Post-translational modifications of proteins play very important roles in regulating RNA metabolism and affect many biological pathways. Here we mainly summarize the crucial functions of small ubiquitin-like modifier (SUMO) modification in RNA metabolism including transcription, splicing, tailing, stability and modification, as well as its impact on the biogenesis and function of microRNA (miRNA) in particular. This review also highlights the current knowledge about SUMOylation regulation in RNA metabolism involved in many cellular processes such as cell proliferation and apoptosis, which is closely related to tumorigenesis and cancer progression. Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-like post-translational modifiers are well conserved in eukaryotes . Up to nSUMOylation is a reversible process of adding SUMO to substrate proteins at specific lysine residues as a post-translational modification (PTM) . In mammvia an isopeptide bond between its C-terminal glycine G) and the ε-NH2 group of a lysine residue in the target protein  residues of target proteins  protein . SUMOyla protein . The fir protein . Second, protein . The SUM protein .In general, the most common target proteins of SUMO modification are to have a common motif ψ-K-X-E/D , but there are also some proteins without classical motifs that have been more reported recently . In usuaImportantly, SUMOylation plays an important role in RNA metabolism, including messenger RNA (mRNA) and non-coding RNA (ncRNA). Abnormal RNA metabolism caused by SUMOylation is often the main reason affecting the occurrence and development of tumors. On the one hand, mRNA metabolism regulated by SUMOylation involves in transcription, processing , mRNA translation and quality control. On the other hand, SUMOylation of regulation on ncRNA metabolism is mostly and broadly focused on microRNAs (miRNAs). In addition, researches on cellular processes compartmentalized by membrane-less organelles (MLOs) have become a hotspot. MLOs typically contain disordered proteins and RNAs by liquid-liquid phase separation (LLPS) . MLOs foThis review provides insights into the role of SUMOylation in regulating RNA metabolism in the pathophysiology of diseases, especially cancers. We summarize the regulation of RNA metabolism by SUMOylation from the following aspects: 1) RNA transcription; 2) mRNA processing; 3) RNA stability; 4) RNA editing and RNA modification; 5) Non-coding RNA metabolism.via SUMO conjugating to histones, histone-modifying enzymes, transcription factors, transcriptional co-regulators and transcriptional machinery-related proteins. The mechanisms underlying SUMO-mediated transcriptional regulation includes alterations in subcellular localization, DNA binding, protein-protein interaction, stability and enzymatic activity  such as CREB-binding protein (CBP) and p300 while it is removed by histone deacetylase (HDACs). CBP and p300 are co-regulators of many sequence-specific DNA binding proteins in response to a variety of signaling , and areOn the other hand, HDACs reverse chromatin acetylation by removing acetyl groups and alter the transcription of oncogenes and tumor suppressor genes governing cancer initiation and progression . HDACs fA wide range of transcription factors and co-transcriptional regulators have been reported as SUMOylated proteins. In most cases, SUMOylation is usually associated with transcriptional repression . Here, wAlthough SUMOylation of most transcription factors and transcriptional co-regulators plays a negative role in transcription, it also activates several transcription factors, such as tumor suppressor p53 , the G2/There are also some evidences show that SUMOylation of transcriptional machinery-related proteins can promote transcriptional activation. SUMO1 marks the chromatin on the promoters of many housekeeping genes, which encode translation factors and ribosomal subunit proteins, to promote the transcription of these specific RNAs . The C-tThe transcription profiling reveals that SUMOylation represses the global transcription by inhibiting transcriptional elongation. SUMOs and MYC oppositely control global gene expression by regulating the dynamic SUMOylation and deSUMOylation of CDK9. CDK9 is the catalytic subunit of the P-TEFb kinase and is essential for productive transcriptional elongation. SUMOylation of CDK9 represses global transcription, while MYC amplifies global transcription by antagonizing CDK9 SUMOylation  Figure . The oncIn response to stresses, the transcription of housekeeping genes is usually downregulated in cells. Stress-induced SUMOylation of negative elongation factor (NELF) is required for global transcriptional repression by promoting the binding of NELF to the promoters, which impairs RNAPII elongation. NELF forms nuclear condensations in a dephosphorylation-dependent and SUMOylation-dependent manner under stressful conditions. Biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation  Figure . RNA polMost mRNA precursors (pre-mRNAs) undergo three processing steps: the 5′end is capped by addition of 7-methylguanosine; introns are removed and exons ligated by splicing; and the 3′end is created by an endonucleolytic cleavage followed by addition of a 100–300 nt long poly(A) tail. The evidence linking SUMOylation with the process of RNA capping, splicing and tailing is majorly based upon proteomic analysis . In the in vitro and in cultured cells, including components of U1, U2, U4/U6, U5 snRNPs, non-snRNPs, heterogeneous nuclear ribonucleoproteins (hnRNPs) as well as a wide range of other spliceosome-associated factors, suggesting that SUMOylation may play a role in multiple steps of pre-mRNA splicing . Each snRNP consists of a small nuclear RNA (snRNA) and a large set of associated proteins. In the earliest cross-intron spliceosomal complex, U1 and U2 snRNP bound to the intron to form A complex. Then, the activated spliceosome B Complex generated by the recruitment of U4/U6/U5 tri-snRNP. After numerous RNA and protein rearrangements, including the dissociation of the U1 and U4 snRNPs, and splicing by the DEAH-box RNA helicase PRP2, the catalytic C complex is finally yielded. The C complex catalyzes intron excision and ligation of the exons followed by spliceosome disassembly . Severalsplicing .Serine/arginine (SR) proteins, such as members of the hnRNP family and many splicing factors, are highly SUMOylated in response to various stresses . The SR SAFB, which interacts with the CTD of RNAPII and RNA processing proteins such as SR proteins as mentioned before, is identified as an SUMO1 substrate binding to promoters of highly expressing genes. SUMOylation of SAFB also increases splicing rates of mRNAs encoding ribosomal proteins, suggesting a role for SUMOylated-SAFB in coupling transcription and RNA processing . SAFB isIn vitro polyadenylation assays show that SUMOylated PAP has lower poly(A) synthesis activity, indicating that SUMOylation of PAP inhibits polyadenylation activity to profoundly affect gene expression , CFII, CPSF (cleavage and polyadenylation specificity factor) and CstF (cleavage stimulation factor) and additional protein factors . The 3′epression . Dysfuncpression . Converspression . Thus, rgadd45α mRNA stability, which in turn increases apoptosis under arsenic exposure  is one of the most common modifications in mRNA, rRNA, tRNA, microRNA and long non-coding RNA  rapidly form thus transiently storing mRNAs and RNA-binding proteins (RBPs) to limit protein synthesis. SGs are associated with various diseases including cancers, viral infections and neurodegenerative diseases . It has 6A is the most prevalent internal RNA modification in eukaryotes. It is a dynamic and reversible modification that occurs primarily in 3′untranslated regions (3′UTRs) and near the stop codons  is another important posttranscriptional modification of RNA. The modification m5C in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2 and specifically recognized by the mRNA export adaptor ALYREF . NSUN2 iADAR1, an RNA-editing enzyme also undergo SUMOylation to regulate RNA metabolism. ADAR1 binds to double-stranded RNA and converts adenosine to inosine, resulting in changes in the amino acid coding that alter protein sequence/function . ADAR1 i.Non-coding RNAs (ncRNAs) are a class of RNA molecules that do not encode proteins but can function except mRNA, tRNA and rRNA. miRNAs are small non-coding regulatory RNAs ranging from 20–25 nucleotides, which binding to partially complementary sequences within the 3′-untranslated regions of target mRNAs inducing translational repression or mRNA degradation . miRNAs .). The hnRNP K homology (KH)-type splicing regulatory protein (KHSRP) is one major component of the DROSHA complex. KHSRP can promote the biogenesis of miRNAs, such as let-7 family, whose pri-miRNAs harboring short G-rich stretches in their terminal loops (TL). SUMO1 modification of KHSRP at K87 can be induced upon the microenvironmental hypoxia while reduced by the treatment with growth factors. SUMOylation of KHSRP probably increases its translocation from the nucleus to the cytoplasm, thus inhibiting its interaction with the pri-miRNA/DROSHA-DGCR8 complex, therefore SUMOylation of KHSRP results in the downregulation of TL-G-Rich miRNAs such as let-7 family, which is linked to tumorigenesis and cancer progression  (Primary miRNAs (pri-miRNAs) are typically transcribed by RNA polymerase II in the nucleus , and thegression   by another RNase III-type enzyme, DICER with its co-factors TRBP , resulting in an RNA duplex of ∼22 nucleotides. The short RNA duplex is bound by an AGO (Argonaute) protein, a component of a multi-subunit complex termed miRISC (miRNA-induced silencing complex) . Finallyigration   are known as important tumor suppressors, which are downregulated in multiple cancers, and associated with increased proliferation and invasion of cancer cells. LIN28 A is a conserved RNA-binding protein that inhibits processing of pre-let-7 miRNAs, thus promoting cancer progression. Our lab showed that LIN28 A is SUMOylated at K15, which is increased by hypoxia but reduced by chemotherapy drugs such as Cisplatin and Paclitaxel. SUMOylation of LIN28 A increases its binding affinity with the pre-let-7 and recruits of terminal uridylyltransferase TUT4 to block processing of pre-let-7. Subsequently, SUMOylation of LIN28 A exacerbates its inhibition of let-7 maturation, thereby reducing mature let-7 production. These effects promote cancer cell proliferation, migration, invasion and tumor growth in DU145 and T47D cell lines  Dou et .Drosophila have demonstrated the role of SUMOylation in primary piRNA biogenesis  and piRNA-guided transcriptional silencing and heterochromatin formation  and long non-coding RNAs (lncRNAs) are also influenced by SUMOylation. Several studies in ormation . Moreoveelopment . HoweverELNAT1 promotes lymphangiogenesis and lymph node (LN) metastasis in bladder cancer (BCa) cell lines by inducing Ubc9 overexpression to catalyze SUMOylation of hnRNPA1 (PSTAR) can bind to hnRNP K and enhance its SUMOylation, thereby strengthening the interaction between hnRNP K and p53, ultimately resulting in the accumulation and transactivation of p53 to suppress hepatocellular carcinoma  can regulate the SUMOylation levels of NAD-dependent protein deacetylase sirtuin-1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α)/peroxisome proliferator-activated receptor-α (PPARα), specificity protein 1 (SP1)/HDAC4 in cardiac hypertrophy . LncRNA  hnRNPA1 . LncRNA  hnRNPA1 . LncRNA  hnRNPA1 . Oncogen hnRNPA1 . LncRNA  hnRNPA1 . LncRNA  hnRNPA1 . LncRNA arcinoma . Interesarcinoma . In addiarcinoma . These fIn the past decade, SUMOylation research has found a large set of SUMO target proteins, which regulate the different stages of RNA metabolism. In this article, we summarize the regulatory roles of SUMOylation in RNA metabolism in cancer cells. A tight control of mRNA metabolism is crucial to allow cells in response to physiological stresses. SUMO proteome with key proteins/enzymes of mRNA transcription, splicing or nuclear complex is subject to spatio-temporal regulation and cellular stress remodeling to control mRNA metabolism. Since most of SUMOylation substrate proteins are localized in the nucleus, the effect of SUMOylation is mainly to inhibit the global transcription activity. When SUMOylation sites are located within the inhibitory or repressive domains, mutations of these sites can dramatically enhance the transcriptional activity.MiRNA biogenesis is closely regulated by SUMOylation, thus affecting tumorigenesis and cancer progression. SUMOylation has no uniform effect on miRNA biogenesis process and miRNA-induced RNA silencing efficiency. Considering that the activity and abundance of core proteins and their partners in different stages of miRNA biogenesis are not consistent in different cancers, there is no comprehensive understanding and definite conclusion on how SUMO specifically affects miRNA metabolism in specific types of cancer. Moreover, a lot of lncRNAs mediate SUMOylation of target proteins, which plays complex and precise regulatory roles in cancer progression. However, the role of SUMOylation in the transcription, processing, stability and functions of lncRNAs is limited and unclear. Finally, understanding the impact of SUMOylation on RNA metabolism may provide new therapeutic strategies for cancer treatment.


