .In the presence of nitric oxide, the soluble guanylyl cyclase (sGC) is activated to catalyze GTP to cyclic-GMP (cGMP), the second messenger for various downstream signaling for proliferation, synaptic plasticity, vasodilation, and platelet aggregation. Bolin and Cardozo-Pelaez investigated the effect of 8-oxo-GTP on sGC . They ob2O2 treated HA1 hamster fibroblast cells was transfected, production of proinflammatory cytokines, IL-6, MCP-1, and type I interferon were found lower than those of control mtRNA isolated from untreated cells. In contrast, when oxidized mtRNA derived from H2O2 treated THP-1, human monocyte cells were transfected to the differentiated THP-1 induced by PMA, the inflammatory response to induce IL-6 and TNF-α was more activated relative to that of control mtRNA  polymerase 1 (PARP1) cleavage after H2O2 treatment leading to a higher survival rate. In addition, PCBP1 mutant in KH1 domain showed deficiency both in 8-oxo-G binding and PARP-1 cleavage  , Y box-bcleavage . In contcleavage . Togethec catalyzes peroxidation through the heme iron. To this end, we have reported that cyt c readily oxidized tRNA to form a cross-link in the presence of H2O2  is long known as the endodeoxyribonuclease which cleaves off DNA strand specifically at the 5′ side to the AP site during the process of DNA base excision repair. Later, this enzyme was found to be able to cleave c-myc mRNA  and was r tissue . These rN-glycosidase activity . HoweveA duplex . These fThe scheme in in vitro and in vivo, observed with H2O2 treatment (The level of oxidized cellular RNA is higher than that of genomic DNA under oxidative stress conditions, reatment , UV irrareatment , or treareatment . These ereatment  and the reatment . On the reatment . PNPase reatment , and latreatment . MutT/MTreatment . Moreovereatment . Therefo


A signal analysis of the complete genome sequenced for coronavirus variants of concern—B.1.1.7 (Alpha), B.1.135 (Beta) and P1 (Gamma)—and coronavirus variants of interest—B.1.429–B.1.427 (Epsilon) and B.1.525 (Eta)—is presented using open GISAID data. We deal with a certain new type of finite alternating sum series having independently distributed terms associated with binary  TThere are thousands primary sources available in GISAID that warrant urgent investigation on biometric analyses, comparisons and characterization of these sequences of different coronavirus lineages responsible for the ongoing pandemic. In particular, an analytical study of emerging SARS-CoV-2 variants (sharing mutations) is highly needed since some variants appear to be more persistent and contagious. This is a good reason for increasing genomics surveillance on the emerging variants by the development of new tools that can detect and catalog these strains in a timely manner .Similarity between biological sequences forms the basis for determining whether there is sequence homology, as defined in terms of shared ancestry between them in the evolutionary history of life . AlthougA)denine, (C)ytosine, (G)uanine and (T)hymine (or Uracil RNA genome for single strand folded onto itself) according to their progression along the genome sequences. Our mapping into four binary projections of the coronavirus sequence follows previous studies on the three-base periodicity characteristic of protein-coding DNA sequences [N nucleotides on the order of 30,000 base pairs (bp) in length. This novel finite and alternating sum series of binary projections is applied to most variants of SARS-CoV-2 so far sequenced. It is shown that our approach provides additional information to conventional genomics similarity computations by alignment methods [In this work, we propose a new quantitative method for the examination of distinctive patterns of complete coronavirus genome data. We deal with a certain type of alternating finite series having terms converted to binary values equences ,10. In t methods ,12.T is denoted as U. As for the severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 complete genome (Accession ID: MN908947) [By this new method we uncover distinctive signals of the intrinsic gene organization revealed by the genome sequences of the single-stranded RNA coronaviruses. In particular, we analyze the genome sequences for coronavirus variants of concern—B.1.1.7 (Alpha), B.1.135 (Beta) and P1 (Gamma)—and coronavirus variants of interest—B.1.429–B.1.427 (Epsilon) and B.1.525 (Eta). The present new method could be useful for the surveillance and dynamics of genome variants in general. The coronavirus genome is RNA, not DNA, and the nucleotide base T, store instructions to assemble and reproduce every living organism. In DNA, nucleotides of one strand are complementary to those of the opposite strand according to the pairing rules A-T and C-G. Similarity plots of SARS-like CoVs and bat SARS-like CoVs complete sequences of T revealed apparent recombination events, useful to understand the probable coronavirus pandemic origins [The encoded genes in a sequence of four nucleotides, represented by the symbols  origins ,2,3,4. Gorted in .Similarity plots based on the full-length nucleotide sequence of gene variants of coronavirus are presented. In Note: As of this writing, new labels have been given by WHO for SARS-CoV-2 variants of concern: B.1.1.7 (Alpha), B.1.135 (Beta) and P1 (Gamma). New labels have also been given for variants of interest: B.1.429–B.1.427 (Epsilon) and B.1.525 (Eta). A SARS-CoV-2 isolate is a