Even after complete resection, non-small cell lung cancer (NSCLC) shows preferential recurrence in the mediastinal lymph nodes, lungs, brain, bone, liver, and adrenal gland. However, the relationship between clinicopathological factors and recurrence patterns after resection has not been well-evaluated. Among 688 NSCLC cases with complete resection between 2004 and 2016, 233 cases recurred at our institute. On multivariate analyses, NSCLCs with lymph node metastasis and pulmonary metastasis at surgery commonly recurred in the mediastinal lymph nodes and lungs, respectively. Young age, adenocarcinoma, and vascular invasion were correlated with brain metastasis. Although no variable was associated with bone metastasis, vascular invasion was correlated with postoperative liver and adrenal gland metastasis. Pathologically proven stage II or III NSCLC, adenocarcinoma, and the presence of lymphatic permeation would result in multiple metastases. Vascular invasion, larger invasive size, and advanced stage were independent risk factors of early recurrence. Considering survival, vascular invasion, elderly age, and non-adenocarcinoma were unfavorable prognostic factors after recurrence. Some clinicopathological variables were correlated with organ-specific metastasis and post-recurrence survival. Particularly, vascular invasion was a biomarker of brain, liver, and adrenal gland metastases and a prognostic marker after recurrence among completely resected NSCLC. This information is useful for more frequent patient follow-up and identifying organ-specific distant metastasis. Owing to the use of high-resolution computed tomography (HRCT), small and resectable non-small cell lung cancer (NSCLC) are increasingly being detected worldwide ,2. NeverSolid tumors have great variations in the patterns of metastatic organ tropism. In other words, a particular cancer tends to relapse to one particular organ or multiple specific organ sites. In general, NSCLC is likely to metastasize to some organs, including the mediastinal lymph nodes, lungs, brain, bones, liver, and adrenal glands ,12,13,14Between January 2004 and December 2016, a total of 688 patients with NSCLC underwent complete resection and systematic lymph node dissection at our institute. For diagnostic work-up, all patients underwent chest contrast-enhanced HRCT and positron emission tomography (PET) CT. When nodal metastasis was suspected, additional examinations were performed, such as endobronchial ultrasound-guided transbronchial needle aspiration or biopsy using mediastinoscopy. All therapies, including surgery, adjuvant chemotherapy, and post-recurrent therapies, were discussed and decided at a multi-disciplinary team conference. Among them, 223 patients had a postoperative recurrence of NSCLC. Patients were excluded if they underwent sublobar resection and/or neoadjuvant chemotherapy and had NSCLC with positive intraoperative pleural lavage cytology. The detailed data of these enrolled patients are shown in Data collection and analyses were approved by our institutional review board  in January 2020 . As the research was a retrospective review of chart data and specimens, and no personally identifiable information was included, the need to obtain written informed consent was waived.We evaluated all the pathology slides of resected specimens. After fixing the specimens with 10% formalin and embedding them in paraffin, serial 4 μm sections were stained with hematoxylin/eosin and elastica van Gieson to assess the degree of pleural and vascular invasion. When required, we also stained the sections with D2-40 to evaluate the extent of lymphatic permeation. The cases were reviewed according to the 4th edition of the World Health Organization histological classification and staged according to the 8th edition of the TNM classification of the Union for International Cancer Control.All patients were followed-up at our outpatient department quarterly in the first two years after resection and semi-annually thereafter. Contrast-enhanced CT scans of the chest and upper abdomen were routinely obtained during every scheduled outpatient visit for follow-up. CT or magnetic resonance imaging (MRI) of the brain were also routinely performed semi-annually. Such radiological examinations were also performed when neurological symptoms occurred or when clinical suspicions were raised. Once any metastasis was discovered, a routine examination such as PET was performed to determine the presence of other metastatic sites, if any.Local recurrence was defined as tumor recurrence in contiguous anatomical sites, including the ipsilateral hemithorax and mediastinum after resection. Distant metastasis was defined as tumor recurrence in the contralateral lung or outside the hemithorax and mediastinum after resection. If both local and distant recurrences were noted within three months at the time of initial recurrence, these cases were classified into the distant metastasis group or the multiple metastatic group in number analysis (single vs. multiple). The location of recurrence at the time of final recurrence was defined as all organs where metastatic tumors were identified by the time of death or the last follow-up, regardless of the treatment effect. For patients who did not undergo resection or biopsy to confirm tumor recurrence, judgment was made according to the clinical course, progression, or aggressive clinical behavior.p < 0.1 on univariate analysis were entered into the multivariate forward-backward stepwise model. Statistical significance was defined as p < 0.05. All analyses were performed using SPSS software .The length of organ-specific metastasis-free survival (time to recurrence) was defined as the interval between the date of resection and the date of the specific organ sites of metastasis  or the last follow-up. The length of survival after recurrence was defined as the interval between the date when recurrence was identified and the date of death or the last follow-up. Survival curves were drawn using the Kaplan-Meier method. The log-rank test was used to perform comparisons. To investigate the association between clinicopathological factors and specific organ sites of metastasis, clinicopathological factors were analyzed using the chi-squared/Fisher tests and multivariate logistic regression test. For specific organ site metastasis-free survival, univariate and multivariate analyses were performed using the Cox proportional hazards model. All variables with n = 688) and recurrence cases (n = 223) are shown in p = 0.001; and HR: 4.59, p = 0.001 in all cases, HR: 2.98, p = 0.001; and HR: 2.45, p = 0.002 in recurrence cases, respectively), and NSCLCs with pulmonary metastasis at surgery relapsed significantly more often in the lungs . On evaluating typical distant organ metastasis, the factors correlated with brain metastasis both at initial and final recurrence included young age , adenocarcinoma , and tumors with vascular invasion . Although no variable was associated with bone metastasis in recurrence cases, vascular invasion in surgical specimens was associated with postoperative liver metastasis both at initial and final recurrence  and adrenal gland metastasis   and multivariate (multiple logistic regression tests) analyses in all cases (ctively) .p = 0.012). On evaluating all recurrence sites at the time of final observation, adenocarcinoma  and the presence of lymphatic permeation  were risk factors for multiple recurrence  at the time of initial recurrence were not significantly correlated . Howevercurrence .p = 0.008), larger invasive size , and advanced stage  were independent risk factors of early recurrence among recurrent cases In addition to time to recurrence, vascular invasion was an unfavorable prognostic factor even after recurrence on multivariate analysis (HR: 1.61, The results of the current study demonstrated a strong correlation between some perioperative clinicopathological factors and recurrence patterns including the location and number of metastases at the time of initial and final recurrence. Although a couple of studies have shown various predictors of brain metastasis after complete resection, few studies have shown that NSCLC preferentially relapses to other organs including mediastinal lymph nodes, liver, and adrenal gland on systemic evaluation of recurrence sites ,16,17,18The mediastinal lymph nodes, lungs, brain, bones, adrenal gland are the most common organ sites of metastasis of resected NSCLC ,12,13,14While the liver was not the most common organ site of metastasis of lung cancer, liver metastasis was observed during follow-up after surgery. Few studies have evaluated liver metastasis after the resection of NSCLC. Vascular invasion was an independent risk factor for liver metastasis as well as brain and adrenal gland metastasis. Small malignant nodules in the liver are usually difficult to differentiate from benign liver cysts. Hence, if follow-up CT reveals a liver nodule after the resection of NSCLC with vascular invasion, additional examination is required.Regarding numbers of recurrence sites, even though tumor relapsed postoperatively, single recurrence site was found more frequently among non-adenocarcinomas, NSCLCs without lymphatic permeation, and those with lymph node metastasis at surgery when observed for long period as shown in Among metastatic cases of NSCLC, vascular invasion, large invasive size, and advanced stage were independent risk factors of early recurrence. Although these factors and others—such as lymphatic permeation and pleural invasion—are known prognostic factors for recurrence-free survival, the risk factors for early recurrence have not been evaluated previously ,6,7,8,9.The current study has some limitations and biases. Because the study was retrospective and performed at a single institute, patient selection bias and time trend bias were inevitable. Genetic information such as driver mutations and immunogenic status including programmed cell death 1/programmed cell death 1 ligand 1 status were not evaluated since not all cases had this information. In addition, the study might have had diagnostic bias, as conventional imaging including full-body CT could not always identify all metastatic lesions. There was also bias in distinguishing second primary lung cancer from recurrent NSCLC. Last, although cases with postoperative radiation for prevention of local recurrence were not included, clinical effect of postoperative systemic chemotherapy was not considered because systemic treatment would not change preferential patterns of recurrence.In conclusion, the results of the current study demonstrate that some clinicopathological factors were associated with organ-specific metastasis, number of metastasis sites, and post-recurrence survival. This information is important for determining which patients need follow-up with shorter period systemic evaluation and further studies regarding the molecular mechanisms that lead to organ-specific metastasis in NSCLC.