I2, with unit-cell parameters a = 95.70, b = 79.60, c = 117.44 Å, β = 95.57°. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1. In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 8HKW).53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group  Specifications Table•The significance of importin-α oligomerization has been debated in nuclear transport field. The data reported here extends our knowledge on importin-α oligomerization and will benefit researchers studying the structure and function of nuclear transport receptors.•The purification procedure, crystallization condition, and a photograph of the crystal used in this work serve as a useful reference for researchers interested in structural study of importin-α-cargo complexes.•Although the quaternary structure reported here could be an artifact of the crystallization process, the data enriches structural database of what can happen during crystallization of protein-peptide complexes, and will benefit researchers interested in protein crystallization.•The molecular mechanism of interactions between nucleoporins and nuclear transport receptors is not fully understood, and remains an important issue in nuclear transport field. The data that convincing electron density corresponding to Nup153 was not observed in the crystal structure reported here is a negative data useful for researchers studying nucleoporin-importin interactions.1The initial objective of this work was to gain structural insights into how Nup153 interacts with importin-α and promotes nuclear import of NLS-containing proteins such as 53BP1. However, this work serendipitously led to a discovery of a novel quaternary structure of importin-α-NLS complexes that can be formed at least in the crystal.2X-ray diffraction dataset up to 1.9 Å resolution was collected from a single crystal of the ARM-repeat domain of human importin-α3 bound to the bipartite NLS of 53BP1 -tagged 53BP1 NLS  were co-expressed in the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene) at 20°C from pRSFDuet-1 (Novagen) and pGEX-TEV To prepare the ARM-repeat domain of importin-α3 in complex with the 53BP1 NLS, untagged importin-α3  and glutathione 3.21459GTSFSGRKIKTAVRRRK1475) was synthesized by GenScript. A plate-shaped crystal was obtained using hanging drop vapor diffusion at 20 °C with 1.5 μl of a protein solution (0.58 mM importin-α3–53BP1 NLS complex and 0.71 mM Nup153C in buffer C) mixed with 1.5 μl of mother liquor (0.2 M LiNO3 and 25% PEG3350) over a reservoir of 600 μl of mother liquor. The crystal was briefly soaked in a cryoprotection solution  for less than 30 s, and flash-cooled in liquid nitrogen. X-ray diffraction datasets were collected at 95 K at the Photon Factory beamline BL-17A using an EIGER X 16 M detector at a wavelength of 0.98 Å. Diffraction data were processed using iMOSFLM and CCP4 programs A synthetic peptide (referred to as Nup153C) corresponding to the extreme C-terminus (residues 1459–1475) of human Nup153 (This work meets the ethical requirements for publication in this journal. This work does not involve human subjects, animal experiments, or any data collected from social media.Yoshiyuki Matsuura: Conceptualization, Investigation, Writing – Original Draft, Funding acquisition.Yoshiyuki Matsuura: Conceptualization, Investigation, Writing – original draft, Funding acquisition.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. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


The most differentially abundant transcripts were enriched in mRNAs encoded by the mitochondrial genome. We did not detect any differences in the plasma RNA profile between patients who developed HF compared with those who did not. In summary, we show that mRNAs, lncRNAs and circular RNAs can be reliably detected in plasma by deep RNA-Seq. Multiple coding and non-coding transcripts were altered in association with CAD, including several mitochondrial mRNAs, which may indicate underlying myocardial ischaemia and oxidative stress. If validated, circulating levels of these transcripts could potentially be used to help identify asymptomatic individuals with established CAD prior to an acute coronary event.Advances in RNA sequencing (RNA-Seq) have facilitated transcriptomic analysis of plasma for the discovery of new diagnostic and prognostic markers for disease. We aimed to develop a short-read RNA-Seq protocol to detect mRNAs, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in plasma for the discovery of novel markers for coronary artery disease (CAD) and heart failure (HF). Circulating cell-free RNA from 59 patients with stable CAD  and 30 controls was sequenced to a median depth of 108 paired reads per sample. We identified fragments from 3986 messenger RNAs (mRNAs), 164 long non-coding RNAs (lncRNAs), 405 putative novel lncRNAs and 227 circular RNAs in plasma. Circulating levels of 160 mRNAs, 10 lncRNAs and 2 putative novel lncRNAs were altered in patients compared with controls (absolute fold change >1.2,  Coronary artery disease (CAD) is a major cause of death worldwide . The proAdvances in RNA sequencing (RNA-Seq) technologies have facilitated transcriptomic analysis of plasma for the discovery of new diagnostic and prognostic markers for disease. RNA molecules are released into the circulation from healthy, apoptotic and necrotic cells and may provide insight into the health status of solid tissues that cannot be readily biopsied . CirculaH19 [CoroMarker [SMILR [HOTAIR [LIPCAR [H19, CoroMarker and LIPCAR have been associated with the presence of CAD [LIPCAR have also been associated with increased risk of cardiovascular mortality in patients with HF [SMILR have been associated with atherosclerotic plaque instability and inflammation [HOTAIR appears to be protective of cardiomyocytes, and lower circulating levels have been associated with acute myocardial infarction [To date, approximately 65 lncRNAs have been associated with CAD or HF ,11,12,15H19 ,19, CororoMarker , SMILR [r [SMILR , HOTAIR  [HOTAIR  and LIPC [LIPCAR  are notae of CAD ,19,20,23ammation . In contfarction .To extend the list of candidate biomarkers for CAD and HF, this study aimed to develop a short-read RNA-Seq protocol to detect mRNAs, lncRNAs and circRNAs (including putative novel lncRNAs) in human plasma. This protocol was applied to plasma from patients with established stable CAD (samples collected ~4 months after an acute coronary event) and healthy controls to screen for candidate mRNA, lncRNA and circRNA biomarkers associated with the presence of coronary artery disease and the progression from CAD to HF. We hypothesised that circulating levels of non-coding RNAs in patients with stable CAD may reflect progression of atherosclerotic disease or adverse myocardial remodeling and may help identify patients at risk of future cardiovascular events. Here, we demonstrate that mRNAs, lncRNAs and circRNAs can be reliably detected in human plasma by deep RNA-Seq, and we report novel candidate biomarkers for the presence of CAD.n = 2140) admitted to either Christchurch Hospital or Auckland City Hospital, New Zealand, were recruited into the Coronary Disease Cohort Study (CDCS) [From July 2002, patients (y (CDCS) . Inclusin = 3358) [Volunteers randomly selected from the Canterbury, New Zealand electoral rolls and age- and sex-matched to existing Christchurch Heart Institute acute coronary syndromes, MI and HF patient cohorts were recruited into the Canterbury Healthy Volunteers Cohort between 2003 and 2013 ( = 3358) . ParticiBlood was taken from an indwelling intravenous cannula placed 30 min prior to sampling, with the patient remaining semi-recumbent. Peripheral whole blood was collected into EDTA tubes and centrifuged within 30 min of collection at 4 °C. Plasma was stored at −80 °C. de novo HF within 3 years of their index hospital admission and had at least 5 mL plasma available  and a matched group of patients who remained free of HF over a median 5.0 years follow-up . HF− patients and controls (n = 31) were matched to the nearest HF+ group matched for age, sex, ethnicity, body mass index, smoking status and activity. In addition HF− patients were matched to the nearest HF+ group matched for heart rate, LVEF, E/e’, creatinine, previous medical history , severity of disease  and medications . Matching was performed with the MatchIt package  in R [2ust 2018 . Details of RNA extraction and quantitation are provided in the ® Stranded Total RNA-Seq Kit v2–Pico Input Mammalian . All libraries were normalised and equimolarly pooled before being paired-end sequenced on an Illumina NovaSeq 6000 S1 flowcell using v1.5 chemistry, generating ~100 million reads per sample at 2 × 100 bp read length. Library preparation was performed by the Otago Genomics Facility , and sequencing was performed by the Ramaciotti Centre for Genomics . Details of total RNA-sequencing library preparation are provided in the https://github.com/zoeward-nz/PhD, accessed on 20 August 2018. Briefly, libraries were de-multiplexed, adapter- and quality-trimmed using Trimmomatic [The bioinformatics pipeline was designed to detect mRNAs, lncRNAs and circRNAs and generate data on putative novel lncRNA and circRNA transcripts  are summarised in p = 0.666). The median number of uniquely aligned reads was 22 million reads per sample (19%), ranging from 3.4 to 64 million reads per sample (3.3–73.9%). Clinical characteristics of patients ~4 months after index admission (when patients were stable) and controls are shown in RNA-Seq generated a median of 108 million reads per sample. Principal component analysis identified three outlying samples . While there was no correlation between sample storage time and total read depth , sample storage time was weakly negatively correlated with the number of uniquely mapped reads per sample , suggesting that RNA integrity in stored plasma declines over time. However, the number of uniquely aligned reads did not differ between patients and controls , suggesting that the difference in sample storage time between patients and controls would be unlikely to adversely influence our findings.Plasma sample storage time was approximately 5 years longer for patients compared with controls  overlapped (n = 7) or were in very close proximity  to CCCTC-binding factor (CTCF) transcription factor binding sites, suggesting they may have a potential gene regulatory role. No transcripts were found to be differentially expressed between HF+ and HF− groups.A total of 405 multi-exonic putative novel lncRNAs were detected in plasma . Putativp < 0.01 after adjustment for multiple comparisons), although two circRNAs were borderline significant , these transcripts could potentially be used as markers to help identify individuals at impending risk of an acute coronary event.Sequencing total RNA from human plasma is technically challenging due to the low abundance of RNA, with only a few studies reported to date ,16,17,40Among the 20 mRNA transcripts most strongly associated with the presence of CAD, 13 originated from the mitochondrial genome. In patients with CAD, myocardial ischaemia and oxidative stress can promote mitochondrial apoptosis of cardiomyocytes, leading to release of damage-associated molecular patterns into the circulation, including cell-free mitochondrial RNA . This isH19 [CoroMarker [SMILR [HOTAIR [LIPCAR [H19 