variant of interest “if, compared to a reference isolate, its genome has mutations with established or suspected phenotypic implications”, and is variant of concern when, through a comparative assessment, it has been demonstrated to be associated with the “Increase in transmissibility, virulence or change in clinical disease presentation; or Decrease in effectiveness of public health and social measures or available diagnostics, vaccines, therapeutics”.It is important to note that in all figures illustrated below, the samples for a given country correspond to data from a given variant. For example, the difference between the 33 and 19 samples from Brazil in github.com/wrpearson/fasta36 (accessed on 22 June 2021)). The command line used is: In these calculations we installed and ran, under the Linux Ubuntu O.S., the current version 36 of the FASTA sequence comparison software, which includes lalign36. It produces multiple non-overlapping alignments for protein and DNA sequences using the Huang and Miller sim algorithm for the Waterman–Eggert algorithm  of the different coronaviruses isolated with the first SARS-CoV-2 Wuhan-Hu-1 sequences, shown in f of the different coronavirus sequences with N nucleotides  as depicted in T nucleotides, binary values can be assigned to those sequences in order to apply Discrete Fourier Transform methods. Genetic sequences generate inherent signals since they are functions of an independent variable X, denoting the occurrence of a particular nucleotide in position k of the sequence. This technique has been broadly used in the literature to search for periodicity in DNA sequences [Let us analyze next the power spectrum as a function of a discrete ‘frequency’ equences ,10,14.In For all variants considered, our plots reveal a distinct peak above noise level at around frequency rganisms ,10.In the past, the two methods of similarity and power spectrum have been developed for computing different types of protein features. We have seen that when applied to the genome of coronavirus variants, both of these analyses do not give much information on variant characterizations themselves. We shall show next how an alternative method can provide additional insights to conventional studies, which may help to characterize variants of the current pandemic.In the present paper we deal with the simplest alternating sum of the typeudies in ,10. In oT’s imprints for the genomics strand of prevalent coronavirus variants reported from different countries for a number of samples. As reference, we illustrate complete genome sequences of SARS-CoV-2 from the city of Wuhan in China and the bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses: bat-SL-CoVZC45 and bat-SL-CoVZXC21 [Analyzing genomics sequencing via Equation  allows oEquation  providesudies in  suggest In particular, we note distinctive trends, especially around the nucleotide region of the S-Protein. The base sequence series for Cytosine in The positive and negative terms in the sums in Equation  for our N intervals. Plus and minus signs are chosen sequentially starting with To recap, we can conclude the following. Our method is effective and easier to apply in protein sequence comparison. It is motivated by the need to identify genetic mechanisms involved in coronavirus spreading. The added value of the alternating sums of the type in Equation  is to haata sets ,17.We have shown that these alternating sums provide additional information to conventional similarity comparisons and power spectrum approaches. We also verified that the Discrete Fourier transform of the complete alternating sum series (not shown) leads to a peaked structure at a ‘frequency’ equal to 16,6666 meaning for the sums a 100/6 characteristic periodicity. Distinctive trends have been identified, e specially around the nucleotide region of the spike protein for all variants studied. These emerging variants seem to have only a few mutations, i.e., no more than a dozen amino acid changes out of the 1200 building blocks that make up the spike protein. They give a selective advantage for their replicating capacity ,18.n-moment calculations We downloaded worldwide sequencing data of coronavirus variants from GISAID and verified multiple deviations from the originating first Wuhan sequences identified over a year ago. Our statistical representation of coronavirus genome variants, taking sums with both signs, can reveal signals from future genome evolution at the level of nucleotide ordering. These observations lie at the heart of future studies. One could investigate further the present numerical results to study the


RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications. Bacteria are remarkably versatile organisms, which can survive and grow in numerous environmental niches on the planet. They modulate the expression of their genes to respond and adapt their growth to environmental stress such as temperature and pH shifts, nutrient availability, antimicrobials or dangerous chemical reactive species. This is particularly the case for pathogenic bacteria, which must adapt their metabolism to the host environment and ensure their survival facing the human/animal immune systems and antibiotic therapies. Bacteria can sense the environment directly via the effect that physical and chemical stresses might have on different macromolecules, or via signaling systems, very often two-component systems (TCS), transducing the stress signal from a specific