Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.  Small non-coding RNAs are a class of non-coding RNAs that are shorter than 200 nucleotides (nt). In recent years, small RNAs have been characterized as key regulators of several fundamental transcriptional and post-transcriptional cellular mechanisms ,2,3. NexTo monitor and analyze the expression of known small RNAs, however, a microarray and reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) become the methods of choice. Whereas microarray approaches allow for large-scale expression profiling studies of known sequences, RT-qPCR quantitation remains the most widely used, relatively simple, straightforward and least expensive method to routinely monitor and quantitate the levels of known small RNAs of interest in research laboratories. The RT-qPCR quantitate expression of specific RNA sequences by exponential amplification mostly uses a fluorescent reporter, either via a DNA intercalating agent (SYBR green) ,5 or usiThere are two types of quantitation based on RT-qPCR: while relative quantification lies on the ratio of target RNA found in unknow samples compared to a control sample, absolute quantitation consists of calculating the number of RNA copies using a standard curve established with known amounts of the synthetic form of the RNA of interest. In every case, quantification can be normalized and improved by addition to the samples of a synthetic small RNA sequence absent from the studied cells, tissues or species (known as a spike-in) , or usinOver the recent years, the use of NGS, microarray and RT-qPCR provided the research community with the tools that allowed researchers to improve their grasp upon the diverse classes of small RNAs and allowed a better understanding of their function. This is exemplified by an increasing number of studies demonstrating the functional role and biological significance of non-coding RNA fragments, such as transfer RNA (tRNA)-related RNA fragments (tRFs)  or rRNA-There is, currently, no method available to specifically detect and quantitate known small RNAs shorter than 16 nt. If it were to exist, such a method would greatly facilitate the study of unusually short RNAs, accelerate the pace of research in that field in both prokaryotes and eukaryotes, improve our understanding of their biology and anticipate their possible use as biomarkers or in therapeutics. Such a method would need to be designed to detect and quantitate the levels of specific RNA sequences in a specific and sensitive manner, even if or when these small RNAs derive from abundant and longer RNA precursor sequences .Recently, a small RNA-Seq analysis of 11 samples derived from six different species revealed a new family of RNAs shorter than microRNAs. The two main sequences found were one of 12 nt, which we named dodecaRNA (doRNA), and its 13-nt variant, which differs only by an additional Cytosine at the 5′ end and was named C-doRNA . These tThis section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.The short doRNA (12 nt) and C-doRNA (13 nt) are the two most abundant sequences of the family of dodecaRNAs (doRNAs) that we discovered recently in human and mouse small RNA transcriptomes (please refer to the manuscript submitted as a separate Standard Paper) . The onlIn order to take up this technical challenge, we set-up a new detection method, based on RT-qPCR, but preceded by a 5′ adapter ligation step aimed at (i) lengthening doRNA and C-doRNA to the size of a microRNA, and (ii) positioning the discriminating 5′ C at the center of the adapter-ligated sequences . In thisAfter ligation, the adapter-ligated RNA products were polyadenylated to introduce a poly(A) tail at the 3′ end, followed by reverse transcription using a poly (dT) oligonucleotide containing a Universal primer (UP) in its floating tail, and a 3′ degenerated anchor. This step was crucial, since it differentiated our RNAs of interest from their precursors elongated on their 3′ side, which complemented the splint-mediated ligation of the adapter that specified and discriminated their 5′ side.The final step consisted of the qPCR quantitation of our RNAs of interest using LNA oligonucleotides ,18,19. TThe efficiency of doRNA and C-doRNA-splinted 5′ ligation of an adapter was a major determinant of the specificity and sensitivity of our detection method. In order to optimize the splinted 5′ ligation step, we optimized the buffer composition and the sequence of the splint. First, the adapter was always added in excess during ligation, in order to avoid adapter dimers while ensuring a high ligation efficiency. The adapter:target RNA ratio gave equivalent ligation efficiencies; however, ligation using the ratio of ~five adapters to one target gave slightly better results , a 3′ hydroxyl on the acceptor RNA (adapter RNA) and ATP . The synOur assays suggested that the splint yielding the most efficient ligation was the 4:2 splint for doRNA and for C-doRNA .In order to identify the most efficient workflow for the doRNA and C-doRNA-splinted 5′ ligation RT-qPCR, we analyzed the Cq results from qPCR obtained from analyzing total RNA extracts of mouse neuronal cells, the N2a. These were used because of their high levels of doRNAs. We, first, determined that pre-annealing the splint with the adapter, before adding them to the reaction, improved doRNA and C-doRNA ligation (v/v) DMSO may also optimize our splinted 5′ ligation RT-qPCR detection method by improving the efficiency of the 5′ ligation step  components that were tested: DMSO, PEG8000, ATP, T4 RNA ligase buffer, RNase inhibitor, annealing buffer, adapter and splint . DMSO is2, 1 mM DTT pH 7.5) proved to be essential to the reaction   from N2a cells, which already contained doRNA and C-doRNA, supplemented either with water (control) or 6.02 × 10C-doRNA) A. C-doRNR2 > 0.9983, p < 0.0001), allowing their quantitation from a number of copies as low as 240 . The correlation was stronger for the 22-nt microRNAs than for the 12 and 13-nt doRNAs, which may be explained by their difference in length and, for microRNAs, by the detection of the authentic sequences without the need of adapter ligation.We applied our splinted 5′ ligation RT-qPCR method to validate the small RNA-Seq profile of doRNA, C-doRNA, miR-25 and miR-30a in the same total RNA samples derived from eight (8) primary and cultured human and mouse cells and tissue that were analyzed. The difference between doRNA/C-doRNA and miR-25/miR-30a levels, when compared by small RNA-Seq, was less pronounced when assessed using our splinted 5′ ligation RT-qPCR method, which was expected to be more quantitative than small RNA-Seq. We observed a strong correlation between the levels of these four small RNA species, when the data expressed in terms of copy numbers versus RPM were plotted  and affordable methods are necessary to provide accurate and complete information on very small RNA profiles and functions. In this study, we designed a novel splinted 5′ ligation-based RT-qPCR that allowed the detection of a new family of doRNAs half the length of microRNAs. The crucial step of the protocol involved splint-mediated ligation of an RNA adapter, a highly efficient and low-bias approach that may improve ligation efficiency and specificity. High ligation efficiency is important to increase yield and allow for low input samples.Adapter ligation to the 5′ end of small RNAs is particularly well described and used in small RNA library construction involved in small RNA-Seq. Several comparative studies on 3′ or 5′ ligation of total RNA exist ,27,28, bThe strong correlation of the microRNA data obtained by splinted 5′ ligation RT-qPCR versus small RNA-Seq suggests that our method allowed the detection of doRNAs without adding a bias in microRNA detection. Splinted 5′ ligation RT-qPCR also demonstrated its utility when assessing the differential expression of small RNAs between several different cell or tissue samples, which is commonly used in clinical diagnostic and more fundamental research. It may also enable the discovery and study of the gene targets or processes possibly regulated by these unusually short RNAs and provide clues on their role in cellular and molecular biology in health and diseases. The splinted 5′ ligation RT-qPCR protocol may be easily adapted and optimized to detect any RNA sequences of interest shorter than microRNAs, certainly down to 12 nt in length, as shown in this Method paper, and possibly even a little shorter, albeit expectedly with a variable specificity and sensitivity. The discovery of doRNAs, containing a core sequence of only 12 nt, may open a new area of research, and the splinted 5′ ligation RT-qPCR detection method that we set-up may be instrumental to achieve significant advances that would otherwise not be possible. We showed here that our method could detect two small RNAs shorter than 16 nt and did not interfere with the detection of two microRNAs using regular microRNA oligonucleotides. However, the use of our approach remains to be validated for other unusually short RNA sequences. The splinted 5′ ligation RT-qPCR method that we established, depicted in This new method is relatively simple and straightforward to establish, perform routinely and scale-up in a molecular biology laboratory, and may help researchers explore and understand the biology and importance of RNA species shorter than microRNAs ,30. All synthetic RNA and DNA oligonucleotides used in this study were synthetized by Integrated DNA Technologies Inc. , and their names associated with their sequences, as summarized in http://www.faculty.ucr.edu/~mmaduro/random.htm) (accessed on 3 August 2021) to generate various 10-nt sequences. Then, we selected sequences with the lowest capacity to bind to human or mouse transcripts. To that end, we reverse-complemented different potential adaptors and used BLAST nucleotide (blastn) searches from NCBI to verify if there were sequences in the human or mouse transcriptome that could correspond to the sequence of our adaptor. No significant similarity was found. Through blastn, we first searched for any correspondence with the “AATGGTTGTC” sequence (the reverse complement of our adapter) or the “CTGTTGGTAA” sequence (the complement of our adapter). Then, we selected the “Genomic + transcript databases” using the “Human genomic plus transcript (Human G + T)” or the “Mouse genomic plus transcript (Human G + T)” to verify in both organisms. The program used was MegaBLAST (optimized for highly similar sequences). An RNA adapter was used to lengthen unusually short RNAs of interest (the doRNAs doRNA and C-doRNA) in their 5′ end. We selected an RNA adapter sequence that differed from the precursors of doRNAs and did not map to the human or mouse transcriptomes. We ascertained that adapter-ligated doRNA or C-doRNA sequences did not create a pre-existing RNA in human or mouse. Briefly, we used an online random generator (https://www.mirbase.org/cgi-bin/blast.pl) (accessed on 3 August 2021) and tRFdb (http://genome.bioch.virginia.edu/trfdb/search.php) (accessed on 3 August 2021) and found no similar sequence.We carried on the same analysis for the extended doRNA and