fragments was 1.4-fold higher in patients than controls, and although this did not reach statistical significance, this fold-change is consistent with previous findings [CoroMarker, SMILR, HOTAIR, LIPCAR and other circulating lncRNAs previously associated with CAD, possibly due to their low abundance. Relatively few of the lncRNAs previously associated with CAD or HF  and ranked among the top 10 most differentially abundant lncRNAs. While our findings support a potential role for RMRP in stable CAD, the direction of association in our study contrasted to that previously observed in the human left ventricle, where patients with ischaemic heart failure had significantly higher expression of RMRP compared to controls [RMRP into plasma, leading to higher levels in tissue and lower levels in plasma compared to controls. Alternatively, lack of concordance between the two studies might also reflect differences in the pathology of CAD and HF or the timing of sample collection in the disease course. In contrast to RMRP, we did not detect differences in the abundance of the other lncRNAs previously associated with CAD or HF  between patients and controls. While these lncRNAs may have important roles in the pathophysiology of CAD and HF and may be potential therapeutic targets, our findings suggest they are unlikely to have strong potential as biomarkers for stable CAD. In addition to es . controls . The appNotably, the majority of lncRNAs associated with the presence of CAD in our study overlapped or were in close proximity to CTCF binding sites, suggesting a potential regulatory function. CTCF is a transcription factor that, along with the cohesion complex, creates large loop domains within nuclear DNA called topologically associated domains. DNA contacts within each topologically associated domain are strong enabling enhancers and promoters to be brought into close contact to regulate gene expression . Severalp-adjusted <0.01, our finding that circUBAC2 ranked as one of the most promising candidates supports a previous report that showed higher circulating levels of circUBAC2 patients with myocardial infarction compared to healthy controls and, when combined with four other circRNAs, had good sensitivity and specificity for the diagnosis of myocardial infarction [circUBAC2 may also be elevated in patients with stable CAD. To date, very few studies have investigated the plasma ‘circRNAome’ using RNA-Seq . CircRNAfarction . Our finOur study has three major limitations. First, the relatively small sample size limited our power to detect differences in the circulating RNA profile between groups. Consequently, we may have missed some important associations with CAD, and we were unable to detect associations with subsequent HF. This may have been compounded by the close matching between patients who subsequently developed HF and those who did not. Second, we cannot completely exclude the possibility that our findings may be confounded by the effects of medications or sample storage time. Third, we have been unable to identify a large, well-characterised cohort of patients with blood sampling ~4 months after an acute coronary event in which to validate our findings. Consequently, further work is needed to confirm the specificity of our findings for CAD and establish their potential clinical utility. In summary, we have demonstrated that mRNAs, lncRNAs and circRNAs can be reliably detected in human plasma by deep RNA-Seq and have identified several promising candidate mRNA and lncRNA biomarkers for the presence of stable CAD. While RNA-Seq in human plasma is technically challenging due to the low abundance of RNA, this can be overcome by ultra-deep sequencing (>100 million paired reads per sample). In future, advances in library preparation kits, enrichment techniques and sequencing technologies will improve the detection of cell-free RNA in plasma and facilitate widespread use of RNA-Seq for the analysis of the plasma transcriptome.


Molecular Therapy Nucleic Acids has addressed a major question of whether artificial circular RNAs (circRNAs) escape immune-surveillance or not.in vitro circularization. Transcriptome analysis was performed after transfecting A549 cells with circRNAs in order to examine innate immune system activation. Findings revealed minimal innate immunity activation (observed only after treatment with high doses of the circRNAs) as well as no transcriptional up-regulation of RNA sensors. On the contrary, circRNAs-ds not only up-regulate genes involved in innate immune responses but also up-regulate PKR, a key RNA sensor. The authors proposed that factors like secondary-structure elements, sequence composition, or liposome-based transfection may play a role in the induction of the innate immune response apart from contaminants. For the exclusion of contaminants of transcription reaction, stringent purification was performed via gel extraction. The authors concluded that a polyacrylamide-urea gel purification approach appears to be the most efficient method for the purification of circRNAs. In vitro synthesis of immunogenicity-free circRNAs have potential clinical relevance and biomedical applications. This study will make a significant contribution in the field of RNA therapeutics by providing the scientific community with a strategy for reducing the immunogenicity of produced RNA circles. One of the important points that needs to be addressed here is whether the same phenomena will be observed for longer circRNAs, as T4 RNA ligase has limited circularization capacity when the RNA is longer.A recent paper by Breuer et al. in Several studies have investigated the potential immunogenicity of circRNAs relative to linear RNAs and have reached varying conclusions . FindingAnabaena pre-tRNA group I introns, circRNAs approximately 1,200 nt in length, modifications done (m1ψ), cell types used for transfection , transfection reagent used (Lipofectamine MessengerMAX), amount of circRNA transfected (40–200 ng), phosphatase treatment, RNase R digestion, and HPLC to purify circRNAs, dose of circRNA (350 ng), and route of inoculation ;Anabaena pre-tRNA group I introns as well as with T4 RNA ligase, circRNAs approximately 336, 410, and 522 nt in length, modifications done (none), cell types used for transfection , transfection reagent used (Lipofectamine MessengerMAX reagent), amount of circRNA transfected (200 ng), and denaturing PAGE to purify circRNAs;in vitro circularized RNAs.Findings of all these studies are debatable, which further highlights the fact that several additional factors could impact the observed immunogenicity of artificial circRNAs apart from contaminating RNA species, e.g., the study design of Chen et al. included synthesis of circRNAs with permuted group I introns from phage T4 td gene, circRNAs approximately 1500 nt in length, modifications done (none), cell types used for transfection , transfection reagent used (Lipofectamine 2000), amount of circRNA transfected (500 ng), and RNase R treatment used to purify circRNAs;in vivo should be tested and are therefore still required.In summary, it is still difficult to make definitive conclusions about circRNA immunogenicity, and there are still several unanswered questions that need to be addressed before reaching a final conclusion, e.g., does only purification strategy matte in evading RIG-I detection? What is the role of differences in secondary structures, tertiary structures, and sequence length in the immunogenic potential of circRNAs? How could the extra fragments of pre-tRNA and td genes impact the immunogenic potential of circRNAs? Keeping all the work done on the immunogenic potential of artificial circRNAs and in order to answer all of the aforementioned questions, further in-depth investigations of cellular immune responses to circRNAs with varying lengths and shapes produced by different approaches in different cell lines and


As a common complication of epithelial ovarian cancer (EOC), malignant ascites contributes to the peritoneal metastasis of EOC. CircRNAs play essential roles in tumor metastasis. However, no circRNAs have been reported to be involved in EOC peritoneal metastasis via ascites.Total of 22 samples from 9 EOC patients containing primary lesions (T), tumor cells from ascites (ASC), and metastatic lesions (M) were included for RNA sequencing to identify differentially expressed circRNAs and mRNAs among different tumors. Bioinformatic analyses, including single-sample Gene Set Enrichment Analysis and soft cluster analysis, were performed to find circRNAs potentially correlated with ascitic metastasis. Wound healing and transwell analysis were performed to evaluate tumor cells metastasis in vitro. Quantitative real-time PCR and western-blot were used for gene expression evaluation.According to transcriptomic analysis, ASC showed mesenchymal phenotype while T and M showed epithelial phenotype. 10 circRNAs were differentially expressed among ASC, T, and M. Among them, hsa_circ_0000497 and hsa_circ_0000918 were significantly up-regulated in ASC. Functional analysis showed that both hsa_circ_0000497 and hsa_circ_0000918 promoted metastasis of EOC via epithelial-mesenchymal transition (EMT) in vitro. The regulatory network construction identified 8 miRNAs and 19 mRNAs, and 7 miRNAs and 17 mRNAs as potential downstream target genes of hsa_circ_0000497 and hsa_circ_0000918, respectively, which may play pivotal roles in EOC ascitic metastasis.circRNAs (hsa_circ_0000497 and hsa_circ_0000918) contribute to metastasis of EOC via ascites by regulating EMT. These circRNAs may serve as novel potential therapeutic targets or prognostic biomarkers for EOC peritoneal metastasis.The online version contains supplementary material available at 10.1186/s12967-022-03404-9. Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. Five years survival of EOC is only 29% due to its high invasive and metastatic feature . ApproxiAscites contains tumorigenic cytokines, proteinases, chemokines, and growth factors and function as a metastatic tumor microenvironment , which pCircular RNA (circRNA), a novel class of endogenous RNAs, was found to be involved in multiple biological processes, including tumor development and metastasis. Increasing evidence showed that circRNAs promoted cancer cell metastasis by regulating EMT , 17. TraIn the present study, we found two circRNAs, hsa_circ_0000497 and hsa_circ_0000918, significantly up-regulated in ascites tumor cells from EOC patients via transcriptomic analysis. Expression of hsa_circ_0000497 and hsa_circ_0000918 were up-regulated in ascites tumor cells compared with primary and metastatic tissues. The overexpression of these circRNAs promoted tumor cell metastasis via EMT in vitro. In addition, we constructed a regulatory network including miRNAs and mRNAs for each circRNA and identified the potential downstream targets of these circRNAs, which may contribute to the promotional role of circRNAs in tumor cell metastasis. These findings indicated that hsa_circ_0000497 and hsa_circ_0000918 contribute to peritoneal metastasis of EOC via ascites by regulating EMT of tumor cells, which can be used as novel therapeutic targets or prognostic biomarkers of EOC peritoneal metastasis.4 sets of paired samples, including primary tumor lesions (T), ascites tumor cells (ASC), and metastatic lesions (M), and an additional 5 pairs of T and M were obtained from 9 EOC patients for RNA-sequencing. And 8 sets of paired samples, including T, ASC and M were included for validation. All EOC patients were informed at the Department of Gynecology and Obstetrics of Tenth People`s Hospital of Shanghai. And the histopathological diagnosis of EOC was based on the World Health Organization criteria .This stu2 and at least 95% humidity in RPMI1640  supplemented with 10% FBS . Both cell lines were qualified by STR  before further analysis.EOC cell lines SKOV3 and OVCAR3 were cultured under a 37 °C with 5% CO−△△Ct method with GAPDH reference control. All primer sequences were listed in Additional file Total RNA of samples was extracted using RNAiso Plus  according to the manufacturer's instructions. The concentration and purity of all RNA samples were subsequently measured by NanoDrop2000 . 