sensor histidine kinase to a corresponding response regulator . ResponsIn bacteria, the rate of translation is modulated by multiple signals present in mRNAs affecting translation initiation, elongation, termination, and ribosome recycling ,4,5,6. Tsyn/anti conformations with relatively greater ease [The number of newly detected modifications increases regularly . They cater ease . The dynter ease  have genter ease . The chaIn this review, we illustrate several examples of the involvement of RNA modifications on the expression of virulence genes and in stress responses in different pathogenic bacteria. Even though these studies provide mechanistic explanations for only few cases, they already reveal a multitude of strategies developed by pathogenic bacteria to survive, persist, and fight against host immune defenses based upon the modulation of RNA modifications.A prerequisite to analyze the functions of RNA modifications is obviously to be able to detect the modified nucleosides, to quantify them, to map their localization within specific RNA. A detailed description of the available methods is outside the scope of the review. We will summarize the most employed approaches with their limits and advantages , and detp-toluene sulfonate (CMCT) [3 (MS/MS/MS) analysis [m/z 207.041 for double dehydrated Ψ [Mapping of post-transcriptional RNA modifications using mass spectrometry (MS) existed for 30 years . The pree (CMCT) . The care (CMCT) . More reanalysis  thanks tdrated Ψ .Various high throughput sequencing methods are often used to characterize specific modifications by reverse transcriptase (RT) signatures after chemical treatment. These methods do not need to purify the RNA prior to the analysis but they need bioinformatics data treatment and statistical evaluation of errors. NGS-based methods are often not quantitative, have high false-positive rates , and reqCombination of these highly complementary methods allows a precise localization and characterization of post-transcriptional modifications and their dynamics, a prerequisite to a better understanding of the roles of RNA modifications in bacterial adaptation and virulence.Escherichia coli ribosomes lacking rRNA modifications were severely defective in catalytic activity [The ribosome is a powerful molecular machine and a huge ribonucleoprotein complex (2.3 MDa in bacteria), which performs the crucial task of translating the genetic information into proteins. The ribosome is also a ribozyme with its catalytic centers made by RNAs . To carractivity  and the activity . Numerouactivity ,84, and activity . In bactactivity . Even ifactivity ,87. Thesactivity ,89, , reverse transcriptase extension (RT), RNAseq, and structure analysis. Several high-resolution structures of 70S ribosomes have been achieved both by cryo-EM and X-ray studies. However, cryo-EM structures can be non-uniform in local resolution and confident assignment of modifications is often possible on their most structurally stable core. Assignment of 35 modifications on bacterial rRNAs was first obtained on the cryo-EM structure of solution . This stectively ,94. Moreproposed   increases the flexibility of the nucleotides next to it, and induces 50S maturation delay, slowers subunit association and translocation rate [On the large ribosomal subunit,  of life  whereas observed . Absenceion rate . Surprision rate .S. aureus sub-inhibitory concentrations of the macrolide erythromycin (ermC) responsible for the dimethylation of A2058 at the ribosomal peptide exit tunnel of the 50S subunit [ermC is exposed, allowing translation of ermC [62A2058 are not only erythromycin-resistant, but can also better escape host immune system, avoiding recognition by specific Toll-like receptors [5C1407) and decrease translation accuracy [ksgA gene encoding methyltransferase causes a defect of modifications at A1518 and A1519 in the 16S rRNA and induces kasugamycin resistance [7G527, which is located near the mRNA decoding site has been shown to confer low-level streptomycin and neomycin resistance [The ribosome and more generally the translation apparatus are main targets of antibacterial therapies ,107,108.hromycin D stall r subunit  , trmA (m5U54) and truB (Ψ55), reduces growth rate particularly at high temperature [T. thermophilus, adaptation to low temperature requires maintaining a sufficient flexibility of tRNAs. In this condition, methylation of the ribose of G18 is prevented but only if Ψ55 is present [Shigella flexneri, Ψ55 is linked to the expression of several virulence factors, which are responsible of shigellosis, an intestinal infection causing diarrhea. Deletions of trmH, trmA, and truB genes in S. flexneri are associated with a reduction of the hemolytic activity and a decrease in the secretion system expression [The 5-methylation of U54 and the isomerization of U55 into Ψ55 enhance base stacking and stabilize the tRNA ,135,136.perature . In the  present . Moreovepression . Furtherpression ,140,141.TyrGUA, tRNAAsnGUU, tRNAAspGUC and tRNAHisGUG at the wobble position (G34) in Eukaryotes and Bacteria, permits efficient recognition of both NAC and NAU codons. This Q modification allows fine-tuning of translation and has been correlated with low-level translation of Shigella virulence factors, including the main transcriptional regulator VirF [2i6A37) tRNA modifications. Deletion of either tgt  or miaA  genes leads to a less efficient synthesis of VirF in S. flexneri and