C-doRNA, respectively “GACAACCTTGACTCTTAGCGG” and “GACAACCTTCGACTCTTAGCGG”, on NCBI. We did not find these sequences in the human database, while we found some matches in the mouse. However, the polyadenylation step during the reverse transcription, combined with targeted amplification, permitted to distinguish our extended doRNA or C-doRNA from them. Finally, we tested these sequences on miRBase  was mixed with the splint (10 μM) in annealing buffer . Different annealing protocols were tested, as shown in 2, 1 mM DTT, pH 7.5). The 23 µL of ligation was allowed to proceed for 1 h at room temperature (RT), after which a 1:1 volume of stop ligation buffer  was added. This step lengthened doRNA and C-doRNA to 22 nt and 23 nt RNAs, respectively. Two (2) µL of total ligated RNA were used for RT using the miRCURY Locked Nucleic Acid (LNA)-modified microRNA PCR Assay  and the oligo-d(T) primer with 5′ universal tag included in the miRCURY LNA RT Kit . After cDNA 1/10 dilution, qPCR was performed using miRCURY LNA SYBR® Green PCR Kits  in 96-well plates  using the CFX96 Touch™ Real-Time PCR Detection System  and specific Custom LNA Oligonucleotides for the doRNA  and C-doRNA  amplification. The qPCR temperature cycling protocol was as follows: denaturation step at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s and annealing/elongation at 60 °C for 1 min, with a ramp of 1.6 °C/s. Then, as melting curve was generated by heating from 70 °C to 90 °C with 0.5 °C increments, 10 sec dwell time, and plate read at each temperature. The RT-qPCR data were normalized either using endogenous small nucleolar RNA U6 or the RNA spike-in provided in the miRCURY LNA microRNA PCR Assay kit.The annealed adapter/splint was added to 300 ng of total RNAs and 18 µL of ligation master mix containing 10 units T4 RNA ligase , 10% DMSO, 25% PEG 8000 , 1 mM ATP  and 20 units SUPERase•In RNAse inhibitor  in T4 RNA ligase reaction buffer  values with the corresponding copy numbers were plotted, and the linear curve equation, correlation coefficient (R2) and p value were calculated.doRNA, C-doRNA, miR-25 and miR-30a copy numbers were determined using a standard curve established using the corresponding synthetic RNA oligonucleotides  serially diluted 1/10th to obtain from 6.1 × 10http://www.crchudequebec.ulaval.ca/en/services/sanger-and-sequenom/services/sanger-sequencing/) (accessed on 3 August 2021). All five clones derived from the band amplified with the doRNA primer contained the doRNA-extended DNA sequence  and universal sequence at the other end), whereas all five clones derived from the band amplified with the C-doRNA primer contained the C-doRNA-extended DNA sequence. These sequencing results supported the specificity of our splinted 5′ ligation RT-qPCR method for doRNA vs. C-doRNA detection.The pBluescript II plasmid  was linearized using EcoRV restriction enzyme , inducing a cut within the LacZ gene, followed by dephosphorylation of the blunt ends using Antarctic phosphatase enzyme  to prevent plasmid recircularization. In parallel, doRNA or C-doRNA products amplified by splinted 5′ ligation RT-qPCR were analyzed by 20% polyacrylamide, 8M urea gel electrophoresis, revealed by nucleic acid staining solution  and the bands excised for subsequent gel extraction using QIAquick gel extraction . The purified RNAs were phosphorylated using T4 polynucleotide kinase . The dephosphorylated digested plasmid was then mixed with the phosphorylated doRNA or C-doRNA insert  in a ligation reaction catalyzed by T4 DNA ligase . The ligation products were transformed into bacteria, which were spread on LB plates containing 50µg/mL ampicillin and X-Gal . Bacteria transformed with a plasmid not expressing LacZ gene (containing an insert) grew as white colonies and clones were selected and expanded for plasmid DNA purification. Inserts were sequenced using the reverse or forward T7 oligonucleotides at the SANGER Sequencing platform of the CHU de Québec-Université Laval Research Center (7 copy number) or water (used as control), and total RNA (300 ng) extracted from cultured N2a cells, were prepared in a final volume of 2 µL: doRNA, C-doRNA, mutated C-doRNA (single G2 to U2 substitution), 5′ extended C-doRNA (with 10 nt from the ITS1 sequence), 3′ extended C-doRNA (with 10 nt from the 5.8S rRNA). RT-qPCR data were normalized on small nuclear RNA (snRNA) U6 levels, and the fold change was calculated versus the control sample (water). Each sample was subjected to splinted 5′ ligation RT-qPCR detection of doRNA and C-doRNA. Cq data were considered only when the standard deviation for the technical duplicates was below 0.3. Six (6) samples, each containing one of the following synthetic oligonucleotides  fetal bovine serum , 1 mM sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine in a humidified incubator under 5% CO2 at 37 °C.The human cell lines used in this study were human umbilical vein endothelial cells  and human embryonic kidney 293 (HEK293) cells . The mouse cell lines used in this study were neuronal N2a cells  and NIH/3T3 cells . Cells were cultured in their appropriate medium , supplemA sample of brain cortex  was collected from a two-year-old mouse, using a protocol approved by the Université Laval Animal Welfare Committee and in accordance with the guidelines, regulations and requirements of the Canadian Council of Animal Care for Animals Used for Scientific Purposes. ® or TRIzol LS® reagent  for liquid samples, and contaminated DNA was removed by treatment with DNase I , following the manufacturer’s recommendations.Total RNA samples were obtained using TRIzolhttps://www.arraystar.com/) (accessed on 3 August 2021) and refined using R (Free Software Foundation). Only the reads that were completely identical in both lengths and sequences were assembled as a unique read. Small RNA read counts were normalized as reads per million (RPM) small RNA alignments. The same total RNA samples, derived from the primary and cultured human and mouse cells and tissue mentioned above, were analyzed by small RNA-Seq in the ~8 to 30-nt window of RNA sizes. The RNA-Seq data are described in detail in the manuscript submitted as a separate Standard Paper and were provided through the standard analysis pipeline of Arraystar Inc. . In vitro experiments were conducted in biological triplicates (minimum) with type alpha error set to 0.05 (5%). Statistical significance was determined by one or two-way ANOVA with Holm–Sidak’s post hoc test for multiple comparisons or t-test. Linear regression comparisons were verified by the Fisher test, so the p-value indicated the strength of the linear regression.Statistical analyses were performed using Prism 7 (GraphPad Software, Inc., available at


Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus. Alphavirus genus includes a number of medically important species such as chikungunya virus (CHIKV), Ross River virus (RRV), Mayaro virus, and the encephalitic alphaviruses iewed in ,8).Alphaviruses are small enveloped positive-sense RNA viruses that assemble into highly organized particles with icosahedral symmetry (reviewed in ). Virus The alphavirus Cp selectively packages the gRNA to produce virus particles with a high specific infectivity. During infection the viral RNA replication complex produces both the gRNA and a subgenomic RNA (sgRNA) encoding the viral structural proteins. However, in spite of the presence of a molar excess of sgRNA and cellular RNAs in alphavirus-infected cells, for most alphaviruses packaging strongly favors the viral genomic RNA . RNAs were resuspended in 50 mM Tris pH 7.0, aliquoted, and snap frozen. Then, 50 ng of each RNA was subjected to 15% denaturing Urea-PAGE followed by staining with SYBR Gold  to confirm RNA integrity. Site #1 RNA was previously mapped to nt 5988–6016 in the SFV gRNA . 2, 0.01% Tween-20, 1 µg/µL BSA, 1 mM DTT) to each well. Where indicated, samples contained 200 ng/µL of poly(I:C)  in a 50 µL volume. Samples were preincubated at 37 °C for 15 min with shaking. Alexa488-RNAs were diluted in RNA Binding Buffer, added to each well at a 10 nM final concentration, and incubated in the dark for 30 min at 37 °C with shaking. Plates were magnetized and washed three times with Wash Buffer . Elution Buffer  was then added to each well and incubated in the dark at 70 °C for 10 min with shaking. Plates were magnetized, the supernatant was transferred to a new 96-well plate for measurement, and bubbles were removed. Fluorescence corresponding to bound Alexa488-RNA was measured on a Victor X5 plate reader  using the “Fluorescein 485–535 1s” default protocol. Technical duplicates were performed for all samples. Black, non-binding 96-well plates  were washed twice with Buffer W . MagStrep “type3” XT beads  were equilibrated in Buffer W and added to each well to capture Strep-tagged Cp. Cp was 2-fold serially diluted (4 µM to 3.9 nM) in Buffer W for n + 1 samples, added to its corresponding wells, and incubated with MagStrep “type3” XT beads for 4–6 h at 4 °C with rocking. A no Cp control was included for each RNA sample to assess background for each experiment. Plates were magnetized and washed three times with Buffer W before adding RNA Binding Buffer . This was then multiplied by one hundred to achieve “Fraction bound %”. Technical duplicates were averaged before averaging the 32P-based binding assay [To identify the sequence and/or structural features that Cp recognizes within site #1 RNA, we established an in vitro Cp binding assay using fluorescently labeled RNAs . The assng assay . 32P-labeled RNA assay [We then compared Cp binding to the Alexa488-site #1 RNA vs. to Alexa488-site #1 mutant RNA (here indicated as Mutant) a. The MuNA assay , Cp bounNA assay . The 7meTo test whether Cp preferentially binds the predicted WT* stem-loop, we designed two additional RNAs termed StmLp RNA and StmLpMutant RNA. The StmLp RNA maintains identity with the original site #1 sequence within the WT* stem loop, but is mutated at every other position a,b. The We next tested whether Cp’s recognition of site #1 RNA is conserved among other Cps from the SFV complex. We recombinantly expressed and purified N-terminally Strep-tagged RRV and CHIKV Cps a. We tesWe then tested CHIKV Cp binding to the site #1, Mutant, StmLp, and StmLpMutant RNAs. In the presence of poly(I:C), CHIKV Cp specifically bound the site #1 and StmLp RNAs, but not the Mutant and StmLpMutant RNAs c. This iTo further our understanding of the molecular mechanism of RNA recognition by the alphavirus Cp, we established an in vitro binding assay using fluorescently labeled RNAs and purified SFV Cp. N-terminally Strep-tagged full length SFV Cp was immobilized on beads so that monomeric Cp binding to RNA could be measured in the absence of confounding effects on core-like particle assembly. Using a panel of rationally designed mutated RNAs, we demonstrated that SFV Cp specifically recognized a stem-loop structure within its top genomic RNA binding site, site #1 RNA. This recognition mechanism was conserved across the alphavirus SFV complex, as RRV and CHIKV Cps also specifically recognized the stem-loop structure. Site #1 is predicted to adopt two structures, WT and WT*, and our data indicated that the WT* stem loop structure is specifically recognized by Cp. Both the WT and WT* secondary structures are predicted to form with nearly equivalent free energies, suggesting that they exist in equilibrium. Regulating the equilibrium and formation of the WT* structure could represent a way of regulating Cp binding to site #1 when competing RNAs and proteins are present, as is the case in the cytoplasm of an infected cell. Competing structures could also modulate Cp binding to its other top binding sites on the gRNA, and together these structural features could help to regulate when genome packaging and NC assembly occur. Additional studies will be needed to test this theory. Site #1’s sequence is hyper-conserved between SFV and both RRV (86%) and CHIKV (93%) compared to the overall nucleotide sequence conservation between the respective genomes . A comparable hyper-conservation was also found for a majority of SFV Cp’s top binding site sequences in the gRNA . Thus, aOur in vitro binding data strongly support Cp’s specific recognition of the WT* stem-loop structure within the site #1 RNA. However, the true structure of site #1 RNA within the context of the SFV genomic RNA in the complex cellular environment remains to be defined. Recent RNA structure analyses by SHAPE-MaP have been performed on alphavirus RNAs including that of CHIKV ,25. The


