However, the function and underlying mechanism of Nm modification in regulating viral infection and innate immunity are largely unknown. Here we found that 2’-O-methyladenosine (Am) on poly A+ RNA was increased in virus infected-macrophages. Functional screening identified RNA 2’-O-methyltransferase Fibrillarin (FBL) in facilitating viral infection. Down-regulation of FBL inhibited viral infection through blocking virus entry into macrophages. Furthermore, knockdown of FBL could reduce viral entry by increasing ISGs expression through IFN-I signaling. These results indicated that FBL-mediated Nm modifications of RNA may avoid the innate immune recognition, thereby maintain immune homeostasis. Once FBL is down-regulated, the decreased Nm modifications of RNA in macrophages may act as “non-self” RNA and be recognized by RNA sensor interferon induced with helicase C domain 1 (MDA5), leading to innate immune activation by inducing the expression of IFN-I and ISGs. Therefore, our finding reveals a new role of FBL and its mediated RNA Nm modifications in facilitating viral infection and inhibiting innate immune response, adding mechanistic insight to the RNA modifications in infection and immunity.Type I interferons (IFN-I) play crucial roles in antiviral immune responses through inducing multiple antiviral interferon stimulated genes (ISGs). RNA modifications are emerging as critical post-transcriptional regulators of gene expression programs, which affect diverse biological processes. 2 The i pathway , 3. In t pathway –6. Among pathway , while mne (m6A) –11. We prophages . However’-O methylation (Nm) is one of the most common types of RNA modifications that are found in ribosomal RNAs (rRNAs), transfer RNAs, small nucleolar RNAs and also in messenger RNAs (mRNAs) (’-OH group of RNA riboses and respectively named as 2’-O-methyladenosine (Am), 2’-O-methylguanosine (Gm), 2’-O-methylcytidine (Cm) and 2’-O-methyluridine (Um). Nm endows nucleotides with greater hydrophobicity and affects RNA molecules in a variety of ways including the structure, stability and interaction of RNA, so as to regulate various cellular processes such as translation  confirm that Nm modifications are present not only in the 5’cap of the mRNA, but also in the interior of some mRNAs (RNA 2 (mRNAs) . Nm modinslation , 12–14. nslation , 16. Theelf mRNA , 18. 2’-me mRNAs . However’-O-methyltransferase and a highly conserved protein, which is located in the dense fibrillar component of the nucleolus  is a 34 kDa nucleolar RNA 2ucleolus . FBL maiucleolus . Previouucleolus . Targetiucleolus . Besidesediators . FBL alsediators , 24. How’-O-methyltransferases, in this study we found that FBL inhibits innate immune response by suppressing the expression of IFN-I and ISGs in macrophages, which can promote virus entry into macrophages to facilitate viral infection.By functional screening of eight RNA 2Ifnar1-/-) mice (6-8 weeks old) were obtained from Jackson Laboratory. All mice were bred and maintained under specific-pathogen-free conditions. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Animals Care and Use Committees of the Institute of Laboratory Animal Sciences of Chinese Academy of Medical Sciences (ACUC-A01-2021-040).C57BL/6 mice (6-8 weeks old) were from Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing). The interferon-α/β receptor 1 (IFNAR1)-deficient  and herpes simplex virus type 1 (HSV-1) viruses were used as described previously .’-O-Methyladenosine (591363), Cytidine (119085), 2’-O-Methylcytidine (391517), Guanosine (979688), 2’-O-Methylguanosine hydrate (329290), Uridine (399796), 6-chloropurine riboside (455573), 2’-O-Methyluridine (488001) were obtained from J&K Scientific Ltd.Adenosine (132283), 2These assays were performed as described previously . VSV-G (’s instructions. RNA was reversed-transcribed using the Reverse Transcription System from Toyobo (FSQ 301). Then cDNA was amplified by real-time PCR and analyzed as described previously  according to the manufacturereviously . The pri’s instructions.RAW264.7 and A549 cells were transfected with Lipofectamine™ 3000 Transfection Reagent  or LipoMax DNA Transfection Reagent  for 48 h according to the manufacturer’s instructions. After 48h, the cells were harvested or infected with virus for corresponding hours. siRNAs were designed and synthesized by RiboBio  were transfected into the mouse peritoneal macrophages and A549 cell lines with Lipofectamine™ RNAiMAX Transfection Reagent  for 48 h following the manufacturerFbl-iKO RAW264.7 cells which stably expressed Fbl sgRNA based on above iKO RAW264.7 cells. When Fbl-iKO cell line was infected with this lentivirus, Cas9 expression first induced and then the transient knockout of Fbl with Fbl sgRNA induced.RAW264.7 cells with inducible expression of Cas9 by Cre-loxP system (iKO RAW264.7 cell) were conducted. In these cells, genome was inserted with Cas9 sequence and before cas9 sequence there was transcriptional termination sequences with LoxP sites at both