reduces its pathogenicity [E. coli, it has been shown that ms2i6A is important for translation of RpoS, the general stress response alternative sigma factor, which is particularly rich in UUX-Leu codons over CUX-Leu codons [S. flexneri virF contains a high proportion of UUX-Leu codons [virF translation is not known, it is noticeable that putrescine or a combination of methionine and arginine metabolically related to putrescine, restore VirF expression of S. flexneri tgt mutant [virF could occur in absence of Q. Interestingly, in E. coli polyamine auxotroph mutant, translation rate drastically decreased and concomitantly Q level was reduced [The anticodon loop (ASL) is the other hotspot for modifications, which affect the geometry and physicochemical determinants governing the decoding process ,143. Thetor VirF . Its effgenicity . In E. cu codons . Similaru codons . Althougt mutant . Since pt mutant ,154, it  reduced , suggestgidA and mnmE, encoding two enzymes of the 5-methylaminomethyl-2-thiouridine (mnm5s2U34) modification pathway, significantly reduced the colonization of Salmonella Typhimurium in liver and spleen accompanied by decreased invasion of epithelial cells and compromised ability to survive and replicate inside macrophages [invAEG, spaPQ, and prgHJ, were downregulated in the attenuated mutant strains [S. Typhimurium under the stressful conditions experienced within host macrophages [5s2U34 is also important for virulence in other bacteria. In Streptococcus species, Gram-positive bacteria responsible for a wide range of infections from skin infection to sepsis, GidA/MnmE modification enzymes are essential for acid stress and high temperature adaptation [gidA gene is leading to sensitivity to acidic conditions also in Cronobacter sakazakii, an opportunist pathogen causing neonatal meningitis, enterocolitis, septicemia, bloody diarrhea, and brain abscesses, decreasing its ability to growth in host digestive system [Mycobacterium bovis [5U present at position 34 in tRNAThrUGU under aerobic conditions should be hypermodified leading to either cmo5U or mcmo5U. This hypermodification facilitates decoding of ACG codons, which are particularly abundant in dosR mRNA [5U34 induces a classical Watson-Crick base-pairing geometry involving the wobble position, which allows better stacking between U34 and purine 35, and as a consequence increases the stability of the codon-anticodon interaction.Studies on U34 hypermodifications reveal the central role of wobble U modifications and associated enzymes in bacterial adaptation to environmental conditions and virulence in pathogens. Even if the precise mechanism remains unclear, in the absence of modifications, frameshift occurs resulting in the expression of alternate proteins ,156. Delrophages . This ef strains . In addirophages . The modaptation , for pataptation , reducedaptation . Transpoe system . One of um bovis . When myum bovis , and mycum bovis . In thisum bovis . TranslaosR mRNA . The ratosR mRNA . The prePseudomonas aeruginosa, allowing its survival during infection [P. aeruginosa [In addition to wobble position, 2′-O-methylations of A, C, and U at position 32 by methyltransferases of the Trm family, have been shown to confer resistance to oxidative stress in nfection . Hypometnfection ,171. Thiruginosa .7G46 catalyzed by tRNA guanine-N7-methyltransferase (trmB) is important for decoding efficiency of tRNAAspGUC and tRNAPheGAA. In P. aeruginosa, loss of trmB has a strong negative effect on the translation of Phe- and Asp-enriched mRNAs, including those coding the major peroxide detoxifying enzymes, the catalases KatA and KatB, resulting in oxidative stress-sensitive phenotype [Vibrio cholera revealed specific modifications in various tRNAs that were not described in E. coli tRNAs [Tyr. Although the physiological consequences of these specific features have to be defined, it is tempting to propose that RNA modifications contribute to the speciation of the bacteria and to the adaption of the organism to its specific niches.Finally, modifications of nucleotides in the variable loop contribute to host adaptation. For example, mhenotype . Using tli tRNAs . These mSmall trans-acting regulatory RNAs (sRNAs) belong to a very heterogeneous class of RNAs regulating several processes including virulence gene expression, stress adaptation, and quorum sensing . They ex5U [5U54 in tmRNA is introduced by the SAM-dependent methyltransferase TrmA as it is for the tRNAs [S. Typhimurium infection [The best examples of regulatory RNAs, where modifications have been identified, are tmRNA and Y RNA, both mimicking the tRNA structure . The tmR5U . These mhe tRNAs . Althougnfection , to our Salmonella enterica, expression of YrlA appears to be confined to certain infection stages [S. Typhimurium, rsr and Y RNA genes are located within an “RNA repair” operon including rtcA-rtcB encoding RNA cyclase and RNA ligase, respectively [ΔtruA strain missing Ψ at positions 38, 39, and 40 in the anticodon arm of some tRNAs [E. coli RtcB re-ligates a 16S rRNA 3′ fragment containing the anti-Shine-Dalgarno sequence cleaved by the MazF toxin [The non-coding Y RNAs are present in both eukaryotes and in several bacteria including some pathogens . Bacterin stages . Surprisn stages . Noteworectively . The traectively . tRNA frme tRNAs . Since tme tRNAs . IntereszF toxin . This in6A distribution in P. aeruginosa. Methylation sites were found present in two major sRNAs, RsmY