Here, we found that FTO protein levels, but not RNA levels, were downregulated in CRC tissues. Reduced FTO protein expression was correlated with a high recurrence rate and poor prognosis in resectable CRC patients. Moreover, we demonstrated that hypoxia restrained FTO protein expression, mainly due to an increase in ubiquitin-mediated protein degradation. The serine/threonine kinase receptor associated protein (STRAP) might served as the E3 ligase and K216 was the major ubiquitination site responsible for hypoxia-induced FTO degradation. FTO inhibited CRC metastasis both in vitro and in vivo. Mechanistically, FTO exerted a tumor suppressive role by inhibiting metastasis-associated protein 1 (MTA1) expression in an m6A-dependent manner. Methylated MTA1 transcripts were recognized by an m6A “reader”, insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), which then stabilized its mRNA. Together, our findings highlight the critical role of FTO in CRC metastasis and reveal a novel epigenetic mechanism by which the hypoxic tumor microenvironment promotes CRC metastasis.Fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m Increas cancers –6. There6A modification is involved in cancer pathogenesis and progression through the m6A enzyme system, which is mainly composed of methyltransferases (“writers”), demethylases (“erasers”) and binding proteins (“readers”) [6A enzymes participate in the development of cancers, such as gliomas, leukemia, and breast cancer, by affecting different stages of the RNA life cycle, including pre-mRNA splicing, export, translation, and stability [6A writer methyltransferase like 3 (METTL3) in promoting CRC progression by maintaining the CRC cell stemness phenotype via an m6A-dependent mechanism [6A writers facilitate CRC development in an m6A-dependent manner [6A-specific erasers, AlkB homolog 5 (ALKBH5) and fat mass- and obesity-associated protein (FTO). FTO was first identified as a gene related to obesity and energy metabolism and was then identified as the RNA m6A demethylase [6A demethylase remains poorly understood.Meaders”) , 7. Thestability –12. Receechanism . Meanwhit manner , 15. Theethylase . The rolethylase , 17, 18.ethylase –22. Howe6A level of MTA1 transcripts, resulting in decreased mRNA stability. Moreover, we found hypoxia could induced FTO downregulation mainly through the ubiquitin-mediated protein degradation. We also found serine/threonine kinase receptor-associated protein STRAP) was probably the E3 ligase of FTO and Lysine(K)-216 was its major ubiquitination site. Overall, our findings revealed a critical role of FTO as a predictor of metastasis and recurrence of colorectal cancer and elucidated a new epigenetic mechanism through which the hypoxic tumor microenvironment promotes CRC metastasis.In this study, we first demonstrated that FTO expression was downregulated in CRC tumor tissue and that higher FTO expression was associated with better prognosis in CRC patients. Then, we revealed that FTO inhibited tumor metastasis and progression in vitro and in vivo by decreasing the expression of its downstream target gene, metastasis-associated protein 1 (MTA1). FTO precisely reduced the m6A modification is a dynamic process mediated by enzymes known as writers and erasers. We previously revealed the oncogenic role of the m6A writer METTL3 in CRC [6A eraser expression in CRC, we first analyzed FTO and ALKBH5 mRNA and protein levels in paired CRC tumor and adjacent normal tissues from Sun Yat-sen University Cancer Center (SYSUCC). We found that FTO was downregulated at the protein level but not at the mRNA level in CRC tumor tissues, while ALKBH5 expression did not show any differences  and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Data Portal  [6A eraser FTO is downregulated in CRC and is a potential prognostic indicator in CRC patients.M3 in CRC , and we ces Fig. . Then, w/s/S045) . As expeTo investigate the relationship between FTO and the metastasis-related phenotype, we first detected the protein level of FTO in 11 colorectal cancer cell lines  and chose two cell lines with different FTO levels, HCT116 and DLD1, for further experiments Fig. . We nextTo evaluate the function of FTO in vivo, we applied a nude mouse subcutaneous xenograft model. CRC cell lines with FTO overexpression had a significantly lower tumor growth rate and tumor weight Fig. . HistoloMTA1 level was decreased in FTO-overexpressing CRC cells . We next screened these genes for overlap with a list of 84 human tumor metastasis-related genes , 25 and ls Figs. , S4B. Wels Figs. , S4C. Fuls Figs. , S4D.FigWe next explored whether MTA1 could reverse the effects of FTO depletion or overexpression on CRC cell migration and invasion. We first knocked down MTA1 in HCT116 and DLD1 cells and verified that MTA1 could accelerate CRC cell migration Figs. , S4E. Th6A-dependent roles in tumorigenesis and progression in several cancers. To elucidate the mechanism of FTO-mediated MTA1 regulation, we first predicted potential m6A modification sites on MTA1 mRNA with SRAMP (http://www.cuilab.cn/sramp/) and labeled them as site 1 to site 6 regions.  and around the coding sequence (CDS)-3’UTR junction region  family  and the insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family  [6A readers of MTA1 transcripts. We found that IGF2BP2, but not other readers, specifically bound the MTA1 transcripts in HCT116 cells  , 28. RNAion Fig. . In addills Fig. , and theown Fig. . In TCGAion Fig. . Moreoveort Fig. . The deport Fig. , S6B.Fig6A demethylase ALKBH5 was reported to be induced by hypoxia to participate in the regulation of cancer development [As we mentioned above, FTO was downregulated in tumor tissues, with discrepancies between the RNA and protein expression levels. FTO is also known as alpha-ketoglutarate-dependent dioxygenase FTO, and it relies on oxygen to perform its function . Recentlelopment , 30. Theelopment , and con6A demethylase, has been shown to be involved in the occurrence and development of various cancers through different downstream genes. For example, FTO promoted breast cancer progression by increasing the RNA degradation of the pro-apoptosis gene BNIP3 via m6A demethylation and a YTHDF2-independent mechanism [6A regulatory machinery [6A modification and m6A machinery on the tumorigenesis, development and metastasis of CRC.FTO, as an machinery . Howeverachinery . FTO is achinery , 21. In In this study, the colon cancer cell lines DLD1 and HCT116 were used for phenotypes experiments and downstream gene screening. DLD1 cells are derived from Duke C colon cancer, positive for p53 in the presence of S241F and show strong Wnt signal. HCT116 cells are derived from Duke D colon cancer, with high expression of EMT signatures, and show higher aggressive behavior and higher in-vivo metastasis rates than DLD1 , 34. Our6A reader to perform further functions. In our study, IGF2BP2 directly binds to MTA1 mRNA, and its binding site was consistent with the m6A modification site controlled by FTO. Moreover, IGF2BP2 inhibition restrained MTA1 expression in the case of FTO deficiency, suggesting a FTO-IGF2BP2 m6A regulatory mechanism of MTA1 expression in CRC. Here, we discovered a new epigenetic mechanism that regulates MTA1 expression and provides a novel therapeutic strategy for targeting MTA1 in CRC. According to previous reports, including work in our group, IGF2BP2 facilitated colorectal cancer progression [Methylated RNA requires an mgression , 40, 41,6A modification was a crucial effector of the hypoxic response [6A demethylases ALKBH5 and FTO belong to the same AlkB family dioxygenases, it has been found that they exhibited contrary expression tendencies in primary neurons after oxygen deprivation/reoxygenation, and FTO was decreased under hypoxia [Hypoxia is one of the most important characteristics of the tumor microenvironment and it plays an important role in promoting mesenchymal transformation and metastasis of tumor cells , 43. Preresponse . Hypoxiaresponse , 30. Tho hypoxia . However6A-dependent gene regulatory mode in the epigenetics of hypoxia-induced cancer metastasis. However, there are limitations in this study. The specific interactions between hypoxia, FTO, and STRAP are not elaborated and the effects of hypoxia tumor microenvironment on cellular m6A modification levels mediated by other m6A writers, erasers, and readers need further exploration.Here, we first reported that hypoxia could decrease FTO protein expression but not RNA level in CRC cells. Furthermore, we revealed that the hypoxia-induced FTO depletion was mainly cause by ubiquitin-mediated protein proteasome-associated degradation and STRAP was necessary for FTO degradation as the E3 ligase. Knocking down STRAP could reverse the downregulation of FTO under hypoxia. Previous research showed post-translational ubiquitination on Lys-216 directed FTO protein degradation . Similar6A-dependent mechanism. In addition, we found that hypoxia can decrease FTO in CRC cells, mainly by increasing its ubiquitin-mediated protein degradation. We revealed a novel regulatory mode of RNA demethylase FTO degradation exerted by the tumor hypoxic microenvironment, indicating that FTO is a promising predictive factor for CRC metastasis  samples from 369 stage I–III CRC patients who underwent radical surgery at Sun Yat-sen University Cancer Center were collected. Of these, paired tumor-adjacent normal tissue specimens were available in 240 cases. The comprehensive clinicopathological information of patients was obtained from medical records. Informed consent was obtained from each participant, and the study was approved by the Institutional Review Board of the Sun Yat-sen University Cancer Center.2 cell culture incubator. For hypoxia exposure, cells were cultured under 