N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of  Adenovirus transcripts contain N6-methyladenosine (m6A) modification. Here the authors profile m6A modification sites on adenovirus mRNAs using Illumina meRIP-Seq and nanopore direct RNA sequencing, and showcase a role for m6A in splicing of viral late mRNAs. To maximize coding capacity of the 36 kilobase genome, adenovirus employs a tightly regulated gene transcription process. Early genes and subsequent late genes are produced from both strands of DNA using cellular RNA polymerase II and the spliceosomal machinery to generate capped, spliced, and polyadenylated messenger RNAs (mRNA). Besides the four canonical ribose nucleosides, adenoviral RNA is also known to contain RNA modifications3. RNA modifications comprise a family of over one hundred chemical modifications of nucleic acid that can play important roles in both RNA biogenesis and function5. In eukaryotic messenger RNA, N6-methyladenosine (m6A) is the most prevalent RNA modification besides the 7-methylguanosine cap6. This mark has been implicated in regulating multiple processes of RNA maturation, including splicing, polyadenylation, export, translation, and ultimately decay12. Current understanding suggests that m6A is added to messenger RNAs co-transcriptionally in the nucleus by recruitment of a writer complex composed of METTL3, METTL14, WTAP, and other accessory proteins to RNA polymerase II15. These modified mRNAs are bound by reader proteins such as the YTH family 17, various hnRNPs19, and the IGF2B20 family of RNA binding proteins, which affect various downstream fates of the modified mRNAs. This mark is reversible through the action of erasers, and FTO and ALKBH5 have been proposed to demethylate m6A22.Adenovirus is a nuclear-replicating DNA virus with a linear double-stranded genome that is dependent on the host cell machinery for productive infection6A in cellular RNAs, RNAs from several diverse viruses such as adenovirus, Rous sarcoma virus, simian virus 40, herpes simplex virus, and influenza A virus were also shown to contain m6A in internal regions26. In particular, adenovirus serotype 2 was shown to contain m6A at sites away from the cap2. These marks were added to nuclear pre-mRNA and retained in the fully processed cytoplasmic RNA3. These two studies also predicted adenovirus RNAs to contain on average four m6A modifications per transcript, along with low levels of methyl-5-cytosine, for a total of 1/400 (0.25%) of modified viral nucleotides3. However, no subsequent studies have elucidated the functions of m6A modification in adenovirus. With the advent of high-throughput m6A sequencing methods, interest in viral RNA modifications has been rekindled. RNA viruses such as HIV, influenza A, picornavirus, and various Flaviviridae including Zika, dengue, and hepatitis C virus are influenced both positively and negatively by m6A added via METTL3, and many of these viral RNAs are bound by cytoplasmic YTHDF proteins34. In hepatitis B virus, m6A at the same site can both stimulate reverse transcription, as well as reduce mRNA stability35. For DNA viruses such as SV40 and KSHV, deposition of m6A on viral RNA transcripts can enhance viral replication39. Interestingly, multiple labs have published conflicting functions for m6A within the same viral transcript of KSHV, which suggests cell type specific roles39. Of note, recent work using human cytomegalovirus also implicates m6A in controlling aspects of the interferon response, thereby indirectly regulating viral infection41. Since adenovirus is reliant on cellular polymerases and mechanisms to generate and process its viral RNAs, adenovirus infection provides an excellent opportunity to study the consequences of co-transcriptional m6A addition.Shortly after the discovery of m6A have relied on antibody-based immunoprecipitations to enrich for methylated RNA within a relatively large nucleotide window 43. These techniques are indirect, because antibody-precipitated RNA has to be converted to cDNA before sequencing. Although other RNA modifications can be located due to mutations or truncations resulting from reverse transcription46, these events are not generated in the case of m6A due to efficient base pairing with thymine and uracil. Several techniques have circumvented some of these limitations, such as photo-crosslinking assisted m6A sequencing (PA-m6A-Seq)47, m6A individual nucleotide resolution crosslinking and immunoprecipitation (miCLIP)49, and RNA digestion via m6A sensitive RNase (MAZTER-seq)50. In general, these methods are labor intensive, and require either specialized chemical addition to cell culture, large amounts of input material, or higher unique read counts than meRIP-seq. Furthermore, the antibodies used to precipitate m6A may themselves have sequence or structure biases, and cannot distinguish between m6A and the similar modifications m6Am51. To this end, the ability to sequence native RNA molecules directly using nanopore arrays provides a new approach to locate RNA modifications. While detecting modified DNA nucleotides is possible using both PacBio and Oxford Nanopore Technologies platforms53, detection of RNA modifications has proven much more challenging. Recently, two groups have shown detection of m6A using nanopores in yeast total RNA and in human cell lines55. In addition to detecting RNA modifications directly, production of long reads by these platforms provides distinct advantages in the study of gene-dense viral genomes, which encode complex and often overlapping sets of transcripts56. To date, the ability to use direct RNA sequencing to map full-length transcripts and their RNA modifications unambiguously has not been realized.Until recently, sequencing methods to map m6A-interacting enzymes but instead concentrates these host proteins at sites of nascent viral RNA synthesis. While meRIP-Seq was able to identify numerous methylated regions on both early and late kinetic classes of viral mRNA, the complex splicing structure and overlapping nature of the adenovirus transcriptome precluded unambiguous transcript assignments and m6A localization by this method alone. To overcome this limitation, we developed a method to predict sites of m6A modification at single-base resolution within full-length RNA by direct RNA sequencing and used this technique to predict m6A specific to transcript isoforms. While we found that both viral early and late genes are marked by m6A, expression of viral late RNAs in particular decreased dramatically with loss of the cellular m6A writer METTL3. This late gene-biased effect was primarily mediated by decreased RNA splicing efficiency in the absence of METTL3, and could be extended to all of the multiply spliced adenovirus late RNAs. Overall, these results highlight a new technological advancement in long-read RNA sequencing, and reveal that m6A influences the splicing and expression from a viral pathogen.In this study, we found that adenovirus infection does not alter expression of m6A, the impact of infection on cellular components involved in writing, reading, or erasing m6A is unknown. The majority of m6A on messenger RNA is installed co-transcriptionally in the nucleus by a writer complex composed minimally of METTL3, METTL14, and WTAP that associates directly with RNA Pol II15. To examine whether m6A-interacting enzymes are altered during adenovirus infection, we performed immunoblot analysis over a time-course of infection with adenovirus serotype 5 (Ad5) in A549 lung adenocarcinoma cells  to demarcate viral replication centers -selected RNA from A549 cells that were mock-infected or infected with Ad5 for 24 h , as well as the positive control cellular transcript MALAT1. These data indicate that viral early and late transcriptional units contain m6A, and that this modification is added by the cellular writer complex that includes METTL3. However, the exact location of the m6A mark could not be assessed by this approach.We next asked whether viral RNAs were m6A-sequencing platforms, we sought a technique that would provide both single nucleotide resolution, as well as long read length to allow for unambiguous assignment of m6A sites to specific adenovirus mature transcripts. Nanopore sequencing has been used to call DNA modifications directly using differences between measured and expected current values as nucleotides travel through the pore53. It is well established that signal deviations during direct RNA sequencing can result from the presence of one or more base modifications, and that this leads to an increase in base-call error rate around the modified base61. The likelihood of a given nucleotide being modified  can be assessed by a 2 × 5 contingency table to examine the distribution of base-calls between two datasets  at a given genome position, as was recently demonstrated62. Here, a G-test on the distribution of A, C, G, U, and indels provides a score and p-value that requires subsequent (Bonferroni) correction for multiple testing .To address shortcomings of short read-based ming Fig. . This ap6A positive and negative datasets, we generated METTL3 knockout A549 cells using CRISPR-Cas9 with a strategy that included regulated expression of a nuclease-insensitive transgene  dataset, we filtered for a one-fold or greater increase in the ratio of mismatch:match base-calls compared to the M3KO (m6A negative) dataset. This reduced the number of putative sites to 191–217 , we next calculated the distance between each candidate site and its nearest neighbor candidate in a strand-specific manner . Most significant candidate sites filtered for mismatch:match rates (94.2–99%) were located within five nucleotides of an AC motif  produced secondary alignments for many reads and supplementary alignments for a smaller subset of reads  to predict modified nucleotides65. Using the same datasets as inputs, we identified 204 putative m6A sites at the isoform level: 93 of these were also reported by DRUMMER in all four comparisons, while a further 69 were also reported by DRUMMER in 1-3 comparisons. This left just 41 sites predicted by NanoCompore that were not identified by DRUMMER compared to 42 sites predicted by DRUMMER but not NanoCompore  or type-III interferon (IFNL1) transcripts are induced by Ad5 infection or METTL3 depletion, whereas poly(I:C) transfection induces these transcripts several hundred-fold. Furthermore, expression of interferon-stimulated genes such as MX1 and OAS1 was blocked downstream of poly(I:C) transfection by JAK inhibition but not induced by viral infection or METTL3 knockdown. In addition, blocking the interferon pathway with JAK inhibition did not alter the specific decrease in viral late gene we report here  labeling approach67. After 24 h infection of A549 cells with Ad5 following knockdown of METTL3, nascent RNA was labeled with 4sU for the last 10 min of infection. Total RNA was harvested, nascent RNA conjugated with biotin, and precipitated for analysis by qRT-PCR with primers to viral transcripts. We examined unspliced regions of viral RNA near the early E1A and E4 regions, as well as within the tripartite leader of the Major Late Promoter (MLP) that precedes nearly all adenovirus late genes. While relative transcription of the E1A promoter was increased two-fold after METTL3 knockdown, a separate early promoter (E4), and the viral late promoter itself showed no significant change  and Fiber (late gene) as representative of their kinetic classes. Of note, Fiber is the only late gene that can be incorporated into this type of qPCR assay, since no other intron amplifying primer set within the late gene transcriptional unit is specific to a single gene. Upon knockdown of METTL3 or WTAP, the splicing efficiency of the E1A gene did not change, whereas the splicing efficiency of Fiber significantly decreased  sites, the major late transcriptional unit has over 25 splice acceptors and 5 major sites of polyadenylation, leading to over 20 functional open reading framescts Fig. . We examsed Fig. . METTL3-sed Fig. . The effsed Fig. . Fiber RDC1 Fig. . This deDC1 Fig. . A similDC1 Fig. .6A in cis on viral transcript splicing we turned to a transgene splicing assay in which we can ablate m6A sites , or with all 15 DRACH sites present in both exonic and intronic regions silently mutated to ablate deposition of