and RsmZ [Another example of sRNA modification in pathogenic bacteria derives from a transcriptome-wide profiling of mand RsmZ , which sand RsmZ ,204. TheS. aureus, an opportunistic pathogen causing a large variety of infections, has been shown to contain at its 5′ end a peculiar modification which is co-transcriptionally introduced [S. aureus transcripts containing this 5′ cap, including RNAIII. In Gram-negative bacteria, NAD has been reported to have a stabilization effect protecting the RNA from the 5′ processing enzyme RppH, which produces monophosphate at the end of RNAs that are substrates of RNase E [B. subtilis, NAD protects RNAs from the exonucleolytic activity of RNase J1/J2 [S. aureus, the presence of NAD in RNAIII does not induce its stabilization nor affects its structure, but it has important consequences on pathophysiology. By a yet unknown mechanism, the NAD modified RNAIII leads to a decreased expression of δ-hemolysin and reduced cytotoxicity [Recently, one of the best characterized sRNA in troduced . The exptroduced ,207. Thitroduced . This st RNase E . In B. sse J1/J2 . In S. atoxicity .7G modification of eukaryotic mRNAs, other modifications have been described, including the 6-methyladenosine (m6A), the 5-methylcytosine (m5C), inosine (I) derived from adenine deamination and pseudouridine (Ψ). These modifications, which are present within 5′- and 3′-untranslated regions (UTRs) and the coding sequences of mRNAs, contribute to fine tune gene regulation [E. coli mRNAs and is thought to modulate mRNA stability and translation efficiency [B. subtilis spores, a dormant state developed in response to different stresses [E. coli [Besides the classical 5′-cap mgulation ,215,216.ficiency ,217. Recstresses . Its rol[E. coli . Interes[E. coli  and oxid[E. coli .E. coli, Ψs have been detected in some mRNAs. They can be located at specific codons and as a consequence they alter translation speed or mRNA decoding [6A, which has been detected in genes involved in energy metabolism in Gram-negative bacteria, including pathogenic P. aeruginosa [6A reduces both sense and stop codon reading accuracy [6A forms less stable base-pairing with uridine (U) and destabilizes local RNA structures and short duplexes [Bacterial mRNAs can also contain post-transcriptional modifications. In decoding , and at decoding ,224. Ψs decoding . Anotherruginosa . Very reaccuracy ,227. Theaccuracy . Indeed,duplexes ,229. It Arg [E. coli and in the two pathogenic bacteria Klebsiella pneumonia and Yersinia enterocolitica, recoding tyrosine (UAC) to cysteine (UGC) has been reported in mRNAs encoding self-killing toxins like HokB [hokB mRNA could promote non-genetic cell heterogeneity [Adenosine deamination is most probably the major modification in mRNAs, which can directly influence the activity of the synthesized protein. Adenosine-to-inosine (A-to-I) mRNA editing is catalyzed by the tRNA adenosine deaminase enzyme TadA, which recognizes stem-loop structures resembling the anticodon arm of tRNAArg . Becauseike HokB . The preike HokB . By doinike HokB . Since bike HokB , the hetogeneity .thrS mRNA [The review has presented several examples which unveil the diversity of functions of RNA modifications in translation and RNA degradation in bacterial pathogens. Because technologies are constantly improving, it is expected that the whole set of modifications will be mapped in RNA molecules as well as their quantifications. This will be the prerequisite to better study their dynamics upon stress or during bacterial infection. Further works correlating the modifications with the characterization of their protein co-factors  will be another step necessary to get an in-depth overview of their functions and the physiological consequences on bacterial pathogens. Many questions still remain to be addressed: what is the extent of chemical modifications diversity and complexity among evolutionary distant bacteria? What are the consequences of RNA modification defects on bacterial physiology and pathogenesis? Are there specific classes of bacterial sRNAs and mRNAs that could be modified extensively as it is for tRNAs and rRNAs? Many mRNAs carry regulatory tRNA-like elements such as found in the T-Box regulatory elements  or in thhrS mRNA . As it wSome of the data suggested that some tRNA modifications are species-specific. Hence, it is expected that modifications might contain some metabolites issued from specific metabolic pathways resulting from the bacterial adaption to its ecological niches. Meanwhile during the infection process, the pathogens have to face metabolic burden and should counteract the host defenses mechanisms. It would not be so surprising that some of the modifications might be acquired or disrupted during the infection process with consequences on the bacterial proteome. The functional studies of the modification enzymes might also lead to biotechnological applications, to design gene reprogramming or new tools for RNA studies, or to select novel anti-microbial strategies. Many of these studies will require the development of more simple technologies, such as the direct RNA sequencing methodologies, to facilitate the mapping and the discovery of RNA modifications among bacterial species. Although many studies have been done on tRNA and rRNA modifications, we are still far to fully appreciate the impact of modifications on RNA functions. We are only at the beginning of the tip of the iceberg.