1% O2, 5% CO2, and 94% N2 using the Anoxomat™ Mark II anaerobic system . All cell lines were authenticated by STR fingerprinting at the Medicine Lab of the Forensic Medicine Department of Sun Yat-sen University  before use.The human CRC cell lines HCT8, HCT116, HCT15, DLD1, SW480, SW620, RKO, Ls174t, CW-2, LoVo, and HT-29 were purchased from American Type Culture Collection  and Shanghai Cell Bank of Chinese Academy of Sciences . The cells were cultured in RPMI 1640  medium with 10% fetal bovine serum  and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a 5% COSiRNAs for gene expression knockdown were synthesized by RiboBio Co., Ltd. , and the sequences are listed in Supplementary information: Table 6 suspended in 100 µL PBS) were subcutaneously injected into each mouse. Tumor size was measured every four days with a caliper, and tumor volume was determined by the standard formula V = ½ (Length × Width2). At the end stage, tumor weight was measured.For the tumorigenesis experiments, 4- to 5-week-old female BALB/c nude mice  were used. Randomization was conducted and mice were treated by an unblinded manner. FTO-overexpressing and control cells , and the metastatic nodules in the lungs were counted under a microscope. All animal experiments were performed in accordance with protocols approved by our Institutional Animal Care Committee.For the metastasis experiments, two xenograft models were established as described in our previous study . For the6A antibody  or rabbit IgG was added to the beads and incubated by gentle rotation at 4 °C for 2 h. Beads were washed and then mixed with the RNA samples in RIP immunoprecipitation buffer on a rotator at 4 °C for 3 h to overnight. RNA was recovered by the addition of TRIzol after proteinase K digestion, followed by chloroform extraction. Eluted RNAs were precipitated with glycogen and 3 M sodium acetate in 100% ethanol at −80 °C overnight. The immunoprecipitated m6A RNAs were reverse transcribed into cDNA and quantified by qPCR according to the procedure described above. Each sample was analyzed with normalization to the input. The primers for MeRIP-qPCR are listed in Supplementary Information: Table Total RNA was purified from CRC cells using TRIzol reagent. More than 50 μg of total RNA was used for subsequent RNA fragmentation and immunoprecipitation. The total RNA was fragmented into ~100-nt-long fragments, and 1/10 of the RNA was saved as the input control. PierceTM Protein A/G Magnetic Beads  were washed and resuspended in IP buffer, and 5 μg of anti-mMTA1 mRNA for each group was determined by qRT-PCR assay as described above. Levels of MTA1 mRNA at each time point were normalized to GAPDH (a highly stable transcript and was independent of FTO activity). Percent mRNA remaining was plotted and determined by nonlinear regression analysis with Prism 8.0 software .Actinomycin D (ActD), a transcription inhibitor which intercalates into DNA, is widely used for measuring mRNA decay rates . CRC celRNA was transcribed in vitro using the MEGAscript T7 Transcription Kit  and labeled with desthiobiotin using the Pierce RNA 3′ End Desthiobiotinylation Kit . RNA pulldown assays were carried out with the Pierce Magnetic RNA-Protein Pulldown Kit  according to the manufacturer’s protocol. Then, 50 pmol of labeled RNA was added to 50 μL prewashed streptavidin beads, mixed gently and incubated at room temperature for 30 min. A 100 μL master mix of RNA-protein binding reaction with 2 mg protein lysate was prepared and added to the RNA-bound beads. After incubation for 30–60 min at 4 °C and washes, the streptavidin beads were heated with 50 μL elution buffer for 5–10 min at 95–100 °C, and the retrieved protein was detected by standard immunoblotting assays.TM RNA-Binding Protein Immunoprecipitation Kit . Briefly, cells were harvested and incubated with magnetic beads which coated with anti-IGF2BP2 antibody  or mouse IgG antibody overnight at 4 °C. Then washed the beads six times and incubated with proteinase K to digest the protein. Phenol-chloroform-isoamyl alcohol reagent was used to extract the RNA in the immunoprecipitates and inputs. RT-qPCR was conducted to quantify MTA1 mRNA. The relative expression was normalized to input and IgG was used to test the specificity of RNA–protein interactions.RIP assay was performed by the Magna RIPt test was used to analyze the differences between groups. Survival curves were constructed for each group using the Kaplan–Meier method and compared statistically by the log rank test. Univariate and multivariate analyses were conducted using Cox regression analysis. Pearson’s chi-square test was used to estimate the correlation between two variables. All analyses were carried out using SPSS  version 18.0  and GraphPad Prism version 8 . When the P < 0.05, the results were considered to be statistically significant.Data and bars represent the mean ± standard deviation (SD) of three independent experiments. The sample size determination was account on the need for statistical power. The experiments about patient samples was performed by a blinded manner. A two-tailed Student’s For further details, see the online Supplementary Information Methods section. Antibodies used in this study were listed in Supplementary Information: Table Supplementary Information


In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.  Currently, the characterization of the SARS-CoV-2 genome has become a priority for global healthcare in order to identify more suitable vaccines and therapeutic drugs . The useEven though cDNA sequencing is considered the gold standard for the analysis of critical materials , DNA polFor directly sequencing the coronavirus genome from clinical samples, such as nasopharyngeal or oropharyngeal swabs, the suggested protocol by Oxford Nanopore is the PCR tailing of SARS-CoV-2 . It has The PCR tailing of the SARS-CoV-2 protocol is based on a multiplex primers scheme that allows coverage of the whole genome. However, sequencing protocols taking advantage of target enrichment through the primers set, such as the PCR tailing of SARS-CoV-2, might be difficult in recovering individual mutations in underrepresented amplicons. This aspect may be considered relevant for the exceptional genetic plasticity of the genomes of RNA viruses. Indeed, the high mutation and recombination rates during genome replication by viral RNA-dependent RNA polymerases lead to populations of closely related viruses, so-called “quasispecies.” .Conversely, the direct RNA sequencing (direct RNA seq.) of SARS-CoV-2 has the advantage of displaying its native sequence, In this study, we investigated the suitability of Nanopore Oxford MinION Mk1B , a thirdAfter the collection of ten oropharyngeal swab samples from Italian patients with COVID-19, we assayed the concentration and fragmentation level of the extracted RNA to arrange it for sequencing, as recommended by Oxford Nanopore in the Input DNA/RNA quality control guidelines . Next, wSamples. For this study, we collected RNA samples from ten Sicilian patients between 19 and 23 March, 2020. The patients tested positive in the 2019-Novel Coronavirus (2019-nCoV) Real-Time rRT-PCR Panel (Centers for Disease Control and Prevention (CDC) Atlanta, GA 30333, USA). Furthermore, to compare the nanopore sequencing results, we included two samples from uninfected individuals as negative controls. In order to assay the suitability of our custom primers and to avoid possible PCR artifacts derived from high fragmented and very low concentrated RNA, such as that extracted from the swab, we used negative controls of high-quality RNA extracted from brain biopsies of two patients who underwent surgery before the beginning of the epidemic in Italy. The biopsies were made available from the Department of Experimental Biomedicine and Clinical Neurosciences, School of Medicine, Neurosurgical Clinic, University of Palermo.RNA extraction. The swab buffer kit was assayed using the QIAamp Viral RNA Mini Kit in Automated purification of RNA on QIAcube Instruments  according to the manufacturer’s instructions.Assessment of concentration and fragmentation level. The quality of the fragmented sample was assessed with an Agilent Bioanalyzer 2100  using the Agilent RNA 6000 Pico assay . We assayed 1 µL of each pure sample. The RNA extracted from the samples showed high fragmentation and the lowest concentration levels with an RIN index between 2.6 and 2.1 and concentration between 19 and 829 pg/µL.The samples were organized in two pools (A and B), each with a final volume of 10 µL. Pool A included samples from three patients: two with a PCR cycle threshold (Ct) value of 18 and one with a Ct value of 20. To elaborate, we pooled 8 µL of samples from the first two patients (4 µL from each) and 2 µL from the last patient. Pool B included RNA samples collected from ten patients (1 µL from each patient): three samples were common in Pool A, while the other seven had decreasing Ct values and were collected from two patients with a Ct value of 21, two with 22, two with 23, and one with 24.Library preparation, priming, and commencement of a sequencing run. Both pools were sequenced using a MinION Mk1B sequencer with the SQK-RNA002 protocol. After preparing the sequencing libraries A and B, as previously described, to ligate retrotranscription adapters (RTA) to the 3′ end of the RNA molecules, we combined each library with 6 µL of a mix comprising 3 µL of NebNext Quick Ligation Reaction Buffer, 1.5 µL of T4 DNA Ligase, 1 µL of RTA, and 0.5 µL of 110 nM RNA CS (RCS). Each reaction was incubated for 10 min at 22 °C. Next, the reactants were mixed with 9 µL of nuclease-free water, 8 µL of 5× first-strand buffer, 4 µL of 0.1 DTT, and 2 µL of 10 mM dNTPs. For cDNA synthesis, we added 2 µL of SuperScript III, following which both reactions were incubated under the following conditions: 50 °C for 50 min, 70 °C for 10 min, and 4 °C in Mastercycler Pro S  before proceeding to the next step.The subsequent wash step was performed according to the procedure for the specified protocol.Next, we measured the concentration of 1 µL of each library using a Qubit 3.0 Fluorimeter and the dsDNA HS assay kit . The concentrations were 1.2 ng/µL and 0.6 ng/µL for Libraries A and B, respectively. Next, we performed the attachment of the 1D sequencing adapter step according to the manufacturer’s instructions and recorded final concentrations of 0.9 ng/µL and 0.3 ng/µL for A and B, respectively. Lastly, 20 µL of sample from each library was mixed with 17.5 µL of nuclease-free water and 37.5 µL of RNA running buffer (RRB) in a final volume of 75 µL. The assessment of the R9.4 Flow Cell, which was performed prior to the loading of the libraries, revealed that 1575 active pores were available for sequencing. We performed a new MinKNOW  . At this point, we paused the process and loaded the B loading mix in the flow cell. Next, the run was resumed and continued till the number of reads generated was unvaried at 397 k . At the end of the sequencing process, we obtained the “Fast5” raw data files.. We developed a computational pipeline to analyze the output from the Oxford Nanopore MinION device. The first step