1 ug of total RNA was used for cDNA (Complementary DNA) synthesis using PrimeScript RTMaster Mix . Quantitative real-time PCR (qRT-PCR) was performed on a QuantStudio Dx  using SYBR Premix ExTaq kit . The relative expression was calculated by the 2The protein extraction was completed with RIPA buffer  with 1% General Protease Inhibitor Cocktail  on ice and centrifuged at 4 °C, 12,000 rpm/15 min. The concentration of the supernatant was evaluated using the BCA Protein assay kit , and then the extracted proteins with 6 × SDS loading buffer  were denatured by heating (95 °C) for 10 min. The sample was transferred to the PVDF membrane after SDS-PAGE electrophoresis, blocked with 5% skim milk solution for 100 min, and incubated with primary antibodies at 4 °C overnight. Then, the membranes were incubated with the secondary antibody at room temperature for 1 h. Immobilon ECL substrate  and Amersham Imager 600  were used for signals detection and image acquisition. The antibodies used in this study were listed in Additional file The pLCDH-ciR  was used to create hsa_circ_0000497 and hsa_circ_0000918 overexpressing plasmids. Small interference RNAs (siRNAs) targeting the junction sequence of hsa_circ_0000497 or hsa_circ_0000918 and control siRNA , and the relative migration rate was calculated according to the formula below [2.SKOV3 cells were seeded in 6-well plates with 1 × 10la below .\documenCell invasion and migration assays were performed using 24-well transwell plates  coated with or without Matrigel™ Matrix . 24 h after transfection, tumor cells (1 × 105) for invasion assay and (5 × 104) for migration assay were trypsinized and washed twice with PBS, suspended in 200 μL serum-free RPMI1640, slowly dripped into the pre-coated insert, and incubated in a 24-well plate containing 600 μL RPMI1640 with 20% FBS per well for 48 h. The matrigel and cells on the upper surface of the membrane were then wiped off. The invasive cells that migrated through the membrane and adhered to the lower surface, and were fixed in 4% paraformaldehyde for 18 min and stained with DAPI . The number of invasive cells was photographed and counted using an inverted microscope imaging quantification field system .4 cells per well. 48 h after transfection, EdU was applied at 20 μM. The cells were then fixed with 4% paraformaldehyde and stained with Alexa Fluor 555 and DAPI. The cell proliferation was photographed and counted using an inverted microscope imaging quantification field system .The cell proliferation was detected using BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 555 . SKOV3 cells were seeded in 24-well plates with 7 × 102 transformations were performed on normalized read counts. To avoid the log of zeroes, all read counts were increased by 1 before taking the log transformation. The circRNAs were detected and identified by the find_circ algorithm with default parameters [The next-generation RNA-sequencing was performed in . Briefly, total RNA was isolated with the RNAiso Plus . The quality was checked using Agilent Bioanalyzer . RNA integrity number (RIN) for all samples was more than 7. The Illumina TruSeq Stranded RNA Sample Preparation kit  for library preparation. Then the libraries were sequenced using Illumina NovaSeq 6000  with paired-end  was utilized to emphasize variation and similarity among the 22 samples using R packages DESeq2 [grepel, ggplot, and pheatmap packages. The venn diagram was employed to take the intersection of DECs and DEGs.Differentially expressed circRNA (DECs) and differentially expressed genes (DEGs) were identified using R packages DESeq2  with folhttp://geneontology.org/)  was applied to evaluate the enrichment score of EMT gene signature in each sample. Signatures of epithelial n = 31) and mesenchymal (n = 54), used for ssGSEA, were connected from previous literature and GO databases  was applied to perform soft clustering analysis of circRNA and mRNA from T, ASC, M expression matrices [The matrices . RNAs wehttp://www.miranda.org/) was used to identify binding miRNAs of the circRNA candidates. Interactions between miRNA (microRNA) and mRNA were analyzed based on the TargetScan (http://www.targetscan.org) and miRDB databases (miRNA target prediction database) (http://mirdb.org). DEGs recognized in both databases were considered as candidate mRNAs of circRNA-miRNA regulation elements. The circRNA–miRNA–mRNA regulatory network was constructed through combination analysis of circRNA–miRNA pairs and miRNA–mRNA pairs and visualized using Cytoscape 3.8.2 [Cancer-Specific CircRNAs Database (CSCD) is utilized for visualizing the structure of circRNAs. The circBase . p < 0.05 was considered to be statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001 and **** indicates p < 0.0001.http://geneontology.org/) to conduct ssGSEA analysis [To investigate the dynamic states of epithelial-mesenchymal transition (EMT) during epithelial ovarian cancer (EOC) ascitic metastasis, we performed a comprehensive analysis of gene expression profiles of 22 samples, including 9 primary tumor lesions (T), 4 ascites (ASC), and 9 metastatic lesions (M) from 9 EOC patients. Principal component analysis (PCA) was performed to analyze the pattern of mRNA expression profile. Our results showed that ASC metastasis was distinguished remarkably from T and M, while T basically overlapped with M, indicating that the common essence showed between T and M as solid tumor tissue  and 71 circRNAs (including 57 up-regulated and 14 down-regulated) were found differentially expressed in T-ASC group and ASC-M group, respectively . Hsa_circ_0000497 consists of 5–13 exons of its host genes SLAIN1 with the length of 788 nucleotides (Nt) and locates at chr13:78,293,666–78,327,493 , respectively. After validation of the overexpression Fig. a, tumor http://www.circbase.org/). 85 and 23 miRNAs were found to potentially bind with hsa_circ_0000497 and hsa_circ_0000918, respectively  and TargetScan (http://www.targetscan.org/vert_72/) were used to identify targeting genes of candidate miRNAs. Total of 5875 mRNAs for hsa_circ_0000497 targeting miRNAs and 4897 mRNAs for hsa_circ_0000918 targeting miRNAs were obtained from both databases. Among these mRNAs, 36 genes were found differentially expressed in ASC based on our transcriptomic analysis compared with T and M . So they may also function via encoding proteins since circRNAs containing initial AUG followed by ORF can directly encode polypeptides [Serval mechanisms underlying the regulatory function of circRNAs had been reported previously . CircRNApeptides . HoweverWe found up-regulation of hsa_circ_0000497 and hsa_circ_0000918 in ASC and identified their role in the promotion of EOC metastasis. There are still some limitations to our study. First, the sample size should be further expanded to verify the expression of hsa_circ_0000497 and hsa_circ_0000918 in ascites, primary and metastatic lesions of ovarian cancer. Second, only tumor cell lines were included for function analysis. Whether these circRNAs promote metastasis in vivo remains unclear. Xenograft mice models including patient-derived tumor xenograft (PDX), will be applied for further research in vivo in our future study. As for the underlying mechanism, only a regulatory network was constructed based on transcriptomic data. Whether or not are these downstream genes regulated by circRNAs need to be assessed via gain or loss of function assays in vitro and in vivo models. In addition, the capability of these circRNAs used as a therapeutic target or prognostic biomarkers and so on also needs more investigation.In summary, we found two circRNAs, hsa_circ_0000497 and hsa_circ_0000918, specifically up-regulated in ASC from EOC patients. Overexpression of these circRNAs enhanced the metastatic capability of EOC tumor cells via EMT. We also constructed the downstream regulatory network of these circRNAs and found out the miRNAs and differently expressed mRNAs potentially contributed to the role of circRNAs in EOC metastasis. These findings indicated that hsa_circ_0000497 and hsa_circ_0000918 contribute to intraperitoneal metastasis of EOC via ascites by regulating EMT of tumor cells. And they should be used as novel therapeutic targets or prognostic biomarkers of EOC intraperitoneal metastasis.Additional file 1:Table S1. The sequences of primers and siRNAs used for experiments in this study. Table S2. Antibodies used in this study. Table S3. Detailed information for EMT related gene sets. Table S4. Predicted miRNA of candidate circRNAs. Table S5. ORF of candidate circRNAs. Figure S1. CircRNAs differentially expressed in ascitic metastasis of ovarian cancer. Figure S2. Silencing hsa_circ_0000497 and hsa_circ_0000918 inhibit the cell invasion and migration of OVCAR3 cells. Figure S3. mRNAs differentially expressed in ascitic metastasis of ovarian cancer. Figure S4. over-expressing or silencing hsa_circ_0000497 and hsa_circ_0000918 promote or inhibit the cell proliferation ovarian cancer cells