METTL3-dependent m6A  cannot be used to quantify these viral transcripts accurately. Instead, we focused exclusively on the short reads that contained splice junctions, allowing unambiguous assignment to one viral transcript. Since our RNA-seq library preparation was strand-specific, this allowed us to map reads accurately to their strand of origin. Furthermore, we were able to address intron retention of the entire late transcriptional unit by counting only unspliced reads overlapping the major splice donor exiting the conserved third exon of the tripartite leader . This strategy is schematized for top-strand viral genes  selection employed for sequencing in capturing mature transcripts.To determine if mnes Fig. . When wenes Fig. . Of thesWhile short reads targeting splice junctions provided high depth quantitative results, we turned to long-read sequencing with unambiguous transcript mapping as an orthogonal technique to validate these results. We counted full-length RNA transcripts after infection of METTL3 knockdown A549 cells normalized to the read depth of the experiment Fig. . Confirm6A sites to unique RNA isoforms, we asked if any specific features of these RNAs correlated with the loss of expression we observed after METTL3 knockdown. The total number of potential m6A sites per transcript was not correlated with change in expression , total number of exons, or the longest intron within a single transcript (data not shown). While these correlations might hold true for specific cellular genes that have features similar to the viral transcriptome , global correlations appear confounded by the many diverse roles m6A modification can have on any single transcript.Utilizing the power of our long-read dRNA sequencing approach to map m6A methylation in both cellular and viral transcripts during infection with adenovirus. Using a conventional antibody-based approach to detect m6A, we discovered that both viral early and late kinetic classes of mRNA were modified. To identify m6A within overlapping viral transcripts, we devised a technique to predict METTL3-dependent methylation sites in full-length RNAs using direct RNA sequencing. This technique identified specific m6A sites at nucleotide resolution within m6A-enriched regions, and revealed adenovirus RNA isoform-dependent sites of methylation. Ultimately, we found that m6A addition in the alternatively spliced viral late transcripts was important for mediating the efficiency of splicing and accumulation of these messages leading to productive infection. The benefit of m6A-mediated splicing efficiency was directly correlated to the intron length of the transcript, implicating a role for m6A in processing of long transcripts with many alternative splicing choices. Taken together, this work demonstrates m6A-mediated control over the splicing of viral transcripts.In this work we have identified sites of m6A detection within complex transcriptomes. Compared to conventional antibody-based approaches, direct detection of m6A modification in RNA provides many advantages. Focusing on adenovirus RNAs, we show that our technique is highly reproducible between biological replicates with a very low false-positive rate (here defined as candidates that did not map within five nucleotides of an AC dinucleotide). In fact, after candidate site masking, greater than 80% of all sites mapped directly to the adenine within an AC dinucleotide, with the rest often mapping within four nucleotides. This is consistent with the fact that nanopores read RNA in a five nucleotide window61. While we demonstrate this method by assaying for METTL3-dependent m6A modifications, theoretically this technique could work for other RNA modifications where the writing enzyme is known, such as methyl-5-cytosine and pseudouridine45. Compared to other single-nucleotide resolution approaches to m6A detection50, direct RNA sequencing avoids the known biases associated with antibody targeting51 and cDNA synthesis49 and, unlike endoribonuclease cutting50, is not specific to a small subset of methylated motifs. Currently, our technique works best with samples of high read depth, and is thus ideally suited to the study of viruses that dominate the RNA processing of their host cell. However, with improvements in nanopore sequencing technologies such as the PromethION which can yield much higher read depth, this technique should be scalable to the study of cellular transcripts as well.We also defined a new method for isoform-specific m6A methylation has hampered the identification of transcript isoform-specific modifications. While our method still does not enable single molecule resolution, the ability to aggregate similar isoforms in bulk using the long reads produced through direct RNA sequencing allows for isoform-level prediction of m6A sites. Indeed, we demonstrate that isoform-level identification was more sensitive for the detection of potential m6A sites in adenovirus mRNA than the aggregate exome-level data by as much as four-fold. While isoform-level alignment and filtering rely on quality annotations, this technique may need additional customization based on transcript structures in the target of choice. Furthermore, we have only shown the efficacy of this design on polyadenylated messenger RNA. Further studies focusing on non-adenylated or nascent RNAs should prove highly informative for defining the role of m6A-mediated regulation within introns and structural RNAs.The lack of long-read assays to map m6A on splicing has been widely reported in both viral and cellular contexts69, this finding has not been without controversy70. One study in particular showed that while m6A can be detected in nascent pre-mRNA, these locations do not change upon RNA maturation and nuclear export15. Furthermore, knockout of METTL3 in mouse embryonic stem cells led to no detectable change in cassette exon splicing15. In general, our findings agree with this as we do not see the global loss of any particular viral splicing event in the absence of METTL3, only a decrease in total amount of spliced transcripts. However, we did find that m6A modifications present in adenoviral late transcripts increase the splicing efficiency of these RNAs. This result is consistent with the results of others that have found that the presence of m6A near splice sites correlates with the rate and efficiency of splicing when studied in a time-resolved fashion in nascent RNA71. Furthermore, it is known that unspliced cellular RNAs resident in the nucleus can be targeted for decay by the nuclear exosome72. Therefore, our hypothesis is that efficient splicing requires m6A and leads to the accumulation of mature mRNAs that would otherwise be destroyed in the nucleus before accumulating in the cytoplasm.While the influence of m6A modification has also been implicated in the regulation of alternative polyadenylation69. While the majority of the adenovirus early transcripts that were insensitive to METTL3 knockdown contain only one cleavage and polyadenylation site, the adenovirus late transcriptional unit contains five poly(A) sites, and usage must be regulated to be able to splice the downstream isoforms. It is intriguing to note that transcripts generated from the L1 region upstream of the first polyadenylation site were the viral late transcripts least affected by METTL3 knockdown. While our quantitative PCR reaction to test the splicing efficiency of the L5 region cannot fully rule out the possibility of fewer transcripts reaching this region due to altered poly(A) cleavage, our splicing reporter transgene assay that does not contain upstream poly(A) sites argues against this hypothesis. Splicing and polyadenylation are thought to happen co-transcriptionally73, and any effect that m6A might have on coordinating these processes remains an interesting topic for future study.The m6A, we only detected a loss of expression within late genes after depletion of METTL3. This was because m6A increased the efficiency of splicing within adenovirus late transcriptional units, where a plethora of potential splice sites leads to an abundance of alternative splice isoforms. Our data show that even when rates of transcription and RNA decay remain constant, increased splicing efficiency can lead to increased transcript abundance, presumably via protection against nuclear decay mechanisms that target unspliced transcripts. This work highlights how RNA modifications can regulate distinct stages of viral gene expression.In summary, we discovered that while both early and late adenoviral genes are marked by m2. All cell lines tested negative for mycoplasma infection and were routinely tested afterwards using the LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich). A549 cells (ATCC CCL-185) were maintained in Ham’s F-12K medium  supplemented with 10% v/v FBS  and 1% v/v Pen/Strep . HeLa cells (ATCC CCL-2) and HEK293 cells (ATCC CRL-1573) were grown in DMEM  were grown in DMEM supplemented with 10% v/v FBS and 1% v/v Pen/Strep.All cell lines were obtained from American Type Culture Collection (ATCC) and cultured at 37 °C and 5% COAdenovirus serotype 5 (Ad5) was originally purchased from ATCC. All viruses were expanded on HEK293 cells, purified using two sequential rounds of ultracentrifugation in CsCl gradients, and stored in 40% v/v glycerol at −20 °C (short term) or −80 °C (long term). Viral stock titer was determined on HEK293 cells by plaque assay, and all subsequent infections were performed at a multiplicity of infection (MOI) of 20 PFU/cell. Cells were infected at 80-90% confluent monolayers by incubation with diluted virus in a minimal volume of low serum (2%) F-12K for 2 h. After infection viral inoculum was removed by vacuum and full serum growth media was replaced for the duration of the experiment.6A-null psiCheck2 reporter plasmid34, where both Firefly and Renilla luciferase genes have all m6A-DRACH motifs ablated by silent mutations. The original 3’UTR reporter was removed from the end of Renilla luciferase using XhoI and NotI restriction enzymes and this site was healed by the insertion of a recombinant multiple cloning site containing XhoI, AgeI, and NotI using two annealed DNA oligos with complementary sticky ends (Obtained from IDT). From this reporter construct lacking significant 3’UTR, the chimeric intron downstream of the SV40 promoter and upstream of Renilla luciferase was excised using StuI and NheI. Using compatible cut sites, a DNA fragment was cloned into this site encoding the third exon of the Ad5 late gene unit , all intervening intron that did not contain other Ad5 genes or splice sites , and the first 482 nucleotides of the Fiber exon and ORF  such that the Fiber ORF would continue in frame to the Renilla luciferase ORF (WT Fiber). Alternatively, the same fragment but with all 15 DRACH motifs silently mutated was used to generate m6A Mut Fiber. Both fragments were excised from plasmids created as a PriorityGENE service from GeneWiz from provided sequences. All DNA oligos can be found in Supplementary Table 1. DNA transfections were performed using the standard protocol for Lipofectamine2000 (Invitrogen).Fiber-Transgene constructs were created from the previously generated mThe following siRNA pools were obtained from Dharmacon: non-targeting control (D-001206-13-05), METTL3 (M-005170-01-0005), METTL14 (M-014169-00-0005), WTAP (M-017323-02-0005), YTHDC1 (M-015332-01-0005), FTO (M-004159-01-0005), and ALKBH5 (M-004281-01-0005). The following siRNAs were obtained from Qiagen: YTHDF1 (SI00764715), YTHDF2 (SI04174534), YTHDF3 (SI04205761), FTO (SI04177530), and ALKBH5 (SI04138869). All siRNA transfections were performed using the standard protocol for Lipofectamine RNAiMAX (Invitrogen). Poly(I:C) was provided pre-complexed with transfection reagent (Invivogen tlrl-piclv), and was reconstituted fresh with molecular grade water before added to cells at a concentration of 500 ng/ml.32 was cloned from pEFTak into the BB72674 entry vector and the NGG Cas9 PAM site at nucleotide position 117 was silently mutated (G to C) via the Stratagene QuickChange Site-Directed Mutagenesis protocol .A549 cells in 10 cm plates were lysed directly in TRIzol (Thermo Fisher) and RNA was extracted and DNaseI treated (Qiagen). Poly(A) RNA was selected with the Poly(A)Purist MAG Kit before being fractionated (Thermo