Ribonucleic acids  have been emerging as a promising new class of therapeutics for bone regeneration. So far, however, research has mostly focused on stability and complexation of these oligonucleotides for systemic delivery. By comparison, delivery of RNA nanocomplexes from biomaterial carriers can facilitate a spatiotemporally controlled local delivery of osteogenic oligonucleotides. This review provides an overview of the state-of-the-art in the design of biomaterials which allow for temporal and spatial control over RNA delivery. We correlate this concept of spatiotemporally controlled RNA delivery to the most relevant events that govern bone regeneration to evaluate to which extent tuning of release kinetics is required. In addition, inspired by the physiological principles of bone regeneration, potential new RNA targets are presented. Finally, considerations for clinical translation and upscaled production are summarized to stimulate the design of clinically relevant RNA-releasing biomaterials. HoweverIn 1965, Marshall R. Urist demonstrated that demineralized bone matrix could stimulate bone growth in ectopic sites . Ever si1.1To avoid loss of bioactivity, safety issues and excessive production costs associated with the large amounts of proteinaceous growth factors that are required because of rapid degradation , gene thOnce in the cytosol, RNAs act by forming complexes with endogenous proteins . siRNA ain vitro synthesis of recombinant proteins in heterologous expression systems, the mRNA-based approach assures correct post-translational modification of proteins, which are often highly challenging to recapitulate during in vitro synthesis , d miRNA) .3)RNA delivery kinetics: Similarly to growth factors, RNA should be delivered in a biologically meaningful time frame, that is, the duration of release and amount of delivered RNA should match the bone healing process as closely as possible to attain optimal therapeutic efficacy [efficacy . The reqefficacy . DeliverSuccessful delivery of RNA requires three key aspects:4.1Based on the current literature on biomaterial-based RNA delivery , three m4.1.1Diffusional post-loading is achieved by impregnating or soaking a pre-made biomaterial after fabrication with an RNA complex-containing solution. Depending on the nanoscale porosity of the biomaterial, the RNA complexes will either penetrate the biomaterial or adsorb onto its surface. This simple loading method has been used for a variety of biomaterials, including metals , polymerin vitro.For example, Huang et al.  soaked aMetridia luciferase (MetLuc) mRNA over 5 days.In contrast, a more prolonged release using diffusional post-loading was achieved by Balmayor et al., who adsorbed mRNA lipoplexes to porous biphasic calcium phosphate (BCP) granules followed by lyophilization. After 24 h, 42% of loaded mRNA was released from the BCP granules, and release was sustained for 7 days. The BCP granules contained 60% hydroxyapatite (HA), a material which has also been used for complexation of RNA . This maIn general, it can be concluded that diffusional post-loading is the simplest loading strategy which enables efficient loading of mRNA onto biomaterials. However, release kinetics are difficult to control because the RNA complexes are associated with the biomaterial rather weakly by non-covalent interactions, resulting in burst-type release patterns and limited control over long-term delivery kinetics.4.1.2Incorporation of RNA complexes into biomaterials can be achieved by adding RNA complexes to the biomaterial before the formation of the final biomaterial matrix. This approach is most often adopted for hydrogels, where the RNA complexes are mixed into the polymer solution before cross-linking the gel. So far, most studies investigated hydrogels made of natural ,97,98 orSteinle et al.  used hydin situ gelation, and the authors demonstrated injectability of the gel through a 23G needle (inner diameter ca. 0.4 mm), albeit without specification of the temperature. Their system showed a relatively fast delivery under physiological conditions (37°C) since 80% of siRNA complexes were released after 3 days. Considering the relatively constant release between the various time points, the authors suggested that the release was governed by hydrogel degradation rather than by diffusion of siRNA complexes.Synthetic polymers offer the specific opportunity to tune release rates by modifying polyplex behavior or network formation characteristics of the hydrogel matrix. Fliervoet et al.  used triSummarizing, incorporation of RNA within a biomaterial matrix can improve control over RNA delivery kinetics. Sustained delivery of RNA has been shown for up to 21 days. Depending on the hydrogel mesh and size of the RNA complex, the release of incorporated RNA complexes is governed by diffusion, hydrogel swelling and/or network degradation. Polymer concentration, molecular weight (MW), cross-linking density, and resultant mesh size are the main factors influencing the release kinetics ,108. If 4.1.3The third main loading strategy involves covalent attachment of RNA complexes to biomaterials after their fabrication using linker molecules. Although this approach theoretically allows for improved control over RNA release kinetics compared to diffusional postloading or incorporation, this loading method requires additional modification steps, such as the synthesis of suitable