PPR035 regulated RNA editing at rps4-926 and orfX-406, while PPR406 regulated RNA editing at orfX-355. The defectiveness in RNA editing at these sites had no apparent penalties in rice respiration and vegetative growth. Meanwhile, the knockout mutants of ppr035 and ppr406 show enhanced drought- and salt tolerance. PPR035 and PPR406 were under the balancing selection in upland rice and highly differentiated between upland and lowland rice ecotypes. The upland-dominant haplotypes of PPR035 and PPR406 shall contribute to the better drought tolerance in upland rice. They have great prospective in the improvement of rice drought tolerance.Mitochondrial RNA editing plays crucial roles in the plant development and environmental adaptation. Pentatricopeptide repeat (PPR) genes, which are involved in the regulating mitochondrial RNA editing, are potential gene resources in the improvement of rice drought tolerance. In this study, we investigated genome-wide mitochondrial RNA editing in response to drought between upland and lowland rice. Responses of mitochondrial RNA editing to drought exhibit site-specific and genotype-specific patterns. We detected 22 and 57 ecotype-differentiated editing sites under well-watered and drought-treated conditions, respectively. Interestingly, the RNA editing efficiency was positively correlated with many agronomic traits, while it was negatively correlated with drought tolerance. We further selected two mitochondrial-localized PPR proteins, PPR035 and PPR406, to validate their functions in drought tolerance.  RNA editing, which is widely observed in plant mitochondria and plastids, is a post-transcriptional mechanism that alters the nucleotide sequence of an RNA molecule after transcription . Plant ROrganellar RNA editing requires an editosome, in which the pentatricopeptide repeat (PPR) family protein is the central molecule . The keyvia respiration  for 24 h) and five phytohormones (100 μm abscisic acid (ABA) for 12 h; 50 μm gibberellin (GA4), 50 μm auxin (IAA), 50 μm cytokinin (CTK), and 100 μm ethephon (ETH) for 4 h). Two or three biological replicates were used .The expression response of a gene to an abiotic stress or a phytohormone can indicate its role in the environmental adaptation and/or growth. It is therefore we have investigated the expression responses of rice ere used . The harjaponica) upland, 60 Geng (japonica) lowland landraces, and 45 wild rice accessions (upland/πlowland and πlandrace/πwild) of the PPR genes were calculated and compared with the genomic average. Meanwhile, the FST of each PPR gene between upland and lowland (U-L FST) or between landrace and wild rice (C-W FST) was also calculated and compared with the genomic average by the single-sample Kolmogorov–Smirnov test. If a PPR gene is highly differentiated between upland and lowland rice, it could be a candidate for rice drought adaptation.We selected 64 Geng . The procedure of the field experiment was described in detail in a previous study . Brieflyth with three replicates for each genotype.For rice landraces, three top leaves from three individuals for each genotype were mixed and harvested on the same day when RWC was measured (August 5th) in the CK and DT fields, respectively. The leaf samples were flash-frozen and stored in liquid nitrogen until RNA extraction. For modern cultivars and wild rice accessions , plants Escherichia coli. Thirty clones were selected and sequenced by the Sanger method to count the ratio of C to T conversions. A good correlation was observed between the editing efficiency estimated by the high-throughput sequencing and that estimated by the Sanger method . cDNA for each sample was obtained from reverse transcription of total RNA using the PrimeScript RT reagent Kit  according to the manufacturer’s instructions. Sixty target regions from thirty-one mitochondrial genes  were selr method .t-test. Second, the ecotype-differentiated site was defined when a significant difference was detected in its editing efficiency between rice ecotypes by independent t-test. Finally, three response patterns of RNA editing at a site were defined: (1) the upregulated site, whose alteration ratio of RNA editing [(Ed−Ew)/Ew] is larger than 0.2; (2) the downregulated site, whose alteration ratio is less than −0.2; and (3) unchanged site, whose alteration ratio is between −0.2 and 0.2. Here, Ed indicates editing efficiency in DT, and Ew indicates editing efficiency in CK. We also compared the RNA editing efficiency between modern cultivars and wild rice accessions by independent t-test.First, the drought-responsive site was defined as its editing efficiency exhibiting a significant difference between CK and DT conditions by paired p < 0.05. The correlation of RNA editing at a mitochondrial gene with a trait was determined by the largest PCC value among all sites of the mitochondrial gene. However, we should point out that the mitochondrial RNA editing is dynamic which means the correlations between RNA editing at the booting stage and the morphological traits measured after harvest  are for reference only.First, we applied the correlation analysis  among all sites based on their editing efficiencies measured from the CK and DT fields, respectively. Second, we applied the correlation analysis between the editing efficiency at each site and each measured trait among rice landraces. The significance of the correlation was determined by the following criteria: |PCC| > 0.2 and PPR genes (PPR035 LOC_Os01g46230 and PPR406 LOC_Os10g30760) were selected for functional characterization. The modified CRISPR-Cas9 method  were identified from T0 transformants by Sanger sequencing at the designed target region . Briefly, 0.3 g well-chopped fresh leaf samples from 15-days-old seedlings were injected into the reaction chamber with 2 ml air-saturated K2HPO4 buffer (20 mm and pH = 6.0). The reaction temperature was set at 25°C, and the speed of the magnetic stirrer was set to 100. The oxygen depletion curve was generated to estimate oxygen consumption rates from a 4-min duration after its slope became stable. Four biological replicates were used for the measurements.The respiration ability of WT, ppr035, and ppr406. A culture dish (8 cm diameter) containing 30 well-matured seeds of plant material was designed as a replicate, and three replicates were involved. The seeds were germinated in a growth chamber (25°C without light) with 12 ml fresh water, 12 ml 20% PEG6000 solution, and 12 ml 180 mm NaCl solution, respectively. To estimate the early growth, at least 10 young seedlings of each material were selected to measure their root lengths and shoot dry weights at 5 days after germination in a growth chamber (16 h light at 30°C and 8 h dark at 25°C).We then tested the germination behavior and early growth of WT, Field experiments were conducted in regularly managed paddy fields at Linshui Experimental Station, Hainan (2019.12–2020.5). Rice seedlings were transplanted into a plot with 4 rows × 8 hills with 20 cm intervals with three replicates (plots) following a randomized complete block design. PH, NT, biomass, 100GW, fecundity, GY, and HI were measured from at least eight plants in each plot.n) was calculated as follows: (WT0−WTn)/(WT0−Wdry). WT0 is the fresh weight of the rice shoot when it was excised (T0). WTn is the weight of the shoots at Tn. Wdry is the weight of the completely dried shoots. The EWR was measured in six 15-day-old seedlings for each plant material.We evaluated the root gravitropism and excised leaf water loss rate (EWR) to estimate drought avoidance. The root gravitropism was quantified using the modified method described in 2O2 content (A064-1-1) was measured before treatment and 4 DAT. The measurements of other physiological traits were performed following the manufacturer’s instructions for their corresponding test kits from Nanjing JianCheng Bioengineering Institute, China.For drought and salt tolerance, 48 rice seedlings of each plant material were grown in a 96-well plate (as a replicate) in the nutrient solution  for 15 dPPR035 and PPR406 using qPCR in the WT. We also tested the expressions of PPR035 and PPR406 in rice seedlings treated with osmotic (20% PEG6000), salinity (180 mm NaCl), oxidative (50 mm H2O2), ABA (10 μm), low temperature (5°C), and higher temperature (42°C) for 2, 4, and/or 8 h. As the expression of genes encoding the alternative oxidases (AOXs) is a molecular feature of mETC dysfunction  in qPCR was Actin and ATP6, respectively.We quantified the spatial and temporal expression patterns of function , expressPPR035 and PPR406, three biological replicates of 15-day-old WT, ppr035-1/2, and ppr406-1/2 plants were sampled for RNA sequencing. The genes differentially expressed between mutants and WT were identified as DEGs in ppr035 or ppr406. Enrichment analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were applied based on the DEGs detected in ppr035 or ppr406. In addition, to determine the common impacts of PPR035 and PPR406 on rice transcriptome, we also defined the core DEG as Type I, a gene determined as the DEG in both PPR035 and PPR406; and Type II, a gene determined as the DEG in at least one mutant line, while its |log2(fold change)| > 2 in all the four mutant lines. The subcellular localization of the core DEGs was predicted using TargetP.OsHAC1; 2, OCPI1, and OsRAA1; 2O2, 15% PEG6000, and 150 mm NaCl in Nipponbare by RNA-seq , salinity stress (180 mm NaCl for 4 and 8 h), oxidative stress (1% H2O2 for 4 and 8 h), ABA (100 μm ABA for 4 and 8 h), low temperature (5°C for 2 and 4 h), and higher temperature (42°C for 2 and 4 h) by qPCR using ATP6 as the reference. Meanwhile, we classified 20 rice landraces as high expression genotypes (HEGs) and low expression genotypes (LEGs) in terms of orfX and rps4 expressions quantified by qPCR. We then tested any differences in the expression of the core DEGs between HEGs and LEGs using an independent t-test. Three biological replicates were involved in qPCR. The primers used in qPCR are listed in via the sub module MEMSAT-SVM (Membrane Helix Prediction) of SECONDARY STRUCTURE PREDICTION  annotated in NCBI.N PSIPRED.orfX was amplified using specific in-fusion primers . A strain transformed with pGEX-6P-1 was used as a control. All of the transformed E. coli BL21 strains were grown in 5 ml cultures at 37°C for OD600 near 0.4–0.6. Then the growth in normal condition was measured by further culturing in 20 ml LB medium containing Ampicillin (100 μg/ml). For abiotic stress treatments, 0.6 M sorbitol and 0.5 M NaCl was added into the medium, respectively. To induce the expression of the target proteins, 0.2 mm IPTG  was also added to the culture solutions. Initial cell concentrations were adjusted to be equivalent in each flask in these experiments. The velocity of the shaker was 200 rpm min−1 and the growth temperature was set at 37°C. The concentration of E. coli cells (OD600) at each time point was determined. Each experiment was replicated three times.Prokaryotic expression system could be used for the verification of some plant stress tolerance genes . We cons primers  and inseUPPR035/UPPR406 and LPPR035/LPPR406, respectively. Any differences in RNA editing efficiencies at the three sites , agronomic traits, and expression levels of the core DEGs between landraces of UPPR035/UPPR406 and LPPR035/LPPR406 were tested by the independent t-test. In addition, protein structures of PPR035U/PPR406U and PPR035L/PPR406L were predicted using Phyre2 with default parameters  called from resequencing. PopART (ver. 17) was used to visualize the haplotype network. The dominant haplotypes in the typical upland and lowland rice were coded as rameters .First, we tested the genome-wide expressions of PPR genes in responses to various abiotic stresses and phytohormones. Among 491 PPR genes predicted across the rice genome, 482 of which were expressed in at least one of our sequenced samples . There wCultivar/πWild and πUpland/πLowland) and genetic divergence (FST(C-W) and FST(U-L)) of the total PPR genes were generally equivalent to the genomic average , including 26 (59.1%) mitochondrion-localized genes, between upland and lowland rice ecotypes , whose editing efficiencies exhibited significant differences between the two rice ecotypes under CK and DT conditions, respectively (ccmC and ccmFc), small ribosomal subunit , and orfX  for functional validation, as they were highly differentiated between ecotypes , predicted and then proven to be targeted in mitochondria , decreased to zero  under the normal and osmotic treatments  among the four mutant lines in total  and KEGG pathways  were highly upregulated in the four mutant lines  related to pollen development and fertility among DEGs  is associated with rice susceptibility to low temperature. However, this