in the process involved the conversion of “Fast5” in “Fastq” format. For this, we used the GPU version of the Nanopore Guppy base caller (v3.4.4) tool [Computational pipeline.4) tool  with theAs a second step, we performed read quality control using the PycoQC (v2.5.0.21) software  with staPycoQC can subsequently provide an overview of the overall quality of the reads. To clean the input data, we filtered the quality and read length using the NanoFilt (v2.7.0) tool . This toThereafter, only the filtered reads were considered in further analysis.At this point, we created an alignment process that mapped reads to a reference genome and identified the exact genomic loci corresponding to each read. Given that, we did not use primers for amplification of the SARS-CoV-2 genome, and we had to remove all the reads that were mapped with other material sequenced from the swabs. In this study, to remove the contaminating sequences, we considered humans, fungal, and bacterial reference genomes, respectively. To elaborate, we used the “Homo sapiens genome assembly GRCh38 (hg38)” from the Genome Reference Consortium  as the hFor each of these reference genomes, we mapped the reads using the minimap2 (v2.17–r941) tool  based onNext, we extracted both unmapped reads and reads with mapping quality lower than 10 using the “view” utility in the samtools (v1.7) library  for eachResultantly, we obtained a subset of the sequenced long reads that did not map with the genomes of humans, fungi, or bacteria.Lastly, we mapped the remaining reads against the SARS-CoV-2 reference genome using minimap2 tools with the same parameters used earlier. We successively analyzed the results of the mapping process using the BCFtools (v1.9) library , a set o® 3130xl System . To enrich the identified mutation region in the N gene, we adopted a nested PCR approach using outer and inner primer sets. Both primer sets are reported in Sanger sequencing. The samples were sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit  with an ABI PRISM 3100 Genetic Analyzer upgraded to the Applied BiosystemsTwo-step RT-qPCR. The ten sequenced samples and the two uninfected control RNAs were retrotranscribed using the ExcelRT™ Reverse Transcription kit . First, 16 µL of each sample was mixed with 2 µL of 50 µM of Oligo (dT)20 primer and 2 µL of dNTP Mix (10 mM each). The mixes were incubated at 70 °C for 5 min and placed on ice for at least 1 min. Next, 8 µL of 5× RT buffer (DTT), 8 µL of DEPC-treated water, 2 µL of RNAok™ Rnase Inhibitor, and 2 µL of ExcelRT™ Reverse Transcriptase was added to each sample. The reactions were incubated at 25 °C for 10 min, at 44 °C for 50 min, and at 85 °C for 5 min, and were subsequently held at 4 °C in Mastercycler Pro S .Next, to amplify the region containing the NC_045512:c.608_609_610delinsAAC mutation, we designed two primer sets to distinguish between a wild type (WT) and a mutated viral strain. The two sets used the same reverse primer, with divergence at the last three bases at the 3′ end of the forward primer. In fact, one forward primer was specific to the mutated sequence, whereas the other recognized the reference sequence.Furthermore, we used two other sets as controls. One set consisted of the primers N2 from the 2019-Novel Coronavirus (2019-nCoV) Real-Time rRT-PCR Panel (Centers for Disease Control and Prevention (CDC) Atlanta, GA 30333, USA), which was used to confirm the presence of the N gene target . The secMoreover, a plasmid vector carrying a synthetic reference SARS-CoV-2 N gene  was used as a reference control.First, both analysis samples and uninfected control cDNA samples were diluted at a 1:2 ratio, while the reference control was concentrated to 0.8 ng/µL. Next, 4 µL of each sample in 20 µL of reaction mixture was assayed by qPCR using the Quantinova SYBR Green PCR kit  according to the manufacturer’s instructions in a Rotor-Gene Q 2plex HRM Thermocycler . The following PCR thermal profile was used: 95 °C, 2 min; 95 °C, 5 s; and 57 °C, 10 s for 35 cycles.To characterize the SARS-CoV-2 mutational landscape, we used RNA extracted from samples collected from five male and five female patients who tested positive for SARS-CoV-2 in the swab test, with the corresponding Ct values ranging from 18 to 24. First, we assessed the concentrations and the fragmentation levels in the samples. The overall samples had significantly low concentrations (in the order of pg/µL) and high fragmentation levels with an RIN index ranging from 2.6 to 2.1. Next, we prepared Pools A and B from the samples. We prepared the former to increase the abundance of sequencing output and the latter to improve the heterogeneity in the data.We set up each pool at a final volume of 10 µL and prepared the libraries for direct RNA seq. After each purification step, the concentrations were 1.2 ng/µL and 0.6 ng/µL in the initial eluates and 0.9 ng/µL and 0.3 ng/µL in the final eluates at the presequencing stage for Pools A and B, respectively. Although the library concentrations were 200 times less than that required for the protocol, we opted to load the libraries and to launch MinKNOW to test the suitability of MinION. To perform a single MinION sequencing run, we loaded Library A in the flow cell at the beginning of the run with 1575 active pores enabled, while Library B was loaded when 1260 active pores remained active. Because the final library concentration was suboptimal, we observed that only a limited number of pores were active at a time, as shown in the MinKNOW duty time plot in Guppy tools and obtained a set of 397,465 reads in the Fastq format file.Next, we launched the base calling of the reads using As stated previously, the overall quality of reads is affected by the adopted sequencing technique, i.e., the direct RNA seq. of samples derived from the swabs. In this experiment, the average read length was lower than that of standard long reads, and the quality of certain reads was below conventional levels. Therefore, we preferred to discard the reads that had low quality (<8), as well as a short length (<500 nt). We obtained a subset of 20,940 good-quality reads.minimap2 tool to filter out reads that mapped with sequences from human, fungal, and bacterial genomes.At this point, we aimed to identify and remove sequences from contaminant species contained in the swab; for this, we used the Using this method, we filtered 97.63% of the reads; details of the composition of these reads are provided in SAMtools and BCFtools libraries.Next, we analyzed the coverage of the mapped reads and the variant calling related to the N gene using the https://www.ncbi.nlm.nih.gov/nuccore/MT457389 (accessed on 12 May 2020).The analysis revealed the existence of the following mutation region with a quality score greater than 94.99: NC_045512:c.28881_28882_28883delinsAAC. The N gene sequence generated is available in the GenBank database and can be accessed at: To determine the frequency of mutation in the samples, we adopted two different approaches: Sanger sequencing and qPCR. Using the former approach, we amplified the region that contained the mutation prior to sequencing it. As shown in In the qPCR assay, we used custom primer sets designed to distinguish between WT and mutated sequences and another pair set. We compared two uninfected controls and the synthetic SARS-CoV-2 N cDNA, which was similar to the positive control of the reference genotype. All the samples yielded positive results with the primer set specified for the mutated sequence  and negaAt last, we investigated whether findings from other studies on SARS-CoV-2 were consistent with our findings. To this end, we considered sequence variations from the China National Center for Bioinformation (CNCB) portal . CNCB isAs shown in Nowadays, next-generation sequencing technologies may be used to analyze whole RNA transcriptomes of complex organisms and whole DNA or RNA genomes of microorganisms, within a short duration and at an affordable cost . The covOxford Nanopore protocols SARS-CoV2 PCR tailing based are suggested to sequence the coronavirus genome directly from nasopharyngeal or oropharyngeal swabs. In order to cover the whole genome, these protocols, exploiting the technology based on an array of nanopores, use a multiplex primers scheme , which generate longer amplicons and tend to produce close-to-finished genomes more quickly than short reads based sequencing platforms . MoreoveOn other hand, these protocols produce a higher percentage of reads that are not analyzable by the bioinformatic pipelines, likely due to a combination of fragmentation of synthesized molecules and prematurely aborted molecules during sequencing . BrejováIn this study, we showed as direct-RNA seq. on an array of nanopores can help to identify the native sequences of the genome of microorganisms at RNA, which could be present in clinical samples such as nasopharyngeal or oropharyngeal swabs. This protocol has the advantage of directly detecting the ribonucleobases passing through the pores without RT or PCR amplification, and this approach is free of possible biases or mis-amplifications introduced during such steps. This direct sequencing also simplifies the gene annotation process, allowing so the identification of more complex or novel transcript isoforms genome wide ,35, and Given the increasing prevalence of SARS-CoV-2 cases confirmed in several countries and the resulting need for unbiased sequencing approaches, our research group performed an Oxford Nanopore direct-RNA seq. experiment on oropharyngeal swab samples of Italian patients bearing SARS-CoV-2 from the Palermo area (Sicily).A similar approach was described on RNA of good quality extracted from cell cultures . In our study, we identified COVID-19 sequences from both high fragmentation (RIN index ranging from 2.6 to 2.1) and very low concentration RNA (in the order of pg/µL), which is directly extracted from the routine oropharyngeal swab.Therefore, in order to improve the abundance and the heterogeneity of the sequencing reads, we chose to sequence two libraries, A and B, in the same experiment, as previously described.Despite these limitations, through an appropriate bioinformatics pipeline used to analyze the Fast5 sequencing data, we detected in all samples used in this study the presence of the NC_045512:c.28881_28882_28883delinsAAC mutation, which is a set of mutations in the SARS-CoV2 phylogenetics already described in the literature.In order to confirm the robustness of the obtained data, we compared the in silico results using Sanger sequencing and qPCR assays, as described in the Results section . These aThe nanopore direct RNA seq. protocol allows carrying out true native RNA sequencing, without PCR request, to explore attributes of native RNA such as modified bases and to remove bias introduced from RT or PCR. Moreover, it might be recommended in the occurrence of RNAs that are difficult to reverse transcribe ,11.Additionally, it could be used for the direct sequencing of the SARS-CoV2 RNA genome, as well as other RNA viral genomes, i.e., in HIV viruses, and it could be also considered advantageous for both surveillance and epidemiologic studies.