linker molecules. So far, only two groups have investigated covalent binding for biomaterial-based RNA delivery.Nguyen et al.  used a dco-glycolic acid) (PLGA) films and siRNA complexed with lipidoids. Less than 5% of siRNA was released from the PLGA films after 21 days, illustrating the strong retention capacity enabled by covalent catechol-amine interactions between catechol groups of polydopamine and amines of the siRNA-lipidoids. Interestingly, successful cellular transfection was already observed after 2 days of culture. Moreover, transfection efficacy of substrate-bound siRNA-lipidoids was higher compared to suspended siRNA-lipidoids , which was attributed to increased cell-siRNA contact. These results also demonstrate that very high effective siRNA concentrations can be achieved through local immobilization.Shin et al.  aimed atGenerally, for covalently linked systems, the RNA release rate is defined by the cleavage rate of the linkage . Surpris4.2Biomaterial matrix degradation is an established tool to modulate release kinetics for any sort of cargo. Consequently, biomaterial degradation mechanisms have been extensively studied and excellent reviews on this topic are available elsewhere ,108,117.Generally, degradation of biomaterials can either proceed heterogeneously from outside to inside or homogeneously throughout the entire bulk material. The biomaterial permeability for the degrading agent  is strongly determining the nature of the degradation mechanism. If this permeability is limited, biomaterials will degrade mainly at the edges, resulting in surface erosion ,108. Dur4.2.1RNA delivery from hydrogels is often enabled by incorporation of the RNA complexes into the biomaterial -degradable cross-linkers for on-demand RNA delivery . The rel4.2.2The degradation of calcium phosphates occurs via either passive chemical dissolution and/or active cell–mediated resorption . IntrinsAlthough calcium phosphate–based materials are widely used in clinical practice, only two studies have tested RNA delivery from these materials so far ,107. Utz4.34.3.1RNA complexes differ in size, surface charge and functional groups depending on the complexation agent used and the ratio between RNA and complexation agent (N/P ratio). The surface charge of RNA complexes and resulting electrostatic interactions with the biomaterial are particularly important in case of diffusional post-loading which depends on electrostatic interactions between RNA complexes and the biomaterial. In case of large RNA complexes or dense biomaterials, the complexes can only be retained at the biomaterial surface ,126. Usu4.3.2Upon incorporation of RNA complexes into biomaterials, release kinetics are modulated by the size of RNA complexes relative to the porosity or mesh size of the biomaterial as well as electrostatic interactions. Carthew et al.  investigAs illustrated previously, size and surface charge both play an important role in modulating the release rate of RNA complexes. However, as both parameters usually vary for different complexation agents, the respective contribution of either one is hard to determine. Also, relevant information may be missing since studies on biomaterial-based RNA delivery often do not report RNA complex characteristics.4.3.3Covalent bonding of RNA to a biomaterial is a very strong—and often irreversible—RNA-biomaterial interaction. However, chemical modification of RNA may impede its cellular internalization, as illustrated by Nguyen et al. , who covAlthough the delivery of covalently linked RNA is mostly governed by the cleavage rate of covalent bonds, the release rate may again also be influenced by complex charge and size through electrostatic interactions and/or hindered diffusion. Importantly, release control via covalent binding requires that biomaterial degradation proceeds slower than bonds are cleaved since release kinetics might otherwise become influenced by matrix degradation as well.4.4A specific requirement for RNA loading through incorporation into biomaterials involves the chemical compatibility between RNA complexes and the biomaterial matrix (except for naked siRNA). RNA complexes are typically formed through charge-driven non-covalent interaction of a positively charged complexation agent with the negatively charged oligonucleotide . Evident4.5in vivo is confronted with the general challenge that any RNA complexes released into the blood stream will, to a major extent, be sequestered by the reticuloendothelial systems and thus accumulate in the liver. The incorporation of RNA complexes into locally applied biomaterials therefore offers the advantage that also the delivery of RNA is local. Ideally, only so much RNA complexes are released at a given point in time as can be locally taken up from cells in the immediate environment so that systemic exposure to the mRNA complexes is minimal.Delivery of RNA in vitro experiments do not reflect the complexity of the physiological tissue environment and do not account for the effects of mechanical loading, perfusion, tissue mobility, and the presence of different cell types.So far, studies on RNA delivery from biomaterials have mostly been conducted in acellular conditions, especially when investigating release kinetics. Moreover, these quantifications of release kinetics are usually performed in simple phosphate-buffered saline (PBS). Although such acellular experiments provide