ends  and VeriKine Mouse IFN Alpha ELISA Kit  according to the manufacturervia qRT–PCR. For virus-entry assays, mouse peritoneal macrophages transfected with the indicated siRNAs for 48 h and then infected with VSV (MOI=3) for 30min in 4°C. After 6 washes with ice-cold PBS and 2% BSA, pre-warmed 37°C medium supplemented with 2% FBS and 15 mM NH4Cl was added to cells. Cells were incubated at 37°C for 1 h to allow the virus to enter cells. Then cells were chilled on ice and incubated with 500 ng/ml proteinase K in PBS at 4 °C for 2h to remove residual plasma-membrane-bound virions. After 6 additional washes with ice-cold PBS and 2% bovine serum albumin. Then cells were lysed and RNA was extracted. And vRNA levels were quantified via qRT–PCR.For virus-binding assays, mouse peritoneal macrophages were transfected with the indicated siRNAs for 48 h and then infected with VSV (MOI=3) for 30min on the ice. Cells were washed 6 times with ice-cold PBS supplemented with 2% bovine serum albumin to remove unbound virions. Then cells were lysed and RNA was extracted. Bound virions were quantified as viral RNA (vRNA) levels ® rRNA Depletion Kit (E6310L) from New England BioLabs (NEB).Poly A+ RNA was purified from total RNA with polyA tail purification using Dynabeads™ mRNA Purification Kit . The remaining rRNAs were further removed using NEB Next1~2ug isolated mRNA or total RNA were digested into single nucleosides by 1U nuclease P1  in 50 μl buffer containing 10mM ammonium acetate, pH 5.3 at 37°C for 12 h, followed by 42°C for 12 h, then mixed with 2 μl 1M ammonium bicarbonate, pH8.3, added 1U Bacterial Alkaline Phosphatase  in a final reaction volume of 100 μl adjusted with water, and incubated at 37°C for 12 h. 100ul chloroform was added to the reaction solution, 80ul supernatant was extracted by centrifugation after vortexing, and 20ul 6-chloropurine riboside (50ug/ml) was added and mixed. High performance liquid chromatography (HPLC) was modified slightly of the published procedures . Briefly’-O-methylation sequencing (Nm-Seq) was performed by CloudSeq Biotech Inc.  by following the published procedures with slight modification (’-end repaired using Antarctic phosphatase (NEB) at 37°C for 30 min. Then, repaired RNA samples were oxidized/eliminated using 10 mM NaIO4 (Sigma) in 200 mM lysine-HCl buffer  in a total volume of 40 µl at 37°C for 30 min. The reaction was quenched by ethylene glycol, samples were further dephosphorylated by Shrimp Alkaline Phosphatase (NEB) at 37°C for 30 min. Eight cycles of oxidation-elimination-dephosphorylation were performed. A final round of oxidation/elimination reaction was performed, excluding dephosphorylation. Then, purified RNA samples were 5’phosphorylated by T4 polynucleotide kinase 3’ phosphatase minus (NEB) at 37 °C for 60 min. Libraries were constructed from treated RNA fragments and untreated input fragments using NEBNext Small RNA Library Prep Set for Illumina (NEB). Sequencing was carried out on Illumina HiSeq4000 according to the manufacturer’s instructions.2fication . Briefly’-O-methylation counts and coverage counts were calculated by bedtools (v2.24) software and in-house scripts, then 2’-O-methylation-ratio (defined as: count/coverage) and 2’-O-methylation-fc (defined as: 2’-O-methylation-ratio/Input-2’-O-methylation-ratio) were also calculated. 2’-O-methylation sites were annotated with gene information by bedtools software. And the 2’-O-methylation sites were visualized in IGV (v2.64) software  software , high qusoftware . Sequenc’s instructions. RNA libraries were constructed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB) according to the manufacturer’s instructions. RNA high throughput sequencing (RNA-Seq) was performed by Cloud-Seq Biotech . Two independent biological replicates were performed for RNA-seq.Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (NEB) following the manufacturer’s t test. The relative gene expression data was acquired using the 2ΔΔCT method. P-value: ns, P >0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.Data are expressed as the mean ± standard error of the mean (SEM) from at least three independent triplicated experiments. The number of individuals and repeated experiments are stated in each figure legend. All data was analyzed using the GraphPad Prism software version 8.4.2. The statistical significance of comparisons between two groups was determined with Two-tailed unpaired Student. We found that the level of Am was increased on poly A+ RNA of RAW264.7 macrophages after VSV infection, while Am level in total RNA remained unchanged after VSV infection .In order to identify RNA modifications that regulate virus infection or innate immunity, we used high resolution mass spectrometry to observe the changed types of RNA modifications in RAW264.7 macrophages induced by VSV infection. To prevent rRNA modifications from interfering with the detection of low RNA modification level on mRNA, we optimized the poly A+ RNA purification method by adding a rRNA