The increase in pandemics caused by RNA viruses of zoonotic origin highlights the urgent need for broad-spectrum antivirals against novel and re-emerging RNA viruses. Broad-spectrum antivirals could be deployed as first-line interventions during an outbreak while virus-specific drugs and vaccines are developed and rolled out. Viruses depend on the host’s protein synthesis machinery for replication. Several natural compounds that target the cellular DEAD-box RNA helicase eIF4A, a key component of the eukaryotic translation initiation complex eIF4F, have emerged as potential broad-spectrum antivirals. Rocaglates, a group of flavaglines of plant origin that clamp mRNAs with highly structured 5′ untranslated regions (5′UTRs) onto the surface of eIF4A through specific stacking interactions, exhibit the largest selectivity and potential therapeutic indices among all known eIF4A inhibitors. Their unique mechanism of action limits the inhibitory effect of rocaglates to the translation of eIF4A-dependent viral mRNAs and a minor fraction of host mRNAs exhibiting stable RNA secondary structures and/or polypurine sequence stretches in their 5′UTRs, resulting in minimal potential toxic side effects. Maintaining a favorable safety profile while inducing efficient inhibition of a broad spectrum of RNA viruses makes rocaglates into primary candidates for further development as pan-antiviral therapeutics. The frequency of infectious disease outbreaks caused by RNA viruses of zoonotic origin in human populations has been rising at an alarming rate in recent years. Prominent examples since the beginning of this millennium are severe acute respiratory syndrome coronavirus (SARS-CoV) (2002–2004), influenza A virus subtype H1N1 (2009–2010), and Middle East respiratory syndrome coronavirus (MERS-CoV) (2012-present). The causes for this intensification are many, including anthropogenic modifications of the environment, encroachment of humans into forested and other natural areas, and overall increased human contact with wildlife benzofuran structure  in complex with RocA, polypurine RNA, and AMP-PNP, a non-hydrolyzable ATP analogue—have revealed that rocaglates reversibly clamp the RNA–helicase complex  65,91,9,991,92.  complex . In sili complex . Ligand- complex ,93.DDX3, the second DEAD-box RNA helicase targeted by rocaglates , interacAt the cellular level, ribosome profiling experiments have revealed the effects of eIF4A inhibition by rocaglates on the cell’s transcriptional program. Several studies showed that the global cellular effects of eIF4A inhibition are limited to about 300 cellular mRNAs ,97. AmonRNA viruses have evolved different ways to capitalize on their host’s translation machinery, making it a challenge to develop a universal strategy for inhibiting viral replication via translation inhibition. However, because of its central role in the translation mechanism, and especially in viral protein synthesis, eIF4A provides an excellent broad-spectrum target in the context of host-targeting antiviral strategies . Next, wArenaviridae, Nairoviridae, Filoviridae are negative-stranded (including ambisense) RNA viruses and the causative agents of diseases such as measles, rabies, influenza, and viral hemorrhagic fevers (VHFs). The latter are febrile illnesses caused by members of various (−)-stranded RNA virus families, including arenaviruses ), nairoviruses ), and filoviruses ) [ (EBOV)) . With fa (EBOV)) . EBOV co (EBOV)) . Indeed, (EBOV)) . Similar (EBOV)) .Coronaviridae are very sensitive to eIF4A inhibition. For example, dose-dependent inhibition by silvestrol and CR-31-B (-) was shown for human coronavirus 229E (HCoV-229E), MERS-CoV, and SARS-CoV-2 replication in vitro, with EC50 values in the range of 1–3 nM [90 of 37 nM in vitro [This large and genetically divergent family of plus-stranded RNA viruses infects a wide range of animals and has significant zoonotic potential, as illustrated by several coronavirus epidemics since the beginning of the century and the current SARS-CoV-2 pandemic  see Tab. Membersf 1–3 nM . In an ef 1–3 nM . A comprf 1–3 nM . Zotatifin vitro . Zotatifin vitro ,108.Togaviridae. The family includes arthropod-borne viruses (arboviruses) that are transmitted by blood-feeding vectors including mosquitos, sandflies, and ticks [Chikungunya virus (CHIKV), an arthropod-borne virus, is a member of the family nd ticks . The  is the causative agent of hepatitis E and a common cause of acute viral hepatitis in humans. HEV requires several host eIFs, including eIF4A, eIF4G, and eIF4E for viral mRNA translation . In vitrPicornaviridae is a family of plus-strand RNA viruses that includes important human pathogens, such as poliovirus (PV) type 1, 2, and 3 and human rhinoviruses (HRV). Picornavirus RNA translation is mediated by class I and II IRES structures and thus depends on eIF4A. In HeLa cells, hippuristanol slowed down PV replication [50 values of 20 nM and 100 nM, respectively [Picornaviridae compared to other RNA viruses, such as members of the family Coronaviridae. The question of whether or not these differences are related to mechanistic differences between cap-dependent and IRES-dependent eIF4A functions and the relevance of DDX3 with its equivalent role to that of eIF4A in class I and II IRES-dependent translation remains to be addressed in further studies.lication , while sectively . The eIFFlaviviridae includes viruses that employ fundamentally different strategies to ensure viral genome translation. One group of viruses, including members of the genera Hepacivirus ), Pestivirus ), and Pegivirus, control viral protein translation strictly through class III IRES elements and are thus resistant to eIF4A inhibitors such as hippuristanol and silvestrol [Flavivirus  and Zika virus (ZIKV)), was widely accepted to employ 5′ cap-dependent translation initiation mechanisms [The family lvestrol ,116. Thechanisms ,118. Trechanisms .Altogether, the broad-spectrum of eIF4A-dependent RNA viruses that are sensitive to rocaglate-mediated inhibition of viral protein translation illustrates the potential of this class of compounds as pan-antiviral treatments in future outbreaks of newly emerging or re-emerging RNA viruses.The a priori development of pan-antivirals has become increasingly urgent in light of the accelerating pace of novel and re-emerging viral outbreaks ,121. AmoTo date, the default rapid response to an outbreak caused by an unknown virus has been to set up large screens of approved antivirals or compounds that have passed phase 1 or 2 (proof-of-concept) clinical trials and have been shown to have favorable safety profiles in humans ,123,124.A framework for the systematic discovery and development of pan-antiviral rocaglates would need to include a large but defined number of in vitro and ex vivo studies showing consistent antiviral effects across viral families and eIF4A-dependent translation mechanisms. Such a framework would provide a solid mechanistic foundation to help predict whether a novel viral strain might be sensitive to rocaglates following an initial sequence-based determination of its putative translation mechanism ,126. TheOnce reasonable in vitro and ex vivo evidence of the pan-antiviral activity of a rocaglate exists, critical in vivo studies need to be carried out in preparation for potential clinical development of the compound . The chaThe need to use a diverse panel of preclinical animal models to test for pan-antiviral activity of a rocaglate means that there will be an equally diverse number of ADMET data that will require careful harmonization to be able to meaningfully extrapolate them to humans. Critical aspects include optimization of the in vivo panel to minimize the number of animal models used and to maximize the similarity of their infection mechanism parameters to humans. If there is for any given virus a choice of model, the model that closest resembles human biology should be used—usually, this would mean to choose larger non-rodent rather than smaller rodent animals. However, this guideline would need to be balanced by how well established and efficient larger infection models are for a given virus, and the ethical, operational, and cost constraints of testing such animals.Once reasonable evidence has been gathered, and the necessary preclinical ADMET data have been collected and evaluated for a range of viral families, the stage is set for carrying out the necessary clinical studies leading to the potential approval of a rocaglate-based antiviral drug. The ongoing SARS-CoV-2 outbreak triggered an unprecedented initial effort to rapidly identify antiviral drugs that could be used to curb infection and/or mortality rates. Although, to shortcut the long time frames required for discovery of novel activities, the immediate focus was on the identification of compounds that had already passed phase 1 clinical studies and for which sufficient safety and potential dosage data in human were available to allow large scale phase 2 and phase 3 clinical trials to take place immediately ,131. A sRocaglates offer an opportunity to expand this approach and develop a novel framework for the development of truly pan-antiviral compounds that could be enlisted immediately upon an outbreak of a novel virus to mitigate its initial spread and effects while more permanent solutions such as vaccines are developed. Critical to this framework would be the confirmation that the compound is safe and effective against a broad-spectrum of existing viruses. Once a novel virus appears, such a drug could be immediately deployed and its effectiveness and possible virus-related adverse effects monitored using the existing framework of a phase 4 or so-called post-marketing clinical study .With Zotatifin eFT226), the first eIF4A inhibitor in the rocaglate family to enter clinical trials as an antiviral, the foundation is set for the further development of rocaglates as potential pan-antivirals [6, the fiBeyond the focus on gathering biological, mechanistic, and clinical evidence of the pan-antiviral potential of a rocaglate, several other critical aspects of drug development need to be considered as well. (i) Manufacturing and scale-up: Rocaglates are complex to manufacture, and as such, the feasibility of manufacturing a lead rocaglate efficiently and in high quantities needs to be determined early in the development process . Key parIt is advisable to initiate conversations with industry partners and regulatory authorities early in the process to explore solutions and optimize the overall process. Pharma partners can contribute both invaluable medicinal chemistry knowhow and the infrastructure and resources for clinical trials, while early contact with regulatory bodies can be key to designing and implementing the clinical trials most conducive to eventual approval of the drug.Finally, before becoming a focal point as potential antiviral drugs, rocaglates were already being extensively researched in the context of cancer ,98. In vRocaglates have emerged as strong contestants in the race to develop broad-spectrum antivirals to deploy against future emerging and re-emerging RNA viral outbreaks. Key to their favorable therapeutic profile is a distinct mechanism of action that provides exquisite target specificity and minimal interference with host biology, resulting in potentially large therapeutic windows.Plasmodium falciparum and P. berghei [Candida auris [It is worth pointing out here that beyond their antiviral and antitumor activity, rocaglates have also been shown to have eIF4A-dependent antiplasmodial effects in vitro and in vivo against  berghei  and an eda auris , furtherWith several rocaglates now entering preclinical development and at least one rocaglate already in early clinical development, a translational path for rocaglates as antiviral and eventually pan-antiviral therapeutics is starting to materialize. Many biological, clinical, and practical challenges remain, but a concerted effort to further develop the many therapeutic possibilities of this promising family of compounds should result in an improved understanding of the biology of eIF4A and of the potential benefits of modulating its activity using small molecules.


RNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human herpesvirus 6B (HHV-6B) infection. The “omics” revolution of recent years has simplified the study of RNA transcripts produced during viral infection and under specific defined conditions. In the quest to find new and differentially expressed transcripts during the course of human herpesvirus 6B (HHV-6B) infection, we made use of large-scale RNA sequencing to analyze the HHV-6B transcriptome during productive infection of human Molt-3 T cells. Analyses were performed at different time points following infection, and specific inhibitors were used to classify the kinetic class of each open reading frame (ORF) reported in the annotated genome of the HHV-6B Z29 strain. The initial search focused on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts was observed in all samples analyzed. The presence of many of these new HHV-6B transcripts was confirmed by reverse transcriptase PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported open reading frames (ORFs), including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome.IMPORTANCE RNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human herpesvirus 6B (HHV-6B) infection. Using six different time points, we were able to identify the presence of differentially spliced genes at 6, 9, 12, 24, 48, and 72 h postinfection. Determination of the RNA profiles in the presence of cycloheximide (CHX) or phosphonoacetic acid (PAA) also permitted identification of the kinetic class of each ORF described in the annotated GenBank file. We also identified new spliced transcripts for certain genes and evaluated their relative expression over time. These data and next-generation sequencing (NGS) of the viral DNA have led us to propose a new version of the HHV-6B Z29 GenBank annotated file, without changing ORF names, to facilitate trace-back and correlate our work with previous studies on HHV-6B. Roseolovirus, within the Betaherpesvirinae subfamily of the Herpesviridae family. HHV-6B is acquired by greater than 90% of the population by the age of three and is the etiologic agent of Roseola, also called sixth disease or exanthema subitum  is a member of the genus  subitum . Roseola subitum . HHV-6B  subitum . As with subitum –6. HHV-6subitum –, 7, 8. Ccontrols .The HHV-6A/B genome comprises a 160-kb linear doubled-stranded DNA molecule , 12. A uInnovative “omics” approaches in recent years have made high-throughput sequencing more available and less expensive, making large genome sequencing and RNA sequencing (RNA-seq) possible. In the last 2 years, a significant amount of data has become available, with the determination of numerous genomes, transcriptomes, translatomes, and proteomes for many species and pathogens. Herpesviruses have proven no exception, with many omics data sets available for cytomegalovirus, gammaherpesviruses, herpes simplex virus (HSV), and even HHV-6A/B. In recent months, a group from Israel reported novel and conserved genomic features for HHV-6A/B using RNA-seq and ribosome profiling (Ribo-seq) . Using tThe objective of this study was to determine the RNA profile during the course of an active HHV-6B Z29 infection in the Molt-3 T-cell line. We also wanted to determine the RNA profile of HHV-6B Z29 infection in the presence of cycloheximide (CHX) and phosphonoacetic acid (PPA), two inhibitors that permit the identification of the kinetic class of each gene. Finally, we defined new spliced variants of several genes, identified new transcripts, and proposed a new annotation for HHV-6B Z29 that is built on the current and widely used reference sequence.To explore the transcriptome of Molt-3 cells infected with HHV-6B, we performed an infection time course study with samples taken at 6, 9, 12, 24, 48, and 72 h postinfection. RNA was extracted and analyzed by RNA-seq. The 72-h postinfection sample was initially used to analyze the read coverage obtained using our RNA-seq protocol. As observed in AF157706). As shown in Using data from across the time course of infection, we generated a heat map showing the relative expression of each ORF described in the annotated HHV-6B genome  were restricted to the MF994829.1 sequence  (X83413) (Our attention then turned to splice variants of the virus transcriptome. We used our RNA-seq data to generate a splice map in relation to the annotated genome . A numbeF157706) . Our RNA(X83413) . To conf(X83413) . As showAF157706, the U12 gene is described as two exons separated by an intron   . From thF157706) . We thenF157706)  24, 25), 25AF157F157706) . Of the F157706) . HoweverF157706)  27, 28), 28AF157F157706) . TherefoF157706)  Table 3AF157706 F157706) . A compaMW536483. In this GenBank record and as shown in AF157706. This mutation was observed in our NGS data on HHV-6B DNA and in all RNA-seq data from the different time points mentioned in this study. However, it is not found in any other HHV-6B sequences in the database. As mentioned in a recent study on HSV-1  were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (Corning Cellgro), HEPES, and Plasmocin at 5 μg/ml . HHV-6B (Z29 strain) was propagated in Molt-3 cells, as previously described .Seventy million Molt-3 cells were infected with HHV-6B at a multiplicity of infection (MOI) of 0.5 for 4 h in 4 ml of supplemented RPMI 1640. Cells were then washed twice with 1× phosphate-buffered saline (PBS). DNA was extracted from 1,000,000 cells, and the remaining cells were resuspended at 500,000 cells/ml in supplemented RPMI 1640. Ten million cells were harvested at 6, 9, 12, 24, 48, and 72 h postinfection, and the cell pellets were stored at −80°C until extraction. DNA was extracted from 1,000,000 cells at 72 h postinfection. Ten million Molt-3 cells were incubated in the presence of 10 μg/ml cycloheximide (CHX)  for 30 min before HHV-6B infection at an MOI of 0.5. Cells were maintained in 600 μl for 4 h, washed twice with 1× PBS, and resuspended at 500,000 cells/ml in supplemented RPMI 1640 containing 10 μg/ml CHX. Cells were harvested at 9 h postinfection, and the pellet was stored at −80°C until extraction. Ten million Molt-3 cells were incubated in the presence of 100 μg/ml phosphonoacetic acid (PAA) (MilliporeSigma Canada) for 30 min before HHV-6B infection at an MOI of 0.5. Cells were maintained in 600 μl for 4 h and washed twice with 1× PBS. DNA was extracted from 1,000,000 cells, and the remaining cells were resuspended at 500,000 cells/ml in supplemented RPMI 1640 containing 100 μg/ml PAA. Cells were harvested at 72 h postinfection, and the pellet was kept stored at −80°C until extraction. DNA was extracted from 1,000,000 cells at 72 h postinfection in the presence of PAA. Ten million uninfected Molt-3 cells were also harvested as a negative control.DNA was extracted from cells using the QiaAMP DNA blood minikit as described by the manufacturer (Qiagen Inc.) and analyzed by ddPCR as previously described . In brieLinear HHV-6B viral DNA was isolated as described above from cell-free viral stocks prepared as previously described . The HHVTotal RNA was isolated using the standard Qiazol protocol . rRNA was removed using a Ribo-Zero gold rRNA removal kit (human/mouse/rat) by following the manufacturer’s instructions (Illumina Inc.).Libraries were generated for all conditions using the Ion Total RNA-Seq kit v2  by following a standard protocol. Libraries were then sequenced on an Ion Torrent S5 sequencer (Thermo Fisher Scientific).Homo sapiens reference genome GRCh38 and to the Z29 strain of HHV-6B (NCBI accession number AF157706). Alignments were performed using Bowtie2 v2.3.4.1, and junction prediction was determined using TopHat v2.1.1. Transcriptional and gene read counts were obtained with CLC Genomics Workbench v9.5.2 (Qiagen) using the RNA-seq analysis tool and the genome annotations mentioned above.Sequencing returned 94 million reads with greater than 9.4 million reads per condition. Reads were aligned to the Immunofluorescence was performed as previously described . In brieTen million Molt-3 cells continuously infected with HHV-6B were harvested. Total RNA was isolated using the standard Qiazol protocol (Qiagen Inc.) and processed for mRNA analysis by reverse transcriptase PCR (RT-PCR). In brief, 1 μg of total RNA was reverse transcribed in a total volume of 20 μl using the Moloney murine leukemia virus reverse transcriptase. One-tenth of the cDNA was used for the amplification of specific splicing junctions, using the primers specified in Sanger sequencing and next-generation sequencing were performed at the facility located at the CHU de Québec-Université Laval Research Center.MW536483. Sequencing data files have been deposited under the BioProject accession number PRJNA680783.The annotated HHV-6B sequence has been deposited under the GenBank accession number