hypothesis is required further experimental validation.Defects in mitochondrial RNA editing, particularly those at mitochondrial genes encoding proteins of the ETC complex, could always lead to abnormal mitochondria, dysfunction of respiration, and consequent growth and developmental retardation . In this mutants –d and eqts  and PPR406 (SNV001187) can alter the amino acid which are located at the third trans-membrane helix  and the intact (non-edited) orfX had many differences in trans-membrane helixes . The nonne helix . It may PPR035 and PPR046 are located in the genomic region under balancing selection in upland rice , whereas Hap-2 of PPR035 and PPR406 were upland-dominant (coded as UPPR035 and UPPR406). UPPR035 is a wild rice-derived allele that can be detected in wild rice, whereas LPPR035 is possibly a novel mutation obtained during lowland rice domestication as it is absent in wild rice. In contrast, LPPR406 should be derived from its wild ancestor, whereas UPPR406 could be a novel mutation during upland rice domestication. It is noteworthy that RNA editing efficiencies at the three sites regulated by PPR035 and PPR406 were significantly lower in common wild rice than those in modern cultivars  in ppr035 and ppr406 can explain its improved drought/salt tolerance partially by osmotic adjustment.terility . We founvia the endoplasmic reticulum (ER), and finally regulates gene expression in the nucleus  based on the core DEGs detected in ppr035 and ppr406. The widely reported retrograde signals induced by mitochondrial dysfunction are the upregulation of AOXs and/or ROS burst  can be found at: HX and LL designed the experiments. JX, HX, ZL (Zhi Luo), GH, HZ, JL, and ZL (Zhaoyang Li) conducted the field and molecular experiments. HX, JX, and LW analyzed the data. ZL (Zhi Luo), JX, and TL prepared the plant materials used in this study. HX, JX, ZL (Zhi Luo), and LL wrote the manuscript. All authors contributed to the article and approved the submitted version.This work is supported by Shanghai Natural Science Foundation (20ZR1449300), Shanghai Agriculture Applied Technology Development Program (G2016060107), National Key Research and Development Program of China (2018YFE0106200), Shared Platform of Crop Germplasm Resources in Shanghai (18DZ2293700), and Platform for National Crop Germplasm Resources .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N6-methyladenosine (m6A), 5-methylcytosine (m5C), N1-methyladenosine (m1A), 5-hydroxymethylcytosine (hm5C), and pseudouridine (Ψ). The emerging tools of RNA methylation can be used as diagnostic, preventive, and therapeutic markers. Here, we review the accumulated discoveries to date regarding the biological function and dynamic regulation of RNA methylation/modification, as well as the most popularly used techniques applied for profiling RNA epitranscriptome, to provide new ideas for growth and development.Ribonucleic acid (RNA) methylation is the most abundant modification in biological systems, accounting for 60% of all RNA modifications, and affects multiple aspects of RNA . Dysregulation of RNA methylation causes many developmental diseases through various mechanisms mediated by  N1-methyladenosine (m1A), N6-methyladenosine (m6A), N6, 2′-O-dimethyladenosine (m6Am), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), pseudouridine (Ψ), and Adenosine to inosine editing (A-to-I editing), etc.  modification plays an important role in linking deoxyribonucleic acid (DNA) to proteins during the transmission of genetic information. More than 100 post-transcriptional RNA modifications have been identified in important biological processes in viruses, archaea, bacteria, and eukaryotes. Methylation accounts for 60% of the total RNA modifications. RNA methylation was discovered as early as the 1970s , and it g), etc. . Among t studied . Compare studied . Here, wN6-methyladenosine (m6A), the most abundant methylation modification in eukaryotic mRNA, is the most thoroughly studied type of RNA modification  and H = U/A/C (U > A > C) , demethylases (erasers), and binding proteins (readers). The main components of the methyltransferase complex that have been identified include methyltransferase 3 (METTL3), methyltransferase 14 (METTL14), Wilms’ tumor 1-associating protein (WTAP), and KIAA1429 (vir-like m6A methyltransferase associated) (fication . m6A has viruses . In mammnd liver . Althougtil 2011 . With thachieved . The seq> A > C) . In addispecific . m6A metociated) . The demociated) .6A is mainly through the post-transcriptional regulation of RNA by m6A-binding proteins. Currently known binding proteins include the YT521-B homology (YTH) domain proteins  and nuclear heterogeneous ribonucleoprotein HNRNP family proteins   . With thnslation . Furtherertility .6A demethylase gene ALKBH5 accelerates the nuclear export of mRNA  as a methyl donor to methylate cytosine (C) to form m5C  GANGU was consistent with the m5C conserved sequence in archaeal rRNA (5C methyltransferase NSUN2 (NOP2/Sun RNA methyltransferase family member 2) and the binding protein ALYREF (Aly/REF export factor) also proved that RNA m5C modifications have dynamic reversibility , have been a hot field of research in recent years.In addition to m1A methylation was first discovered in non-coding RNAs such as rRNA and tRNA and is widely present in prokaryotic and eukaryotic mRNAs. Unlike m6A methylation, m1A methylation occurs at the N1 position of the adenosine base group. It carries a positive charge by blocking Watson-Crick base-pairing under physiological conditions. Thus, it can drastically alter protein-RNA interactions and RNA secondary structures through electrostatic effects. m1A maps uniquely to positions near the translation start and first splice sites in coding transcripts and correlates with the upregulation of translation  modification was found at the first nucleotide of certain mRNAs (6Am is formed by the combination of 2′-O-methyltransferase (2′-O-MTase) and 2′-O-methyladenosine-N6-methyltransferase. Adenosine is methylated by 2′-O-methyltransferase (2′-O-MTase) to form Am, which can then be methylated by 2′-O-methyladenosine-N6-methyltransferase to form m6Am  + RNAs contained approximately three m6A nucleotides per 105 nucleotides compared to ∼100 m6A nucleotides per 105 nucleotides, revealing 33 times more m6A compared to m6Am  under an AAG motif in vitro and in vivo -family enzymes to hm5C . It is one of the most abundant forms of post-transcriptional RNA modifications, widely present in cellular RNA, and highly conserved among species. ψ is formed by the sequence-specific isomerization of uracil (U) , which i6A methyltransferase holoenzyme complex consists of at least two multicomponent factors of the whole methyltransferase called MT-A (∼ 200 kDa) and MT-B (∼ 800 kDa). However, only the METTL3  protein has been identified, and the subunit alone had no enzymatic activity  experiments and two-dimensional gel electrophoresis analysis showed that METTL14 and METTL3 can form a stable complex at a 1:1 ratio. In vitro experiments on the activity of enzymes involved in m6A formation showed that although a single METTL14 had slightly higher enzymatic activity than METTL3, the heterodimer formed by METTL14 and METTL3 with a strong preference for substrates had the highest enzymatic activity for the RNA of the stem-loop structure, which has no obvious secondary structures. It preferentially methylates GGACU, which is consistent with the conserved sequence of m6A distribution reported previously  experiments , which controls sex determination . Further, immunoprecipitation experiments showed that RBM15 and RBM15B could bind and recruit the WTAP-METTL3 complex to specific target sites. RBM15/RBM15B consists of an RNA recognition domain, as per iCLIP  sequencing and single miCLIP . Sequencing analysis of the base resolution revealed that the RBM15/RBM15B-binding site was significantly enriched near the m6A methylation modification site  mediate the interaction between RBM15/Nito and WTAP/Fl (2)d, thereby promoting m6A modification of Mrna. They also found that Flacc regulates sex determination and dosage compensation in Drosophila by regulating alternative splicing of the Sxl gene. CG7358 gene of Drosophila (named Xio) and its involvement in the sex determination pathway of Drosophila through regulating the alternative splicing of Sxl via m6A modification.Zc3h13, a critical RNA m nucleus . In 2018hylation . KnuckleIn addition to the above-mentioned members, there are other methyltransferase components involved in the selective recognition of methylation sites and division of labor to ensure fine post-transcriptional regulation.5C-specific methyltransferases include the NSUN (NOL1/NOP2/SUN) family proteins and DNMT2 (DNA methyltransferase 2), which belong to the superfamily of Rosman-folded enzymes and are characterized by the conserved cysteine residue SAM. The RNA m5C methyltransferase that has been studied the most recently is the NSUN family, which acts as a methyl donor to catalyze the transfer of methyl groups to cytosine residues of different RNA substrates  and C321 (cysteine 321) sites, where C321 catalyzes the methylation of cytosine by binding to the cytosine pyrimidine ring to form a covalent bond, and C271 mediates the release of RNA. High-throughput sequencing analysis also showed that NSUN2 knockdown significantly reduced mRNA m5C modification   .6A levels in RNA has been observed (6A demethylases. Two novel mRNA m6A demethylases (FTO and ALKBH5) have recently been identified, confirming the dynamic regulation of m6A . As the second identified m6A demethylase, ALKBH5 has m6A demethylase activity comparable to that of FTO and preferentially acts on the m6A site of conserved motifs. Unlike the oxidative demethylation of FTO, ALKBH5 can directly catalyze the removal of m6A without generating intermediate products  domains, including YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, and heterogeneous nuclear ribonucleoproteins (HNRNP). Readers mainly work by directly or indirectly binding RNA. Direct RNA binding is mediated by a reader-specific recognition modification domain such as the YTH family proteins, including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2, which can directly bind to methylation modifications of the target genes in the nucleus and cytoplasm . P-bodies are cytoplasmic ribonucleoprotein (RNP) granules, which are aggregates of multiple functional proteins and RNA in the cytoplasm  was first reported to mediate the degradation of m6A-modified mRNA , which is also a cytoplasmic protein that can promote the translation efficiency of the m6A-mRNA it binds. Ribosomal map analysis showed that YTHDF1 promotes the binding of substrate RNA to ribosomes, which is conducive to RNA translation  experiments showed significant binding of YTHDC1 to RNA containing m6A modifications, while in vivo photoactivatable ribonucleoside enhanced crosslinking and PAR-CLIP sequencing showed that the binding motif of YTHDC1 is consistent with the conserved motif RRACH of m6A. Most of the binding sites were located near the stop codon, which is also consistent with the distribution characteristics of m6A. YTHDC1 can interact with the splicing factor SR protein to regulate the alternative splicing of m6A-containing exons , FMRP translational