Fisher) with RNA Fragmentation Reagents (Thermo Fisher) and ethanol precipitated. For meRIP-qPCR the following protocol was identical but RNA was not poly(A) purified or fragmented. MeRIP was performed using the EpiMark N6-methyladenosine Enrichment Kit (NEB) following manufacturer’s instructions. The following modifications were made following the previously published protocol76 (version 2019-09-12) or STAR (v2.5.4b)77. We called peaks using MACS2 (v2.1.2)78 with the following flags: “-q 0.05 -B –call-summits –keep-dup auto –nomodel –extsize 150”. HOMER (v4.11)79 was then used to identify motifs enriched in the identified m6A-peaks. Metagene analysis and visualization was done using deepTools2 (v3.3.1)80.For meRIP-Seq RNA-seq libraries were prepared from both eluate and 10% input mRNA using the TruSeq mRNA library prep kit (Illumina), subjected to quality control (MultiQC), and sequenced on the HiSeq4000 instrument. Both the IP and input samples were mapped to the GRCh37/hg19 genome assembly and the Ad5 genome using the RNA-seq aligner GSNAPThe following primary antibodies were used for cellular proteins: METTL3 , METTL14 , WTAP , YTHDC1 , YTHDF1 , YTHDF2 , YTHDF3 , FTO , ALKBH5 , β-Actin , GAPDH , and RNA Pol II p-Ser2 . Primary anti-Flag tag antibody was obtained from Sigma-Aldrich . Primary antibodies against viral proteins were obtained from: rabbit polyclonal against adenovirus Hexon, Penton, and Fiber , mouse anti-DBP , polyclonal rabbit anti-DBP , mouse anti-E1A , and mouse anti-E1B55K .For western immunoblotting protein samples were prepared by directly lysing cells in lithium dodecyl sulfate (LDS) loading buffer (NuPage) supplemented with 1% beta-mercaptoethanol (BME) and boiled at 95 °C for 10 min. Equal volumes of protein lysate were separated SDS-PAGE in MOPS buffer (Invitrogen) before being transferred onto a nitrocellulose membrane (Millipore) at 35 V for 90 min in 20% methanol solution. Membranes were stained with Ponceau to confirm equal loading and blocked in 5% w/v non-fat milk in TBST supplemented with 0.05% w/v sodium azide. Membranes were incubated with primary antibodies in milk overnight, washed for three times in TBST, incubated with HRP-conjugated secondary (Jackson Laboratories) for 1 h and washed an additional three times in TBST. Proteins were visualized with Pierce ECL or Femto Western Blotting Substrate (Thermo Scientific) and detected using a Syngene G-Box. Images were processed and assembled in Adobe Photoshop and Illustrator CS6.For immunofluorescence A549 cells were grown on glass coverslips in 24-well plates, mock-infected or infected with Ad5 for 18 h, washed twice with PBS and then fixed in 4% w/v Paraformaldehyde for 15 min. Cells were permeabilized with 0.5% v/v Triton-X in PBS for 10 min, and blocked in 3% w/v BSA in PBS (+0.05% w/v sodium azide) for 1 h. Primary antibody dilutions were added to coverslips in 3% w/v BSA in PBS (+0.05% w/v sodium azide) for 1 h, washed with 3% BSA in PBS three times, followed by secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI) for 1 h. Secondary antibodies were used at 1:500 dilution and conjugated to Alexa-Fluor 488 (Invitrogen A-11001 or A-11008), 555 (Invitrogen A-21422 or A-21428), or 568 (Invitrogen A-11004 or A-11011) in anti-mouse or anti-rabbit. Coverslips were mounted onto glass slides using ProLong Gold Antifade Reagent . Immunofluorescence was visualized using a Zeiss LSM 710 Confocal microscope  and ZEN 2011 software. Images were processed in FIJI (v1.52p) and assembled in Adobe Photoshop and Illustrator 2020.3-Deazadenosine was purchased from Sigma-Aldrich (D8296) and resuspended in Dimethyl Sulfoxide (DMSO) before being added to cells at the time of infection to a final concentration of 25 µM. Ruxolitinub was purchased from Sigma-Aldrich (G-6185) and resuspended in DMSO before being added to cells immediately prior to infection with Ad5 or transfection with poly(I:C) at a final concentration of 4 µM. 5,6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole (DRB) was purchased from Cayman Chemical (Item No 10010302) and resuspended in DMSO before being added to cells at 24 h post infection to a final concentration of 60 µM.HPRT1 and GAPDH.Total RNA was isolated from cells by either TRIzol extraction (Thermo Fisher) or RNeasy Micro kit (Qiagen), following manufacturer protocols. RNA was treated with RNase-free DNase I (Qiagen), either on-column or after ethanol precipitation. RNA was converted to complementary DNA (cDNA) using 1 μg of input RNA in the High Capcity RNA-to-cDNA kit (Thermo Fisher). Quantitative PCR was performed using the standard protocol for SYBR Green reagents (Thermo Fisher) in a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). All primers were used at 10 μM and sequences can be found in Supplementary Table Infected cells were harvested at the indicated time points post infection by trypsin and total DNA was harvested using the PureLink Genomic DNA kit (Invitrogen). DNA quantity was assessed by qPCR and SYBR green reagents using primers for genomic regions of Ad5 and normalized to cellular tubulin (See Supplementary Table g), 4 °C, 5 min. Lysates were serially diluted in DMEM supplemented with 2% v/v FBS and 1% v/v Pen/Strep to infect a confluent monolayer of HEK293 cells seeded in 12-well plates. After incubation for 2 h at 37 °C, the infection media was removed, and cells were overlaid with DMEM containing 0.45% w/v SeaPlaque agarose (Lonza) in addition to 2% v/v FBS and 1% v/v Pen/Strep. Plaques were stained using 1% w/v crystal violet in 50% v/v ethanol between 6 to 7 days post-infection.Infected cells seeded in 12-well plates were harvested by scraping at the indicated time points and lysed by three cycles of freeze-thawing in liquid nitrogen. Cell debris was removed from lysates by centrifugation at max speed , biotin was removed from nascent RNA using 100 mM dithiothreitol (DTT), and RNA was isopropanol precipitated. One µg of total RNA (T) and an equivalent volume of nascent RNA (N) were converted to cDNA and qPCR was performed as described above. Relative transcription rates were determined by the ΔΔCt method to compare nascent transcript levels between control and siRNA treated cells normalized to nascent GAPDH RNA.To assess relative RNA transcription rate, cells were treated with 1 mM 4-thiouridine  for exactly 10 min. Infection was stopped and RNA harvested using 1 ml TRIzol (Thermo Fisher), following manufacturer’s instructions. A fraction of the total RNA was reserved as input, and the remaining 4sU-labeled nascent RNA was biotinylated using MTSEA-Biotin-XX  as previously described76 (version 2019-09-12). The algorithm was given known human gene models provided by GENCODE (release_27_hg19) to achieve higher mapping accuracy. We used R package ggplot2 (v3.3.0) for visualization. Downstream analysis and visualization was done using deepTools2 (v3.3.1)80. Splice junctions were extracted using RegTools (v0.2.0)82 and visualized in Integrative Genomics Viewer.Total RNA from three biological replicates of Control knockdown or three biological replicates of METTL3-knockdown A549 cells infected with Ad5 for 24 h were sent to Genewiz for preparation into strand-specific RNA-Seq libraries. Libraries were then run spread over three lanes of an Illumina HiSeq 2500 using a 150 bp paired-end protocol. Raw reads were mapped to the GRCh37/hg19 genome assembly and the Ad5 genome using the RNA-seq aligner GSNAP61 Sequencing was performed on a MinION MkIb using R9.4.1 (rev D) flow cells (Oxford Nanopore Technologies Ltd.) for 18–23 h (one library per flowcell) and yielded between 1,000,000 – 2,000,000 reads per dataset. Raw fast5 datasets were then basecalled using Guppy v3.2.2 (-f FLO-MIN106 -k SQK-RNA002) with only reads in the pass folder used for subsequent analyses. Sequence reads were aligned against the adenovirus Ad5 genome, using MiniMap2 (v2.15 -ax splice -k14 -uf –secondary = no), a splice aware aligner64, with subsequent parsing through SAMtools (v1.9)83 and BEDtools (v2.27.1)84. Here sequence reads were filtered to retain only primary alignments (SAM FLAG 0 (top strand) or 16 (bottom strand)). Coverage plots  with mapping qualities (MapQ) >0 were retained. Unambiguous transcripts were defined as those that began at a designated 3’ poly(A) tail and extended to within 50 nucleotides of an annotated TSS, or within the third exon of the late transcripts tripartite leader.Direct RNA sequencing libraries were generated from 680 to 1800 ng of poly(A) RNA, isolated using the Dynabeads™ mRNA Purification Kit . Isolated poly(A) RNA was subsequently spiked with 0.3 µl of a synthetic Enolase 2 (ENO2) calibration RNA (Oxford Nanopore Technologies Ltd.) and prepared for sequencing as described previously6A sites on adenoviral RNAs was performed using a new tool, DRUMMER (https://github.com/DepledgeLab/DRUMMER/), which predicts RNA modifications based on comparative profiling of basecall error rates at both exome and isoform level. Briefly, DRUMMER takes sorted.bam files from two datasets as input and parses these individually in a stranded manner using BAM Readcount (https://github.com/genome/bam-readcount) to generate base-call distributions  at each position in the alignment. For each comparison , a G-test was performed on the base-call distributions at each position in each dataset with subsequent Bonferroni correction for multiple testing. Positions with a read coverage of <100× in one or both datasets being compared were excluded from this analysis. To further filter the dataset following the G-test, we calculated the ratio of match:mismatch fractions at each position. Candidate sites were those that gave a statistically significant G-test result (bonferroni adjusted p < 0.01) and had a one-fold or higher reduction in the match:mismatch fraction in the WT dataset compared to the METTL3 knockout dataset. Subsequent masking of clustered candidates was performed by collapsing all sites located within five nucleotides of the same AC motif into a single representative, chosen by picking the site with highest G-test score. Where sites were located within ten nucleotides of each other, but further than five nucleotides from an AC motif, collapsing was performed by selecting the site with the highest G-test score.The identification of putative m6A site prediction using NanoCompore, which predicts RNA modifications based on signal level analyses of current intensity and dwell time for nucleotides translocating through nanopores65. Here, read indexing and resquiggling were performed using Nanopolish and NanopolishComp prior to running NanoCompore SampComp (–logit –sequence_context 2 –sequence_context_weights harmonic) to compare both biological replicates of the WT and METTL3-knockout datasets in a 2 × 2 analysis. Note that NanoCompore processing times were extremely slow for transcripts with >1000 dRNA-Seq reads assigned, so a random subsampling approach (1000 reads) was used in these situations. Putative m6A modified sites were identified on the basis of a GMM logit p-value (context 2) <0.01 and an Logit LOR score >0.5 or <−0.5. Where multiple sites were present with five nucleotides, only the site with the lowest p-value was retained. To calculate the degree of methylation in WT and METTL3-knockout datasets, we parsed the cluster counts column to determine the fraction of reads in datasets with an m6A at a defined sequence position with the m6A depletion estimate representing the difference between these values.To further validate our data, we also performed mFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification. Mapping the epigenetic modifications of DNA and RNA becomes increasingly crucial to understand their diverse biological functions. At least 17 and 160 types of chemical modifications have been discovered in DNA and RNA, respectively  in mammalian genome, which is now widely accepted as the “sixth base”. Tahiliani et al. showed that the ten-eleven translocation 1 (TET1) enzyme catalyses the conversion of 5mC to 5hmC , N6,2’-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), N4-acetylcytidine (ac4C), pseudouridine (Ψ), N7-methylguanosine (m7G), etc.Similar to DNA, cellular RNA is also decorated with diverse chemical modifications, and such modifications participate in all aspects of RNA metabolism. The multitude of modifications in RNA add