On the basis of the efficacy and tolerable safety profiles of immune checkpoints inhibitors (ICIs) in second-line metastatic urothelial carcinoma (mUC) patients, some emerging clinical trials focus on the first-line treatment. Thus, we conducted a network meta-analysis (NMA) to assess and compare the response and toxicity of ICIs in naïve-chemotherapy mUC setting. According to our results, combination therapy (either ICIs plus chemotherapy (CTX) or ICIs plus ICIs) had a higher priority in terms of overall survival. Concerning monotherapy, ICIs are not inferior to CTX in terms of OS. In view of the adverse effect, ICIs are very tolerable, and combination therapy did not lead to a higher incidence of grade 3–5 AEs when compared with CTX.Immune checkpoints inhibitors (ICIs) were considered as second-line treatments in metastatic urothelial carcinoma (mUC) based on better survival benefit and safety profile than chemotherapy (CTX). We aimed to assess different ICIs regimens in the efficacy and safety for front-line treatments in mUC patients. A comprehensive literature search was performed and Phase II-III randomized controlled trials (RCTs) on ICIs for patients with mUC were included. The outcome was evaluated by overall survival (OS), progression of free survival (PFS), objective response rate (ORR), and grade 3–5 adverse events. Network meta-analysis was used to estimate the effect size. Surface under cumulative ranking curves (SUCRAs) were applied to rank the included treatments for each outcome. Results: The survival benefit of a single ICI was non-inferiority to chemotherapy (CTX). Although no superior effects were indicated, combination therapy (either ICIs plus CTX or ICIs plus ICIs) presented better OS compared with CTX alone. In terms of PFS, combination therapy produced a noticeable benefit over CTX. Regarding the SUCRA ranking, atezolizumab plus CTX was associated with the best ranking for OS and pembrolizumab plus CTX was the best in PFS. In terms of safety, a single ICI had better safety profile than CTX and combination therapy had a similar risk of grade 3–5 adverse events with CTX. Conclusions: Our NMA results revealed that combination therapy has better ranking compared with monotherapy in OS and acceptable AEs. ICIs alone present non-inferior OS but a lower incidence of AEs compared with CTX.  Metastatic urothelial carcinoma (mUC) arising from upper or lower urinary tract systems has poor prognosis and short-term survival . CurrentIn recent years, immune checkpoints inhibitors (ICIs) against programmed death 1 (PD-1) and its ligand (PD-L1) have been investigated for mUC which progress despite chemotherapy. Five immune checkpoints inhibitors including two PD-1 inhibitors, pembrolizumab and nivolumab and three PD-L1 inhibitors, as well as atezolizumab, avelumab, and durvalumab have been approved by the Food and Drug Administration for mUC patients with prior chemotherapy or cisplatin ineligible condition . AlthougWith the promising results of PD-1/L1 inhibitors as a second-line treatment for mUC, there are several emerging clinical trials focusing on PD-1/L1 inhibitors for mUC in the first-line setting. However, no head to head RCTs compare the efficacy and safety among ICIs and combination therapy (either ICIs plus chemotherapy (CTX) or ICIs plus ICIs). As ICIs are very expensive and mUC patients have short-term survival, we urgently need to identify the single best available treatment. Hence, we conducted a network meta-analysis (NMA) to investigate and compare the treatment response and toxicity of PD-1/L1 inhibitors versus chemotherapy as a first-line treatment for patients with mUC. The search strategy was presented in Details on the characteristics of three included studies were reported in Results of the quality assessment were presented in Results were presented in Results were presented in Results were presented in Results were presented in Results of the subgroup analysis were presented in Based on Based on Although platinum-based chemotherapy is considered the first-line treatment for UC, it is inevitably associated with some limitations. For example, some patients are unfit to receive chemotherapy due to comorbidities, and some patients progress despite receiving chemotherapy. Recent clinical trials have established the efficacy of atezolizumab as a second-line treatment for locally advanced and mUC, which led to its FDA approval in 2016 ,11. KEYNp = 0.027) and PFS  from IMvigor130 trial.While the results of ICIs are very promising, comparisons among ICIs were currently unknown. Hence, we performed this network meta-analysis on the available Phase III trials  to investigate the role of ICIs and combination therapy (either ICIs plus CTX or ICIs plus ICIs) in the first-line setting for both mUC cisplatin-ineligible and eligible patients ,16,17. SIn our analysis, the addition of pembrolizumab (or atezolizumab) to platinum-based chemotherapy resulted in better PFS and ORR when compared with CTX alone. Although there was no significant overall survival benefit, a better median overall survival was observed in the combination therapy. Chemotherapy has an immunosuppressive effect, but when combined with ICIs, it may impose an immunomodulatory function. By modulating the tumor microenvironment, the body’s immune response may be influenced resulting in a better efficacy upon ICIs . ICIs arIn terms of monotherapy, although a lower response rate was noted upon ICIs, ICIs are not inferior to CTX in terms of OS. Hence, it is reasonable to give ICIs to mUC patients who cannot tolerate the adverse effects of CTX. Given the low response rate of ICIs, combined with another treatment that targets a mutated gene, for example, fibroblast growth factor receptor (FGFR) seems to be a reasonable approach . More reAs expected, our analysis showed that ICIs were associated with a lower incidence of adverse events when compared with CTX. Additionally, no significant differences between ICIs plus CTX and CTX were presented. Adverse effects related to ICIs are termed “immune-related adverse effects (irAEs)” which may affect multiple organs simultaneously or at different time points. However, these irAEs are usually manageable . Even ifUpon further subgroup SUCRA analysis, combination therapy was shown to have better OS in both male or female, young people or elderly, cisplatin eligible or ineligible patients, primary tumor in upper or lower tract. Although ICIs might be better for elderly patients due to its favorable safety profile, an impaired immune system with increasing age may alleviate the efficacy of ICIs ,24. TherThere are several limitations in our study. First, only three Phase III RCTs were included for NMA and the available data is relatively limited. The baseline characteristics of the included patients may be heterogenous across the studies, and the results may be affected. Second, for a more comprehensive comparison, we assessed OS, PFS, ORR, and the severity of AEs in our analysis. However, not all survival rates were analyzed in RCTs and might have an impact on the evaluation of the best treatment modality. Furthermore, upper tract urothelial carcinoma (UTUC) patients comprising of renal pelvis and ureter cancer were also included in this analysis. The molecular characterization between UTUC and bladder cancer may differ and require different clinical management strategies . UnfortuThis study followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) extension statement for the network meta-analysis .Clinical-Trials.gov up to 15 October 2020 without language limitation. Moreover, we searched the abstracts in the main oncology congresses database, such as the American Society of Clinical Oncology (ASCO), European Medical Oncology (ESMO), and American Association of Cancer Research (AACR). In addition, we reviewed the reference lists of the retrieved studies to include more relevant studies. The following search terms were used in the search strategy: Transitional cell carcinoma (TCC), urothelial carcinoma (UC), immunotherapy , and chemotherapy for TCC or UC. The inclusion criteria were as follows: (1) Completed Phase II–III RCTs involving an adult with advanced or metastatic TCC or UC, (2) the RCTs focused on newly diagnosed treatment-naïve patients, (3) the RCTs provided the efficacy and safety comparison between immunotherapy and the standard treatment arm with chemotherapy.We identified eligible studies through a comprehensive search from Medline/PubMed, Embase, Cochrane library (CENTRAL and CDSR), and ClinicalTrials.gov), baseline characters, treatment arm with a subject number, and median follow up duration. We evaluated the quality of included RCTs by the Cochrane Collaboration’s risk of bias (ROB) tool and presented the results by the Review Manager  [Two independent reviewers (H.Y. Li and Y.C. Teoh) performed literature screening, data extraction, and quality assessment. In the case of any discrepancies, a third reviewer (H.L. Chen) was introduced for discussion to reach a consensus. The extracted information included trial name, published year, trial phase, NCT number (a RCT identifier of Denmark) .Our primary outcome was the treatment efficacy, which was evaluated by overall survival (OS), progression of free survival (PFS), as well as the objective response rate (ORR). Our secondary outcome was the safety profile focusing on grade 3–5 adverse events according to the Common Terminology Criteria for Adverse Events (CTCAE) . For binFor the data synthesis, we first generated the network geometry in order to clarify which interventions were compared directly or indirectly. After that, the network meta-analysis (NMA) was applied. NMA is a technique for comparing interventions in a single analysis by combining direct and indirect evidence across the network of available studies. If two particular interventions have never been compared head-to-head, but these two have both been compared to a common comparator (placebo or standard therapy), then an indirect comparison can be evaluated vs. the common comparator. We undertook a network meta-analysis under the frequentist framework by the mvmeta Stata command   to estimConsistency and transitivity were checked for the validation of NMA. Inconsistency was defined as the difference between direct and indirect evidence. The design by treatment inconsistency model was used to check the consistency for each direct treatment contrast based on the difference between the different study designs. Transitivity was evaluated by comparing the distribution of effect modifiers across different comparisons.From this NMA focusing on the first-line treatment of mUC patients, combination therapy (either ICIs plus CTX or ICIs plus ICIs) had a higher priority in terms of OS upon the ranking analysis. Concerning monotherapy, ICIs are not inferior to CTX in terms of OS. The benefit of combination therapy is presented regardless of gender and age upon the subgroup analysis. In view of the adverse effect, ICIs are very tolerable, and combination therapy did not lead to a higher incidence of grade 3–5 AEs when compared with CTX.