regulator 1 (FMR1), leucine-rich pentatricopeptide repeat containing (LRPPRC), and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs).Additionally, studies based on RNA pull-down experiments have detected other potential m5C, two oligonucleotide substrates, with and without m5C, were designed. Through oligonucleotide enrichment combined with protein profiling, the mRNA was identified as a nuclear functional complex component. ALYREF binds to m5C-modified RNA oligonucleotides. EMSA and RIP combined with HPLC and sequencing technology demonstrated that ALYREF’s K171 (lysine 171) mutation can significantly reduce its ability to bind m5C, thereby reducing its ability to bind RNA. Further, fluorescence in situ hybridization technology (FISH) showed that the efficiency of mRNA nucleation decreased with the knockdown of NSUN2 and ALYREF. However, backfilling experiments demonstrated that only the wild-type NSUN2 and ALYREF could reverse the reduction in mRNA nucleation caused by the knockdown of NSUN2 and ALYREF, suggesting that m5C plays an important regulatory role in the process of mRNA nucleation  and their differentiation into neurons , a specific marker for axonal regeneration, were identified as targets for m6A modifications. The local translation of these transcripts is negatively regulated by m6A and can be regulated by FTO in axons , a protein-coding gene, also regulates neuronal development. In a developing mouse brain, the absence of NSUN2 did not affect RG cells but delayed their differentiation into superior neurons. NSUN2 is expressed in early neuroectodermal cells and can differentiate into various region-specific neuronal and glial cell types  diraneously . These mThese high-throughput mapping methods have enabled the characterization of the epitranscriptome in diverse cellular environments. However, further technical improvements are needed to improve the resolution and sensitivity of these methods. Following the recent discovery of more writers, readers, and erasers of the epitranscriptome, as well as the detailed analysis of the known epitranscriptome, new research directions will emerge, which may lead to new treatment strategies. In the future, improved high-throughput technology should help improve spatial resolution, provide chemometric data, and possibly detect new mRNA modifications in the transcriptome.Although wet-lab approaches can obtain relatively accurate mRNA modification information, they are time-consuming, costly, and difficult to conduct. Given that RNA sequence numbers show explosive growth in the post-genomic era, wet-lab approaches are obviously not suitable for systematic and in-depth analysis of the relevant mechanisms and functions of RNA methylation modification. Therefore, many researchers have developed predictive and computational tools for identifying epigenetic modifications , which hN6-methyladenosine sites  and EIIP-PseEIIP encoding scheme. It is important to understand the methylation-related databases involved in developing these tools. We summarized the database information involved in methylation modification  and sort6A, m5C, m1A, pseudouridine, and 2’OMe. Other types of modifications that can be detected but are not clearly defined were not included. We also discussed the advantages and disadvantages of the detecting method based on high-throughput sequencing. It is worth mentioning that the databases related to RNA methylation modification, as well as the prediction and identification tools for RNA methylation sites developed with the help of these databases, have played an increasingly important role in RNA methylation modification research. We found that establishing a powerful method to investigate transcriptome modification is key to understanding post-transcriptional regulation. We emphasized that RNA methylation modification plays a key role in neural stem cells, synaptic functions, nervous system development, and brain development. Future research should focus on the role and mechanism of RNA methylation in neurodevelopmental disorders, which will greatly contribute to the prevention and treatment of developmental diseases.To provide biochemical researchers with the latest progress in RNA methylation, we reviewed the findings to date on the dynamic regulation and key roles of RNA methylation. We found that combining biochemical technology with high-throughput sequencing has made rapid progress in understanding the form and function of RNA modification, especially in mRNA and lncRNA. We mainly discussed mJZ and HL were responsible for gathering information and drawing the illustrations. YZ contributed to information interpretation, editing, and critical revision of the manuscript. WT, Y-QC, JD, S-YB, and Z-XW were responsible for revision of the manuscript. All authors read and approved the final manuscript.


Although the highly effective measles vaccine has dramatically reduced the incidence of measles, measles, and outbreaks continue to occur in individuals who received the measles vaccine because of immunization failure. In this study, patients who have definite records of immunization were enrolled based on measles surveillance in Shanghai, China, from 2009 to 2017, and genomic characteristics regarding viruses retrieved from these cases provided insights into immunization failure. A total of 147 complete genomes of measles virus (MV) were obtained from the laboratory-confirmed cases through Illumina MiSeq. Epidemiological, and genetic characteristics of the MV were focused on information about age, gender, immunization record, variation, and evolution of the whole genome. Furthermore, systematic genomics using phylogeny and selection pressure approaches were analyzed. Our analysis based on the whole genome of 147 isolates revealed 4 clusters: 2 for the genotype H1  and the other 2 for D8 and B3, respectively. Estimated nucleotide substitution rates of genotype H1 MV derived using genome and individual genes are lower than other genotypes. Our study contributes to global measles epidemiology and proves that whole-genome sequencing was a useful tool for more refined genomic characterization. The conclusion indicates that vaccination may have an effect on virus evolution. However, no major impact was found on the antigenicity in Shanghai isolates. Measles is caused by the measles virus (MV), which is one of the most contagious diseases of humans before introducing the measles vaccine. MV is a member of the genus Morbillivirus of the family Paramyxoviridae . The virSince 1978, the live attenuated measles vaccine has been included in the National Expanded Program on Immunization (EPI) plan. In Shanghai, the vaccination procedure for the 2 dose was changed from 8 months and 7 years between 1986 and 2004  to 8 andn = 147), we next performed an in-depth analysis to understand the genetic characteristics of MV.In view of the WHO being set up as a target for endorsing the Western Pacific Regional Plan of Action for Measles Elimination , a high The experiments and protocols in this study were screened and approved by the Ethical Review Committee of the Shanghai Municipal Centre for Disease Control and Prevention (Shanghai CDC). Clinical specimens, including throat swabs and sera, were obtained from the routine MV surveillance program of the Shanghai CDC. Sample collection was approved by either the patients or their parents with prior informed consent.The demographic data of the patients were obtained from the China Information System for Disease Control and Prevention (CISDCP). The definition of suspected measles cases was based on the guidelines of the Shanghai Municipal Measles and Rubella Surveillance Program, confirmed by the WHO case definition for measles . A laboratory-confirmed case was defined by serological (positive serologic test for measles IgM antibody or fourfold rise in measles IgG by standard serologic assay) or virological  evidence of acute measles infection according to the MMS. Measles immunization has been documented since 2009 when the measles surveillance system (MSS) was established. The vaccination statuses of all cases in our study were determined by their vaccination card or memories of patients.The throat swab samples in this study were collected according to the MSS in Shanghai. This study strictly followed the relevant ethical requirements and strictly protected the data security and privacy of the participants. Clinical samples that were positive by PCR were inoculated on cell culture to isolate the virus. The Vero/hSLAM cell line was used for MV isolation from clinical specimens. Vero/hSLAM cells were kept in our laboratory and kindly shared by Dr. Yanagi at Kyushu University, Japan, through the WHO GMRLN. Procedures including the maintenance of cells, sample handling, and virus inoculation were performed as described in the Manual for the Laboratory-Based Surveillance of Measles, Rubella, and Congenital Rubella Syndrome (WHO).The virus genomes of the isolated MV strains were extracted and purified using the QIAamp Viral RNA Mini Kit . The whole genome was amplified with four overlapping long PCR fragments (3.9–5.4 kb) using the high-fidelity enzyme mixture of the SuperScript III One-Step RT-PCR Kit  as described in a previous study , 19 and via pairwise distance mapping using SSE, version 1.2 England, University of Oxford (p-distance in the MEGA program. This distance indicates the proportion (p) of nucleotide/amino acid sites at which being compared are different. It is obtained by dividing the number of nucleotide/amino acid differences by the total number of nucleotides compared.The nucleotide variability of the whole genome was calculated f Oxford . The sca1. The Simplot program  was used for the recombination analysis. The included strains represent the Shanghai MV strains isolated in comparison with reference strains defined by the WHO. Phylogenetic reconstruction was conducted on the nucleoprotein peptide sequences of these strains, as recommended by the WHO for genotype. The same phylogeny was established for the individual genes N, M, F, and H.For the genotyping step of MV isolates, the nucleotides and amino acid sequences were aligned using the MEGA program (version 6 and X) . Phylogenetic trees were constructed by the maximum likelihood method with the best model implemented in the MEGA program. The reliability of phylogenetic inference at each branch node was estimated by the bootstrap method with 1000 replications. Bootstrap values greater than 70% were considered statistically significant for grouping. The nucleotide substitution rates were estimated by a coalescent-based Bayesian method in BEAST (version 2.6.0)p-value ≤ 0.05.Gene sequences of 147 isolates were used for the selection pressure analysis by the Datamonkey server. Codon-based multiple sequence alignments of an individual gene were carried out using the MUSCLE program available in the MEGA X. The ratio of non-synonymous to synonymous substitutions per site (dN/dS) at every codon was calculated using Single-Likelihood Ancestor Counting (SLAC) and Fixed Effects Likelihood (FEL) methods with Since 2001, high-quality pathogenic measles surveillance has been maintained in Shanghai. From 2009 to 2017, the measles incidence in Shanghai was between 0.2/100,000 and 3.82/100,000 annually. According to the requirements of the