a new layer to the gene regulation, leading to the emerging field of “RNA epigenetics” or “epitranscriptomics” He, . RecentlThe interest in understanding the functions of DNA and RNA modifications as well as the related molecular mechanisms has been growing, which drives the progresses in developing chemical and biochemical tools to detect specific modifications within genomes and transcriptomes. On the other hand, the development of new technologies contributes to increased knowledge on modifications of DNA and RNA. In this review, we mainly focus on high-throughput detection strategies for DNA Fig.  and RNA Bisulfite sequencing (BS-Seq), is regarded as the gold standard for 5mC detection. It is based on the differential reactivity of bisulfite between methylated C (5mC and 5hmC) and unmethylated C, in which DNA is treated with bisulfite that leads to the deamination of unmethylated C to Uracil (U) while methylated C is resistant to deamination. In the subsequent PCR amplification, methylated C remains C while unmethylated C can be readout as T. Whole-genome bisulfite sequencing (WGBS), as a whole genome method of BS-Seq, has been widely utilized in DNA methylation profiling, as it can provide single-base resolution with full genome coverage Fig. . The reaRecently, two bisulfite-free whole-genome base-resolution DNA methylation sequencing methods have been developed to replace WGBS. The TET-assisted pyridine borane sequencing (TAPS)  has been developed by New England BioLabs, which first utilizes TET2 and βGT to oxidize and glucosylate 5mC and 5hmC to 5gmC. This provides protection from deamination by the AID/APOBEC family DNA deaminase APOBEC3A in the next step while unmodified C is deaminated to U Fig. . EM-Seq 4) to produce 5fC that subsequently converted to U by bisulfite treatment with an overall 5hmC-to-U conversion rate of 94.5%   is an approach in which 5hmC is firstly modified using βGT, and then 5mC is subsequently oxidized to 5caC by TET1 Fig. , b. TAB-2RuO4) with a conversion efficiency of ~94% and subsequent chemical labeling and conversion of 5fC to T during PCR  is a bisulfite-free method to map 5hmC, which is based on selective oxidation of 5hmC to 5fC by potassium ruthenate   to form a derivative which can not be converted to U during the following BS-Seq. Therefore, the precise genomic locations of 5fC at single-base level can be identified, through comparison of EtONH2-treated BS-Seq and conventional BS-Seq of the same sample. Applying fCAB-Seq, low abundance 5fC at endogenous loci at levels down to only a few percent could be detected. Another bisulfite-based method termed reduced BS-Seq (redBS-Seq) was developed to quantititively detect 5fC in genomic DNA at single-base resolution  followed by BS-Seq  was the first quantitative method to sequence 5fC at single-base resolution in genomic DNA , can quantitatively detect 5fC and 5caC simultaneously at single-base resolution  was introduced based on malononitrile labeling of 5fC , an azido derivative of 1,3-indandione (AI) was used to achieve selectively labelling of 5fC  has been developed to sequence 5caC at base-resolation  consisting of METTL3 and METTL14 as well as additional protein subunits    have been developed, providing examples of antibody-independent m6A sequencing methods  significantly affects alternative splicing  , hepatitis C virus (HCV), influenza A virus (IAV), simian virus 40 (SV40), and human immunodeficiency virus 1 (HIV-1). m6A negatively regulates the infection of ZIKV and HCV  of HIV-1   with antibody against m6A , which can be further methylated by methyltransferase PCIF1 to form m6Am was originally suggested to alter mRNA stability  HEK293T or MEL624 cells does not significantly affect the level of mRNAs with m6Am either   utilizing an evolved reverse transcriptase that reads through m1A more efficiently also reported hundreds of m1A sites, further corroborating its prevalence in mRNA  exploited the formation of covalent bond between C271A mutant NSUN2 and substrate to detect the enriched m5C targets  family enzymes to form hm5C , to allow the pre-enrichment of Ψ-containing RNA through biotin pulldown, which identified about 2,000 Ψ sites in human mRNA  rely on a chemical, N-cyclohexyl-N’-β-(4-methylmorpholinium) ethylcarbodiimide (CMC), which can specifically label Ψ  were highly correlated, suggesting the abundance and thus the sequencing depth certainly influence the modification list. Further comparisons of different methods have revealed other factors that need to be considered, such as varied sequencing depth, different bioinformatics algorithms and cutoffs, distinct cell lines and/or growth conditions, etc  sequencing  deaminase/apolipoprotein B mRNA editing enzyme; AMD-Seq, APOBEC3A-mediated deamination sequencing; APOBEC3A, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like; BER, base excision repair; BS, bisulfite sequencing; C, cytosine; CAB-Seq, chemical modification-assisted bisulfite sequencing; CLEVER-Seq, chemical-labeling-enabled C-to-T conversion sequencing; CMC, N-cyclohexyl-N’-β-(4-methylmorpholinium) ethylcarbodiimide; DHU, dihydrouracil; DIP-CAB-Seq, DNA immunoprecipitation-coupled CAB-Seq; DNATS, DNA methyltransferases; EM-Seq, Enzymatic Methyl-Seq; EDC, 1-ethyl-3-[3-imethylaminopropyl] carbodiimide hydrochloride; EtONH2, O-ethylhydroxylamine; fCAB-Seq, 5fC chemically assisted bisulfite sequencing; fC-CET, 5fC cyclization-enabled C-to-T transition; FFPE, formalin-fixed paraffin embedded; hm5C, 5-hydroxymethylcytosine; hmC-CATCH, chemical-assistant C-to-T conversion of 5hmC sequencing; IP, immunoprecipitation; K2RuO4, potassium ruthenate; KRuO4, potassium perruthenate; lncRNA, long noncoding RNA; lrTAPs, long-read TAPS; m1A, N1-methyladenosine; m6A, N6-methyladenosine; m6Am, N6,2’-O-dimethyladenosine; m5C, 5-methylcytosine; m7G, N7-methylguanosine; MAB-Seq, M.SssI methylase-assisted bisulfite sequencing; MeDIP, Methylated DNA immunoprecipitation; mESCs, mouse embryonic stem cells, mRNA, messenger RNA; NaBH4, sodium borohydride; oxBS-Seq, Oxidative bisulfite sequencing; PCR, polymerase chain reaction; rRNA, ribosomal RNA; RT, reverse transcription; SMRT, Pac Bio Single-Molecule Real-Time; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; T, thymine; TAB-Seq, TET-assisted bisulfite sequencing; TAPS, TET-assisted pyridine borane sequencing; TDG, thymine DNA glycosylase; Tdg KO mESCs, Tdg Knowkout mESCs; TET1, ten-eleven translocation 1; tRNA, transfer RNA; U, Uracil; WGBS, Whole-genome bisulfite sequencing; β-GT, β-glucosyltransferase; Ψ, pseudouridine.5caC, 5-carboxylcytosine; 5fC, 5-formylcytosine; 5gmC, 5-(β-glucosyloxymethyl) cytosine; 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; 6mA, N


Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5′ single-stranded tail in a 5′ to 3′ direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5′-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations. SCV is an enveloped, positive single-stranded RNA virus (or (+) ssRNA virus) with a genome of ~30 kb in length3. Two-thirds of the SCV genome at the 5′-end comprise replicase genes (orf1ab) encoding 16 nonstructural proteins (nsPs). The replicase genes comprising open reading frames (OFR1a and 1b) are translated into two large replicative polyproteins, pp1ab (~790 kDa) and pp1a (~490 kDa), which are involved with and without ribosomal frameshifting into the −1 frame5. These two translational polyproteins are processed autoproteolytically by the major viral cysteine proteases MPRO or 3CLPRO to produce 16 non-structural proteins (nsPs), including RNA-dependent RNA polymerases  and NTPase/helicase (nsP13)8. These viral replicases are the core of membrane-bound replication-transcription complexes that synthesize the entire viral genome and eight subgenomic mRNAs10.Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease caused by a novel coronavirus (SARS-CoV or SCV) that has claimed almost 800 deaths in early 200313. In addition, the inhibition of these targets may interfere with the metabolism of the infecting virus without strong side effects in patients. Several viral helicases have been used as proven drug targets due to the inhibition of helicase activity in animal models of herpes simplex virus (HSV) and in the treatment of hepatitis C15. Therefore, much effort has been spent on the development of small-molecule inhibitors and chemicals as drug candidates to inhibit the function of SARS coronavirus helicase nsP13 (SCV nsP13)20. Helicases are motor proteins that unwind a double-stranded (ds) nucleic acid into two single-stranded (ss) nucleic acids using energy derived from NTP hydrolysis during translocation along a single strand for nucleic acid replication, recombination, and DNA repair24. Helicases are classified into distinct classes depending on whether they can bind ss nucleic acid, unwind dsRNA or dsDNA or both, the polarity of the unwinding (5′ to 3′ or 3′ to 5′), and whether certain signature motifs are present in the primary sequence28. The SCV helicase nsP13 can unwind both dsRNA and dsDNA with a 5′-ss tail along the polarity of 5′ to 3′29, and the enzyme can hydrolyze all deoxyribonucleotide and ribonucleotide triphosphates6. In a previous study, we revealed that the amplitudes of SCV helicase nsP13 are decreased as the length of duplex DNA increased in the presence of ATP30. Moreover, the amplitudes of duplex DNA with a short 5′-ssDNA tail were very low compared with other duplex DNAs with a long 5′-ssDNA tail. Multiple nsP13 monomers bind to more than one site along the ssDNA. SCV helicase nsP13 is derived from (+) ssRNA virus and is essential for the replication of viral RNA. Nevertheless, no study has investigated definite unwinding kinetics of duplex RNA as a substrate by nsP13. Thus, it is highly important to understand the unwinding mechanism of duplex RNA by nsP13 to develop more efficient anti-SCV drugs in depth.Because viral helicase is considered to be essential for subsequent viral replication and proliferation, it is an important potential target for antiviral therapyIn this study, we used partial duplex RNAs and DNAs as a model and control substrate, and the single-turnover kinetics of duplex RNA with a 5′-ss tail of different lengths was investigated according to various times and the concentration of ATP or nsP13. We observed that nsP13 exhibits unwinding results of different patterns in RNAs unlike DNAs. Contrary to expectation, the length of the 5′-ss tail, used as a loading strand of multiple nsP13 monomers, was not significantly related to enhancement of the RNA unwinding process. However, we demonstrated that this result is caused by the substrate specificity of nsP13. We also revealed that the unwinding process of duplex RNA by nsP13 is significantly related to the amount of ATP required for the binding, translocation, and unwinding of substrate although it is not completely clear how ATP and ATPase cycles function in the work performed by helicase nsP13. In addition, we observed that the cooperative translocation by nsP13 is also related to the amount of ATP.27. However, although nsP13 is derived from the (+) ssRNA virus, no mechanistic study exists concerning unwinding using duplex RNA as a model substrate. In this study, we used duplex RNA as a substrate to observe the single-turnover kinetics of duplex RNA unwinding, and the results were compared with the unwinding kinetics of duplex DNA. To investigate the unwinding mechanism of duplex RNA, we purified nsP13 with high purity and activity for in vitro studies as described in the Materials and Methods section , is defined as the probability of the separation of a base pair by helicase in contrast to helicase dissociating from that position on the RNA. It is evaluated as the distance moved by the helicase before dissociating from the RNA. Therefore, to estimate the relative processivity of nsP13 on different duplex lengths, three substrates  unlike dsRNA standard assay. Although weak unwinding was observed due to low concentration of nsP13, the accumulation of unwound ssRNA was slightly higher with an increasing 5′-ss tail  to block the preloading of nsP13 onto the 5′-ss loading strand unlike the standard single-turnover condition used in this study. Also, trap oligo was separately prepared from nsP13. The unwinding reaction was immediately initiated