a first impression of release kinetics, possible interactions with serum proteins and the extracellular matrix cannot be assessed. Similarly, traditional 2D in vitro models can recreate the physiological cell microenvironment more closely to provide more relevant biological information on cell matrix and cell–cell interactions [in vitro models, especially for RNA delivery from flexible biomaterials such as hydrogels.Three-dimensional (3D) ractions  and theractions  used a 3in vivo, but so far, only endpoint measurements  have been reported instead of longitudinal monitoring of the release and distribution of RNA within tissues [in vivo, a major challenge relates to the penetration depth of the modality used to detect the labeled nanocomplexes in deep tissue layers. Bioluminescence and radiolabeling are potential options, although the latter labeling modality might negatively affect the biological activity of RNA through base mutation. Alternatively, conventional fluorescent labels have been used successfully by Wang et al. [in vivo.Several studies have investigated RNA delivery kinetics  tissues ,103,111.g et al.  to invesin vivo but are not accounted for in current in vitro settings. Moreover, current in vivo tests mainly focus on demonstrating therapeutic efficacy rather than unraveling underlying mechanisms of RNA release and biodistribution.In summary, RNA delivery from biomaterials has mostly been studies in models which, by far, do not represent the complexity of the native tissue. Aspects such as perfusion, movement, interactions with extracellular matrix and cell type diversity may have a significant impact on RNA delivery 5Problems related to local delivery of osteogenic GFs (mainly BMP-2) and clinical translation of delivery of osteogenic genes have prompted researcher to explore the opportunity of RNA therapy  for stimulation of bone healing. RNA delivery offers the possibility to transiently express any protein (including intracellular and transmembrane proteins) without the need for genomic integration by using the endogenous protein machinery of the cell. By providing a brief overview of the various stages in bone healing Section  that canin situ hardening materials are the most promising biomaterial candidates. These biomaterials allow for minimally invasive application , which may reduce tissue damage, risk of infection and ultimately help to minimize the health burden for the patient. In this respect, retention of injectable materials at the injection site is crucial. Therefore, future studies should investigate in vivo biomaterial integrity, both directly after injection as well as on the long term after days to weeks of in situ delivery to avoid premature leakage of RNA complexes. To this end, self-healing biomaterials offer the opportunity to maintain biomaterial integrity even after destructive shearing upon injection through narrow needles.Biomaterial-based RNA delivery to stimulate bone healing is still in its exploratory phase. So far, the importance of spatial and temporal control of RNA-based therapeutics from biomaterials is hardly studied. To enable for spatial control over RNA delivery, injectable and Regarding temporal control of RNA-delivering biomaterials, it should be stressed that the optimal kinetics and durations of both biomaterial degradation and drug delivery are not yet known; optimal RNA doses remain to be determined as well. Therefore, dose–response studies, as commonly performed in pharmaceutical research, are required to deepen our understanding of biomaterial-induced RNA delivery.Current biomaterials for RNA delivery do by far not reflect the complexity of the bone healing process. In addition, in vitro conditions used for testing the delivery kinetics of RNA therapeutics from biomaterials differ strongly from the complex, dynamic in vivo environment which is characterized by a highly orchestrated interplay of cells and mechanical and biochemical signals. Furthermore, various processing parameters of biomaterials are mutually dependent, which complicates optimization of these parameters considerably. Therefore, advanced design strategies such as Design of Experiments and computational modeling may help to capture the intrinsic complexity of these systems. In the future, biomaterial-based RNA delivery should be studied in 2D/3D models under dynamic conditions, while longitudinal in vivo monitoring of spatiotemporal delivery kinetics should provide insight into the effect of delivery kinetics of RNA complexes on in vivo bone healing responses. In addition, advanced technologies may allow to better mimic the temporal sequence of factors expressed during bone healing by making use of on-demand release systems and/or co-delivery of multiple biomolecules to create physiological feedback loops.RNA therapy is still in its infancy with only few RNA-therapeutic approved for clinical use. To facilitate clinical translation, preclinical testing should provide proof-of-concepts in clinically relevant  models and their alternatives. Importantly, regulatory compliant upscaling of sterilizable RNA-loaded biomaterials is required to enable clinical translation of RNA-delivering biomaterials with unsurpassed regenerative capacity.10.13039/501100003246Netherlands Organization for Scientific Research  for funding this research.The authors would like to thank The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.