national measles surveillance program, throat swabs are required to be collected for MV separation for every suspected measles case since 2011. A total of 3465 measles cases were confirmed in Shanghai by the laboratory using MV IgM testing or MV RNA testing by real-time RT-PCR assay. Furthermore, 1204 strains were isolated from these cases. According to the MSS, 1288 were unvaccinated cases and 296 cases were vaccinated shown in . In our In this study, 147 complete MV genome sequences were successfully generated from the MV isolates and planned to submit to GenBank. Routine phylogenetic analyses of the carboxyl-terminal 150 amino acids encoded by the N-450 hypervariable region of the nucleoprotein gene, which was shown in A scan of sequence variability across the full MV genome is shown in Furtherly, sequences divided into two groups by vaccine status were analyzed. Sequence similarity including nucleotide and amino acid of the whole genome and individual genes were calculated and analyzed, as shown in KJ755974). Interestingly, the virus isolated from the vaccinated cases accounted for 50% in cluster H1-A and 20% in cluster H1-B. This clustering pattern was also observed when independent trees were generated using single genes H, as well as MF-HVR  and the other 2 for D8 and B3 . Althougs MF-HVR , yet notviz., N, H, and F were also calculated. Varying NSRs observed for N-, H-, and F-genes are 7.92 × 10–4 , 8.43 × 10–4 , and 8.83 × 10–4  substitutions/site/year, respectively  and Nectin-4. Furthermore, H-protein is known to be glycosylated at amino-acid positions 168, 187, 200, 215, and 238, whereas position 416 is an additional potential N-linked glycosylated site. It is found to be completely conserved for these function sites in Shanghai isolates, and none of the amino acids that are encoded by the codons under selection in H-gene are part of the known function sites.China established a National EPI in 1978. Until 2010, after more than 30 years of routine and synchronized nationwide supplementary immunization activities (SIA) , the incTo evaluate the utility and scope of the whole genome sequencing, the WHO GMRLM have formed the “Next-generation. Extended window and Whole genome sequencing working group” (N.E.W.) , which dFor the first time, our study evaluated the genetic diversity and evolutionary dynamics of 147 MV isolates circulating in Shanghai from 2009 to 2015 based on whole-genome sequence analyses, which is a significant addition to MV global data. Thus, this study was conducted to establish the genetic and epidemiological characteristics of the measles cases with immunization records in Shanghai between 2009 and 2017. The virological characteristics of these isolates from these infections are reported for the first time.According to the routine genotyping in our report, only three genotypes have been identified in these cases since 2009, and the most prevalent were H1a and D8 in 2008 and B3 in 2009, which have also been in circulation since the molecular measles surveillance was established in 2001 . However–6 substitutions/site/year to 6 × 10–3 substitutions/site/year (–3), human influenza A (1.8 × 10–3), and slightly higher than the mutation rates reported for dengue virus and human rotavirus . However, there was no significant result. In the selection pressure analysis, no differences were found. The values of NSRs and dN/dS ratios correlate with the structural, functional, and evolutionary constraints operational on these genes. The observed dN/dS ratios also hint at the modest role of immune selection in MV evolution, wherein the highest variability was observed in H-gene, the major target for neutralizing antibodies directed against the virus and binding to host receptors. The lowest ratio was observed in the case of L-gene that encodes RNA-dependent-RNA-polymerase which has been reported to be most refractive to insertional mutagenesis. Thus, complete genome sequencing data of 147 isolates sampled during 2009–2017 substantially increment global genomic data of genotypes H1 isolates. The genetic diversity of China Shanghai isolates was found to be moderate in our report, and our study also underlines constraints on the evolution of MV with no potential impact on antigenicity. This knowledge is valuable for policy decisions regarding MV surveillance and control in the eliminating countries and the areas of measles.Our study, based on viral isolates, provides for the reintroduction of isolation-on-cell-culture as a useful tool for the characterization and comprehensive study of the molecular epidemiology of circulating strains of MV. However, there were also disadvantages, like single nucleotide polymorphisms may be obtained less than directly from clinical samples. Furthermore, amplification to obtain whole genome in our experiment may cause unnatural mutations in the virus genomes. In fact, the high cost made it not applicable on a routine basis in all laboratories . Due to The original contributions presented in this study are publicly available. This data can be found here: BankIT, ON035847–ON035993.The experiments and protocols in this study were screened and approved by the Ethical Review Committee of the Shanghai Municipal Centre for Disease Control and Prevention (Shanghai CDC). Clinical specimens, including throat swabs and sera were obtained from the routine measles virus surveillance programme of the Shanghai CDC. Sample collection was approved by either the patients or their parents with prior informed consent.XZ and CSL designed this study and revised the manuscript. XXC sequenced the samples, analyzed the data, and wrote this manuscript. WT, YYL, YY, and XYC contributed to sample testing. HYC and YL analyzed the data. ZL, YZ, and XDS collected the samples and revised the manuscript. STX and YZ contributed to the design of this study. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The role of methyltransferase-like 3 (METTL3), which participates in catalyzing N-methyladenosine (m6A) RNA modification, in gastric cancer (GC) is unclear. Here, we found that METTL3 was overexpressed in human GC. Functionally, we verified that METTL3 promoted tumor cell proliferation and angiogenesis through a series of phenotypic experiments. Subsequently, ADAMTS9 was identified as the downstream effector of METTL3 in GC, which could be degraded by the YTHDF2-dependent pathway. Finally, the data suggested that METTL3 might facilitate GC progression through the ADAMTS9-mediated PI3K/AKT pathway. Our study unveiled the fundamental mechanisms of METTL3 in GC progression. The clinical value of METTL3 in GC deserves further exploration. GC is the fifth most prevalent gastrointestinal malignancy globally . AlthougIncreasing studies have shown that epigenetic modification strikingly contributes to GC progression, while major studies are focused on the transcriptional level –6. An inAs a core component of the m6A writer, METTL3 is vital for m6A modification and has been identified as an oncogene in some hematological diseases and solid tumors –25. In mA total of 20 specimens, including tumor and adjacent normal tissues (ANTs), were obtained from GC patients receiving gastrectomy at Nanjing First Hospital from July 2019 to July 2020. No patient received antitumor treatment before surgery. The project was approved by the ethics committee of Nanjing First Hospital.http://kmplot.com/analysis/).The public transcriptome data of METTL3 in GC were downloaded from The Cancer Genome Atlas (TCGA) database. The prognostic value of METTL3 in GC was evaluated in the Kaplan–Meier plotter database  targeting METTL3  or YTHDF2 (shYTHDF2) and the control group (shNC) were synthesized . pcDNA3.1  was used to construct pcDNA-METTL3 and pcDNA-ADAMTS9. Lipofectamine 2000  was employed to transfect cells following the manufacturer’s instructions. The sequence of shRNAs is listed in Stable METTL3 knockdown and overexpression models were established by using a lentivirus vector system. Briefly, plasmid-cloned RelB cDNA and siRNA for knocking down METTL3 were transfected into AGC and HCG-27 cell lines according to the manufacturer’s instructions.6 AGS cells mixed in Matrigel  were subcutaneously injected into BALB/c nude mice . Tumor volumes were routinely measured every 5 days according to the following equation: V = 0.5 × (length × width2). One month later, the xenografts were surgically dissected after sacrificing the mice.For the formation of xenograft tumors, 5 × 106 AGS cells in culture medium mixed with Matrigel was deposited on the surface of the CAM. The images were then taken, and the vessel areas were calculated.The chorioallantoic membrane (CAM) assay was employed to estimate the potential role of METTL3 in angiogenesis. The eggs were incubated in a humidified atmosphere of 80% at 38°C for 1 week. On embryonic day 8, 100 µl of a suspension of 1 × 10For RNA-seq, RNA-seq of stable METTL3 knockdown AGS cells and their controls (NC) were performed with HiSeq-2500. The PolyA+ messenger RNA (mRNA) library was estimated with RNA-seq (H/M/R) Library Prep Kit. KEGG analysis was detected with DAVID.For meRIP-seq, RNA fragmentation was performed by using NEBNext Magnesium RNA Fragmentation Module . About 1/10 of the fragmented RNA was used as input control for RNA-seq by Genesky . The others were incubated with m6A antibody, and the enrichment of m6A containing mRNA was analyzed using high-throughput sequencing by Genesky .All the data analyses were performed using SPSS 22.0 . Our data are described as the mean ± S.D. Continuous variables with a normal distribution were analyzed using Student’s t-test. For multiple comparisons, the significant difference test was applied following ANOVA. The correlations were analyzed using Pearson’s correlation coefficients. A P value < 0.05 indicated statistical significance.Cell culture, qRT-PCR, cell proliferation assays, tube formation assays, RNA stability, Western blots, RNA immunoprecipitation (RIP), m6A quantification, RNA-seq, and MeRIP-Seq are listed in In both clinical and TCGA samples, METTL3 was observed to be highly expressed in tumors  to block RNA transcription. As shown in To further confirm the importance of ADAMTS9 in our study, we suppressed ADAMTS9 expression in METTL3-knockdown GC cells  can be found in the article/The studies involving human participants were reviewed and approved by the ethics committee of Nanjing First Hospital. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the ethics committee of Nanjing First Hospital.XW, HX, and XS designed the experiments. NW, XH, and XC performed the experiments. NW, SZ, and XH analyzed and confirmed all data and wrote the manuscript. BZ assessed all content. All authors reviewed the manuscript. XW and HX made a final approval. All authors contributed to the article and approved the submitted version.This work was supported by Grants from the National Natural Science Foundation of China (Grant No. 81773240), The Natural Science Foundation of Jiangsu Province (Grant No. BK20181118), Jiangsu Provincial Special Program of Medical Science (BE2019617), and Nanjing Medical Science and Technique Development Foundation (Grant No. QRX17062).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer YY declared a shared parent affiliation with the authors to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.