by mixing mixture A (nsP13) and mixture B (duplex substrate and trap oligo) without pre-incubation at 37 °C Fig. . This ex40. Additionally, we conducted the gel shift assay using three dsRNA substrates  in a nsP13 dose-dependent manner without trap oligos. In this experiment, the bands with significant retardation were increased depending on the increase in the length of the 5′-ss tail  and nsP13 in a dose-dependent manner Fig. . The ban30, and the essential activity of nsP13 is one-way translocation driven by the ATPase cycles33. Each cycle of the ATPase reaction requires basic steps containing ATP binding, hydrolysis, phosphate release, ADP release, and rebinding of ATP. Therefore, the amount of ATP might influence the unwinding mechanism by nsP13. In a previous experiment using duplex DNA, although there were differences depending on the length of the 5′-ss tail and duplex DNA, nsP13 showed high unwinding activity in the same concentration of ATP. However, in this study, duplex RNA unwinding by nsP13 exhibited very poor unwinding activity compared with duplex DNA of the same size and sequence (except for the presence of U versus T) in the same concentration of ATP. These results might strongly imply that nsP13 has different ATP demands depending on the type of substrate in the unwinding process.The unwinding of duplex substrates by helicase is a stepwise process that comprises helicase binding, translocation, and local separation of duplex substrates. The nsP13 prefers ATP as an energy sourceTo distinguish the ATP amount required according to the type of substrate, two different duplex RNA substrates (20U/25D and 30U/25D RNA) were used in this experiment. For the helicase reaction, 5 nM of duplex substrates and 0.5 µM of nsP13 were used with various ATP concentrations  to assess the unwinding effect by increasing the ATP concentration Fig. . AlthougTo further examine the unwinding effect with an increased ATP concentration, the concentration of nsP13 was determined for the duplex DNA substrates  was used in this experiment. The substrate was incubated at two different nsP13 concentrations (0.5 µM and 2 µM), and the reaction was initiated by the addition of 8 mM ATP, 13 mM MgCl46. We observed that nsP13 could not unwind duplex RNA efficiently compared with that in a previous study using a DNA substrate under the same single-turnover condition although it is a known RNA helicase30. Additionally, we also identified that duplex DNA was unwound almost to completion, but the unwinding of duplex RNA was very poor under the same conditions. These results indicate that RNA helicase nsP13 is more favorable on duplex DNA for duplex unwinding than its duplex RNA counterpart. These findings are similar to the mechanism previously suggested for NPH-II RNA helicase and HCV NS3 RNA helicase47. Despite using a higher concentration of nsP13 and a longer reaction time in this study, nsP13 exhibited poorer processivity in RNA substrates than the previous result using DNA substrates. More importantly, given that nsP13 is essential for the viral RNA replication of the SARS coronavirus, these results suggest that nsP13 requires many non-structural proteins such as RNA-dependent RNA polymerase (RdRp) to enhance its processivity activity toward RNA substrates. Recently, the analysis of single-turnover kinetics exhibited that SCV nsP13 alone has a low processivity for RNA unwinding but a 2-fold higher helicase activity in the presence of RdRp29. Although nsP13 may have different affinities depending on RNA and DNA may be a determining factor for the processivity of nsP13 in duplex unwinding, more intensive experiments to monitor its affinities toward DNA or RNA are needed to understand the difference in the processivity.SCV helicase nsP13 unwinds both DNA and RNA substrates using the energy of ATP hydrolysis with 5′ to 3′ polarity48. However, in this study using duplex RNAs, the unwinding of nsP13 was decreased as the length of the 5′ ss tail increased. nsP13 showed almost no unwinding efficiency on RNA with a 5′-ss tail of 30 nts compared with that on DNA. More surprisingly, we demonstrated that nsP13 is more favorable on DNA in the early stages of reaction initiation and binds DNA substrates faster than RNA substrates of the same sequence. HCV NS3 RNA helicase also showed poor processivity on RNA than DNA31, and some observations revealed that the loading strand of the hybrid substrate was chosen for the unwinding efficiency of the helicase in the single-turnover condition49. For these reasons, although RNA helicase nsP13 can unwind both duplex RNA and DNA, their different affinities for RNA and DNA may also be a key factor to determine nucleic acid unwinding and translocation by nsP13 in unwinding of the duplex. We also demonstrated that although nsP13 interacts with both DNA and RNA, nsP13 shows different substrate specificity for either DNA or RNA. The nsP13 and RNA complexes increased as the concentration of nsP13 increased, and the accumulation of free unwound ssRNA was decreased. By contrast, while free unwound ssDNA increased as the concentration of nsP13 increased, the helicase and DNA complexes were decreased. These results suggest that RNA helicase nsP13 has higher binding affinity to RNA than to DNA. Previous studies demonstrate that binding of ATP to helicase may cause the conformational changes for their substrate binding40. To further understand RNA binding affinity of nsP13, we performed the gel shift assay with three duplex RNA substrates  without trap oligos. In a nsP13 dose-dependent manner without trap oligos, the bands with significant retardation were increased depending on the increase in the length of the 5′-ss tail. This substrate specificity of nsP13 was exhibited as very low ability to unwind and translocate RNA substrates compared to DNA substrates. This similar result was also previously observed for HCV NS351.In a previous study, we demonstrated the unwinding of duplex DNA by increasing the length of the 5′-ss tail. These data suggested that multiple nsP13 monomers binding on the 5′-ss tail are necessary for efficient DNA unwinding. Therefore, the increase in helicase activity with increasing 5′-ss tail may be explained by a model of functional protein interaction of unwinding described in terms of an inchworm model55, it might be important to identify whether the difference in the thermodynamic stability determines the unwinding and processivity. However, a previous study demonstrated that the unique processivity of HCV NS3 helicase on DNA is not caused by the thermodynamic differences between RNA and DNA duplexes31. Most non-ring-shaped (or monomeric) SF1 helicase, such as SCV helicase nsP13, comprises two nucleic acid binding sites, RecA-like domains, in the motor core domains. Helicase exists in open and closed conformations and cycling between the two is necessary for translocation. Eventually, a conformational change in the helicase is caused upon ATP binding. Upon ATP hydrolysis and/or ADP and phosphate release, the helicase relaxes to the initial conformation, resulting in coordinated domain opening and translocation of the helicase56. Therefore, the closed state of helicase is populated upon ATP binding, and the open state is populated when the ATPase products dissociate. These reactions, a cycle of ATP binding and hydrolysis, lead to a stepping movement of the helicase along the direction of unwinding. Previous studies demonstrated that HCV NS3 show a weaker affinity for binding to substrates in the closed state than the open state58. As shown above, we observed that nsP13 exhibits higher binding affinity and lower unwinding activity for RNA substrates than DNA substrates in the same ATP concentration. These characteristics may be explained by different ATP demand of nsP13 depending on the substrate. Also, these results may imply that the open state of nsP13 binds with a higher affinity to RNA than to DNA and hence requires higher concentrations of ATP to drive it into the closed state. We demonstrated that the length of the 5′-ss tail does not enhance duplex RNA unwinding, but the accumulation of unwound ssRNA increases as the ATP increase in a concentration-dependent manner. Moreover, at high ATP concentrations, nsP13 was more active at a high concentration of helicase, implying enhanced cooperative translocation. This result suggests that the unwinding of duplex RNA by nsP13 is a considerably energy-consuming reaction, which also implies the requirement of higher ATP concentration for its open and closed states or stable binding to ssRNA in translocation process. Although additional experiments using ADP is required for measuring DNA and RNA affinity in binding and translocation process, cooperative translocation of nsP13 in ssRNA may be expected to require higher ATPs for stable open and closed state of nsP13 than that of ssDNA. By contrast, although enhancement of duplex DNA unwinding was observed by the increase in the 5′-ss tail, a high ATP concentration was attributable to the decrease in helicase activity, implying that the excessive activity by a high concentration of ATP causes binding instability onto the substrate. Also, these results imply that an unnecessary high ATP concentration in ssDNA leads to excess open and closed structural conformation and cycling between the two, which imply easy slipping of nsP13 from the substrate  and was purified as described previously16.The gene encoding the SCV helicase nsp13 domain was kindly provided by Dr. Huang, J.-D. University of Hong Kong, China. The SCV helicase nsp13 was expressed in 32P]-ATP . The 5′-end labeled single-stranded oligonucleotides were purified using Micro Bio-SpinTM columns . The partial duplex substrates (RNA and DNA) used in this study are shown in Supplementary Table 30.RNA and DNA oligonucleotides were commercially purchased from Integrated DNA Technologies  and were purified by denaturing polyacrylamide gel electrophoresis (PAGE). The single-stranded oligo fragments were radiolabeled at the 5′-end with T4 polynucleotide kinase  and  and mixture B  were preincubated for 15 min at 37 °C, and then the unwinding reaction was initiated by mixing the two reaction mixtures. Additionally, different concentrations of helicase nsp13 were used in duplex DNA unwinding (0.2 μM) and Supplementary Fig. 32P-labeled duplex substrates (5 nM), 5 µM trap oligo (unlabeled bottom strand), 50 mM Tris-HCl (pH 6.8), 50 mM NaCl, 2 mM ATP, and 10% glycerol] and mixture B  were separately made. Mixture A and B were immediately mixed without preincubation and reacted at 37 °C. All the reactions were performed at 37 °C for various times, and the reactions were quenched by the addition of an equal volume of quenching solution . The released single-stranded oligonucleotide (ssRNA or ssDNA) and unwound double-stranded duplex (dsRNA or dsDNA) were resolved by 15% non-denaturing PAGE. The control for measuring maximum unwinding was produced by heating duplex substrates for 5 min at 95 °C and loading immediately on the gel. The gel was exposed to X-ray film, and the band intensities representing the unwound strand were quantified using Image J software. The fraction of unwound RNAs or DNAs was calculated as described previously30, and the single-exponential equation [Eq. 1] was used to fit the unwinding kinetics: F(t) = A (A – exp(–k1*t)) (1), where F(t) is the fraction of unwound at time t, A is the amplitude of unwinding, and k1 is the observed rate constant of the burst phase.For the standard RNA unwinding assay, each equal volume of mixture A , and each equal volume of mixture A and B mixture B  were preincubated for 15 min at 37 °C. After preincubation, the mixture A unwinding reaction was initiated by mixing mixture B at 37 °C for 1 hr. The reaction was stopped by an equal volume of quenching solution . The protein-substrate complexes were then analyzed using the gel shift assay by 15% (with trap oligos) or 10% (without trap oligos) non-denaturing PAGE. The protein-substrate complexes were then analyzed using the gel shift assay by 10% non-denaturing PAGE. The gel was exposed to X-ray film, and the band intensities representing the bound substrate were quantified using Image J software.To conduct gel shift assay, various concentrations of helicase nsP13 were added to mixture A [ɣ-Supplement information.


The current research investigates maximizers’ responses to restrictions of choice freedom during lockdown in the context of the COVID-19 pandemic. Having as a starting point the assumption that for maximizers choice is constitutive of identity, this research proposes that maximizing is associated with search for existential meaning in life. In turn, maximizers’ propensity to search for meaning is associated with a higher susceptibility to experience reactance when their freedom of choice is restricted, which is further associated with higher engagement in online shopping during lockdown presumably as a means to combat reactance and restore choice freedom. Using the lockdown in spring 2020 as a naturalistic context to study consumer responses to restrictions of choice freedom, results of an online study in Austria support these predictions. These findings advance a view of maximizers as “lay existentialists,” who view choice as a meaning-making device that is tightly linked to their sense of identity. As a result, when their choice freedom is threatened, maximizers may respond with higher reactance and engage in restorative actions. In the past two decades, research on individual differences in decision-making has shed much light on how maximizing – the tendency to strive for the best choice – relates to various spheres of life  in maximizing search more for meaning in life driven by a view of choice as paramount to identity construction and expression, then they might also experience more reactance when a situation does not afford them unconstrained choice. A classical finding in social psychology is that when people feel that any of their free behaviors is eliminated or threatened with elimination, they experience an unpleasant motivational arousal, which is called reactance . AlthougFinally, this research also examines an outcome related to consumer behavior. Theory posits that the aversive motivational state of reactance can result in behaviors that attempt to reestablish the freedom that has been eliminated . In thisIn short, the current research examines whether individuals high (vs. low) in maximizing are more likely to (a) view choice as identity, (b) search for meaning in life, (c) experience consumer reactance when choice freedom is limited, and eventually (d) engage more in online shopping during lockdown as a way to restore freedom of choice. One pilot study and one main study test the above predictions.A pilot study first tested the underlying assumption of this research that for individuals high (vs. low) in maximizing choice is more tightly tied to identity.N = 132) recruited from prolific for monetary compensation  and a European student sample (N = 167) recruited from a subject pool of a large Austrian university for course credit . A sensitivity power analysis showed that the respective sample sizes can reliably detect small to medium effect sizes of ρ = 0.21 and ρ = 0.19 (one-tailed) with an alpha level of 0.05 and power of 0.80.The association between maximizing and choice as identity was pilot-tested in two samples: a United States community sample (strongly disagree and 7 = strongly agree). This scale has been recommended as the most suitable measurement of the maximizing construct among the various available alternatives (α = 0.87 in the United States sample and α = 0.80 in the Austrian sample). Then, participants filled out a measure of choice as identity that was devised for the purpose of this research (see strongly disagree and 7 = strongly agree). In both samples, all items loaded on a single factor . Hence, a composite score of choice as identity was created (α = 0.89 in both samples).Both studies were conducted before the COVID-19 pandemic. Participants in the United States took the study online, whereas participants in Austria took the study in the lab. Participants first filled out the Maximizing Tendency Scale . It consrnatives , becausernatives , as the rnatives . In bothr = 0.36, 95% CI = , p < 0.001, and in the Austrian sample, r = 0.34, 95% CI = , p < 0.001. Using data from two different samples, the pilot study provides convergent evidence that individuals high (vs. low) in maximizing are more likely to construe choice as constitutive of identity.Results showed a significant positive correlation between maximizing and choice as identity both in the United States sample, via the university subject pool and took part in the study online for course credit. Two participants failed an attention check (to select a specific answer in one question) and were excluded from further analyses. The final sample comprised 137 participants . A sensitivity power analysis showed that this sample size can reliably detect small to medium effect sizes of ρ = 0.21 (one-tailed) with an alpha level of 0.05 and power of 0.80.One-hundred and thirty-nine undergraduate students of a large Austrian university were recruited α = 0.81) as in the pilot study. Then, they filled out the Meaning in Life Questionnaire (absolutely untrue and 7 = absolutely true): presence of meaning  and search for meaning . Choice as identity (α = 0.88) was assessed with the same six items as in the pilot study . Consumer reactance during lockdown (α = 0.91) was assessed with the following five items on a 7-point scale (1 = strongly disagree and 7 = strongly agree) based on the reactance literature . Participants first completed the same measure of maximizing , p = 0.002; (b) viewing choice as identity, r = 0.26, 95% CI = , p = 0.002; and (c) experiencing consumer reactance, r = 0.17, 95% CI = , p = 0.051  tended to score higher on maximizing than all other participants , t(135) = 1.70, p = 0.091.First of all, inspection of correlation coefficients confirms the main predictions of this study. Specifically, maximizing was associated with (a) search for meaning, χ2(4) = 19.03, p < 0.001, McFadden pseudo-R2 = 0.122. Specifically, results showed that maximizing was associated with viewing choice as identity, B = 0.30, SE = 0.09, p = 0.002, 95% CI = . In turn, choice as identity was associated with search for meaning, B = 0.22, SE = 0.11, p = 0.044, 95% CI = . Search for meaning was associated with consumer reactance, B = 0.26, SE = 0.10, p = 0.008, 95% CI = . Finally, consumer reactance was associated with more online shopping during lockdown, B = 0.58, SE = 0.17, p = 0.001, 95% CI = . The direct effect of maximizing on online shopping after controlling for choice as identity, search for meaning, and consumer reactance was not significant, B = 0.28, SE = 0.29, p = 0.340, 95% CI = . Critically, the indirect effect of maximizing on online shopping via choice as identity, search for meaning, and consumer reactance was significant, B = 0.010, SE (Boot) = 0.008, 95% CI =  was tested . In that respect, people who search for meaning in life might experience stronger consumer reactance because any restrictions to their choice freedom, even in the consumption domain, are perceived as barriers to their search endeavors. An interesting avenue for future research would be to examine whether search for meaning is associated with a higher valuation of choice freedom in general and how this is manifested in various choice domains beyond consumption . Moreover, it might seem paradoxical at first glance that people search for meaning in consumption, given that materialism is known to be associated with lower well-being and meaning in life . But theIt is important to note that these conclusions are based on correlational data in a very specific context of the COVID-19 pandemic. As much as the lockdown might have served as a fitting naturalistic laboratory to study the current research questions, further research in other contexts, times, and populations is necessary in order to draw safer conclusions about the relationship between maximizing, meaning, and reactance. The pilot study, which tested the association of maximizing with choice as identity in two different populations before the pandemic, partly provides some evidence for the robustness of the results. However, the association with search for meaning and reactance needs to be further validated beyond the current historical context. It should also be noted that, although the sample size provided sufficient power to test the predictions, all effects in this research were of small to medium size. Furthermore, given that the use of different scales has been shown to produce strikingly different patterns of results e.g., , future https://osf.io/mx35z/.The raw data supporting the conclusions of this article are available at: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Long non-coding RNAs are emerging as key regulators of gene expression at both transcriptional and translational levels, and their alterations (in expression or sequence) are linked to tumorigenesis and tumor progression. RNA editing has the unique ability to change the RNA sequence without altering the integrity or sequence of genomic DNA, with adenosine to inosine (A-to-I) RNA editing being the most common event in humans. With the ability to change the genetic information after transcription, RNA editing is an essential player in the transcriptome and proteome enrichment; however, when deregulated, it can contribute to cell transformation. In this article, we performed the first deep de novo editing survey in lncRNA, demonstrating that RNA editing is a pervasive phenomenon involving lncRNAs important in the brain and brain cancer. Our study will open a new field of research in which the interplay between lncRNA and RNA editing can add novel insights into cancer.Background: Adenosine to inosine (A-to-I) RNA editing is the most frequent editing event in humans. It converts adenosine to inosine in double-stranded RNA regions (in coding and non-coding RNAs) through the action of the adenosine deaminase acting on RNA (ADAR) enzymes. Long non-coding RNAs, particularly abundant in the brain, account for a large fraction of the human transcriptome, and their important regulatory role is becoming progressively evident in both normal and transformed cells. Results: Herein, we present a bioinformatic analysis to generate a comprehensive inosinome picture in long non-coding RNAs (lncRNAs), using an ad hoc index and searching for de novo editing events in the normal brain cortex as well as in glioblastoma, a highly aggressive human brain cancer. We discovered >10,000 new sites and 335 novel lncRNAs that undergo editing, never reported before. We found a generalized downregulation of editing at multiple lncRNA sites in glioblastoma samples when compared to the normal brain cortex. Conclusion: Overall, our study discloses a novel layer of complexity that controls lncRNAs in the brain and brain cancer. RNA editing is a post-transcriptional mechanism that modifies RNA nucleotides without changing the template genomic DNA ,2. In huLncRNAs play key roles in various biological processes in cell physiology, including imprinting control, cell differentiation, immune response, and chromatin modification ,14. DysrAlu repeats and are particularly abundant  in intronic regions of lncRNAs  . The rea lncRNAs b. Additi lncRNAs c, with c lncRNAs . Of noteabundant 8%—24,256 lncRNAs .p-value < 0.0001) global reduction of editing level in lncRNAs in glioblastoma compared to the normal brain  as detected in a minimum of 10 GBMs and 10 brain cortex samples and with Delta editing medians ≠ 0, thus obtaining a list of 1018 sites to test. We identified a total of 780 statistically significant differentially edited positions in GBM compared to cerebral cortex, as assayed by the two-tailed Mann–Whitney U-test followed by Benjamini–Hochberg multiple test corrections . Of noteWe reported that 704/780 sites (90%) were under-edited in GMB, and 76 sites (10%) appeared over-edited in GBM . The ediSeveral very interesting lncRNA genes emerged from our differential editing analysis, carrying many A-to-I editing sites significantly dysregulated in GBM, some of which, according to the Mann–Whitney test, displayed high significance levels . Among all these genes, the FTX lncRNA, involved the X-inactivation center region , is partAnother long non-coding RNA on which we have focused our attention is MEG3. We found that, similar to FTX, MEG3 is also characterized by the presence of many (11) sites differentially edited in GBM compared to normal brain . All theRecent evidence indicated lncRNAs as versatile molecules capable of acting as oncogenes or tumor suppressors in various tumors, including gliomas. LncRNAs are abundantly expressed in the brain, even if, for the vast majority of them, no role has been functionally characterized.RNA-Seq and bioinformatic approaches have disclosed the pervasive presence of A-to-I RNA editing in the human transcriptome, including mRNA, miRNA, and lncRNA ,35,36, bAlthough A-to-I editing sites have been identified in some lncRNAs, as mentioned above, an exhaustive editing survey in lncRNA has never been attempted. Herein, we applied a new metric for an ad hoc call of editing events in lncRNA, and we performed a de novo editing search within lncRNAs in both normal human brain cortex and GBM. We identified >10,000 novel editing sites and 335 novel lncRNAs undergoing editing that have never been reported before. Moreover, we found that editing signature in lncRNAs can clearly separate GBM and normal brain samples, as shown by the MDS analysis. Comparing the inosinome fingerprint of normal brain and GBM samples, a notable difference between the two sample tissues emerged, with an overall higher editing level in normal brain cortex compared with GBM samples. The editing decrease in GBM in lncRNAs is similar to what was previously reported in whole transcriptome analyses ,26.We also reported that a few highly edited lncRNA sites are present in tumor samples compared to the normal brain, with only two lncRNAs (AC139795.2 and AC006511.7) displaying an editing increase >20% in GBM  for de novo RNA editing discovery were obtained as FASTQ files from the NCBI SRA repository (SRP083311). Libraries were strand-specific and deeply sequenced with Illumina HiSeq2500 in three biological replicates, generating a total of 28.64 million paired-end 150-bp sequencing reads. For differential editing analysis, normal brain controls , with on average 39 million reads (paired ends) per sample, were downloaded from the Genotype-Tissue Expression (GTEx), and primary GBMs (156 samples), with on average 48 million reads (paired ends) per sample, were downloaded from The Cancer Genome Atlas (TCGA). Both datasets, with the same read length (76 bp) and generated from polyA-selected RNA, were downloaded upon authorization from the database of Genotypes and Phenotypes (dbGaP) with accession numbers phs000424.v8.p2 and phs000178.v11.p8, respectively.https://broadinstitute.github.io/picard/).RNA-Seq reads in FASTQ format were inspected using FASTQC program. Adaptors and low quality regions (phred cutoff of 20 for at least 70% of the read length) were trimmed using FASTP (0.20.1) , excludiRNA editing candidates in lncRNAs were called using the REDItoolDnaRna.py script. The first line of command of the REDItools package  is reported. LncRNA transcript annotation was downloaded from GENCODE 34 database . Following the detection scheme as in the protocol published by Lo Giudice and colleagues , initialGene expression levels were calculated in transcripts per million (TPM) units using StringTie 2.1.3  and GENCDifferential editing statistics were calculated with SciPy Python library. Multidimensional scaling (MDS) was carried out in R using the ‘metaMDS’ function of the vegan package, providing as input a Spearman correlation matrix calculated from editing levels for each sample without imposing coverage filters. Plots were generated in R with ggplot2, circlize, ComplexHeatmap, and ggrepel libraries.Herein, we showed the first exhaustive survey of a de novo RNA editing call in lncRNAs in normal brain cortex and glioblastoma. We demonstrated that lncRNAs, among them some oncogenes and tumor suppressor lncRNAs, are deeply modified by A-to-I RNA editing machinery, with editing that is significantly altered in cancer cells.


Both DNA and RNA can maintain left-handed double helical Z-conformation under physiological condition, but only when stabilized by Z-DNA binding domain (ZDBD). After initial discovery in RNA editing enzyme ADAR1, ZDBD has also been described in pathogen-sensing proteins ZBP1 and PKZ in host, as well as virulence proteins E3L and ORF112 in viruses. The host-virus antagonism immediately highlights the importance of ZDBD in antiviral innate immunity. Furthermore, Z-RNA binding has been shown to be responsible for the localization of these ZDBD-containing proteins to cytoplasmic stress granules that play central role in coordinating cellular response to stresses. This review sought to consolidate current understanding of Z-RNA sensing in innate immunity and implore possible roles of Z-RNA binding within cytoplasmic stress granules. Unlike the anti-conformation base arrangement throughout A- and B-DNA, the nucleoside bases in Z-DNA adopt alternating syn- and anti-conformation bases, giving rise to its distinctive left-handed double helical structure with zigzag backbone (thus its name) . The X-r2) . Since t2) –8. Simil2) . Indeed,2) –12. Thern 2 and d(CGGCCG)2, have also been reported to bind to Zα domain , followed by the cascading signaling events culminating in the activation of type I interferon response. Various host sensor proteins such as Toll-like receptors (TLRs), retinoic acid inducible gene- I (RIG-I)-like receptors (RLR), and Nod-like receptors (NLR) can recognize a vast range of viral nucleic acids or other VAMPS upon virus infection –28. ThesCellular stress occurs when the ability to maintain homeostasis balance within a cell is affected. Various factors including viral invasion, heat shock, oxidative stress, nutrient deprivation, DNA damage, can trigger stress response, where cell survival is dependent on successful resolution of the cellular stresses . Upon stWhile initially assumed to be passive repositories of untranslated mRNA, SGs are now thought as RNA triage sites where mRNA transcripts were actively sorted towards decay, storage, or translation reinitiation . SGs funThe phosphorylation of translation initiation factor (eIF2α) by eIF2α kinases catalyses the formation of SGs through stalling of the initiation of ribosomal translation. The recycling of inactive eIF2α-GDP to active eIF2α-GTP is inhibited by eIF2α phosphorylation, thereby disrupting the formation of the essential translation initiator, tRNAiMet . The eIFtRNAiMet .A recent excellent review has highlighted the role of SGs in antiviral response, notably on the mechanisms of viral translational inhibition and counteracting strategies adapted by viruses .SGs formp150 is only induced by type I interferons and is mostly cytoplasmic, whereas the constitutively expressed ADAR1p110 is localized in the nucleus  is within the common dsRBD region  protein family, where their adenosine-to-inosine (A-to-I) RNA editing activity is responsible for a wide range of regulation in gene expression, peptide modification in nervous system, RNA interference (RNAi), protein activation or inhibition . A-to-I  nucleus , 69. ThiD region .p150 in antiviral immune homeostasis and autoimmunity, through MDA5-MAVS sensing pathway  and Bilateral Striatal Necrosis/Dystonia (BSD) have been attributed to ADAR1 dysfunction. Genetic profiling of Adar1 gene among these patients showed that loss-of-function mutation at ZαADAR1 causes the hallmark type I interferonopathy, suggesting that Z-DNA/Z-RNA binding is important to prevent dysregulated interferon response , alternatively known as DNA-dependent activator of IFN-regulatory factors (DAI) or Tumor stroma and activated macrophage protein (DLM1), plays a significant role in innate immune response against viruses or other non–self-agents . ZBP1 haZBP1  activation and host immune response against vaccinia virus infection —a common viral dsRNA mimic  , a major koi herpesvirus infecting common carp, contains an N-terminal Zα domain  have been independently reported to localize to SGs during cellular stresses  have been identified with Zα domain(s). Two common themes stood out among these proteins, where they are important in host-pathogen interaction, and they localize to SGs. The conservation of Zα domain in virus proteins is important for viral pathogenesis and immune evasion, but yet information on Z-DNA/Z-RNA motif within virus genomes is still sketchy. Similarly, while independent studies showed Z-RNA binding is responsible for protein localization to SGs, the bound substrate (Z-RNA) within SGs has not been elucidated. This is largely attributed to the transient nature when nucleic acids adapt Z-conformation that could be dependent on the cellular environment. The condensed mRNA and protein aggregates in SGs may create a favourable environment for B-to-Z transition. Research studies using nucleic acid analogues that are prone to irreversible B-to-Z transition may afford a glimpse into understanding the precise mechanism at work  from Universiti Sains Malaysia. DC is a recipient of Penang Future Foundation Scholarship (1709/CP039). SKN is also funded by Fundamental Research Grant Scheme (203/CIPPT/6711566) from Ministry of Higher Education, Malaysia.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.