removal step, and the rRNA residue in this optimized method was much lower than the traditional purification method of Oligo dT beads . We foun. We founAm is one type of Nm modifications. To provide functional insights into whether mRNAs carrying Nm modification in macrophages are linked to antiviral innate immunity, we performed transcriptome-wide Nm-seq on the poly A+ RNA of RAW264.7 macrophages with or without VSV infection. We identified 6808 Nm sites (fold change (FC)≥4), 5643 of which had a minimal Nm-seq count of ten reads. Next, Gene Ontology (GO) analyses of these methylated genes were performed and showed that the significantly enriched methylated genes were as follows: translation and RNA process for molecular function (MF); extracellular exosome, nucleus and cytoplasm for cell compartment (CC); RNA and nucleotide binding, protein binding for biological process (BP)   via siRNAs-mediated knockdown, we found that knockdown of FBL inhibited VSV infection of mouse peritoneal macrophages, down-regulation of FTSJ1 promoted VSV infection in mouse peritoneal macrophages, and the effects of different siRNA of other enzymes were inconsistent or had no significant effects on VSV infection . FBL is an essential nucleolar protein that participates in pre-rRNA methylation and processing, and also is an extremely well-conserved protein during the evolution from archaea to human  . We further verified that FBL promoted VSV infection by Fbl-iKO RAW264.7 cells . Besides, knockdown of FBL significantly inhibited the infection of DNA virus HSV-1 in mouse peritoneal macrophages , in addition to RNA virus VSV. Consistently, knockdown of FBL inhibited VSV infection while overexpression of FBL facilitated VSV infection in human A549 cells . These results demonstrate that FBL facilitates viral infection.Furthermore, we observed that siRNA-mediated knockdown of FBL decreased VSV protein expression and VSV titers in cell supernatant ethality , FBL kno. Because. Because. Because. Because. Because. Because. Besides, we found that FBL knockdown did not affect the viability of mouse peritoneal macrophages . FBL knockdown significantly inhibited the VSV protein expression in the early stage of infection . Therefore, we hypothesize that FBL may affect the early stages of the VSV life cycle. To test this idea, we performed VSV binding and entry assays in mouse peritoneal macrophages and found that FBL knockdown did not affect VSV binding, but inhibited VSV entry into mouse peritoneal macrophages .IFN-I and ISGs play important roles in antiviral innate immunity. However, the mRNA and protein expressions of IFN-α and IFN-β, as well as the activation of IFN-I signaling pathway in FBL-knockdown mouse peritoneal macrophages were not increased than that in the control cells during VSV infection . Besides. Besides. Besides. Therefore, FBL-promoted VSV entry into macrophages in the early stage of VSV infection was not due to the regulation of LDLR expression in macrophages.It was reported that low density lipoprotein receptor (LDLR) and other members of this receptor family serve as VSV receptors on host cells , 33. We Fbl+/- RAW264.7 cells at the steady state without viral infection . Quantitative reverse transcription PCR (qRT-PCR) analysis also confirmed that the mRNA expressions of Ifi44, Oas2, Ifit3, Ddx58, Ddx60, Oasl1, Bst2, Ifit1 were up-regulated in Fbl+/- RAW264.7 cells . Besides, siRNA-mediated knockdown of FBL in mouse peritoneal macrophages also increased the expressions of above ISGs at the steady state .In order to investigate the underlying mechanism of FBL in facilitating VSV entry into macrophages, we performed RNA-seq and found that mRNA expressions of many ISGs were up-regulated in Therefore, FBL may act as an immunosuppressive factor under physiological conditions. FBL knockdown leads to increased expression of antiviral immune genes, thus inhibiting VSV entry into the “antiviral primed-macrophages”.Ifnar1-/- mouse macrophages, knockdown of FBL could not promote ISGs expression . Meanwhile, we found that FBL knockdown did not affect VSV entry into -/-Ifnar1 mouse peritoneal macrophages anymore . Besides, down-regulation of FBL did not regulate the expression of ISGs directly in macrophages when stimulated with enough IFN-β . Thus, FBL facilitates VSV entry into macrophages depending on the impairment of IFN-I signaling pathway.IFN-I induces the expression of ISGs through interferon-α/β receptor (IFNAR)-JAK-STAT signaling pathway , 3. WhetFbl+/- RAW264.7 macrophages compared to wide type cells . As rRNA is the most abundant type of RNA . We also measured Nm modifications in total RNA, and found that the levels of Am, Gm were decreased on total RNA of Fbl+/- RAW264.7 macrophages compared to wide type macrophages . While overexpression of mouse FBL led to increased Nm levels in Fbl+/- RAW264.7 cells . These results showed that FBL catalyzes the formation of Nm modifications directly.FBL usually catalyzes the formation of Nm modifications on rRNA under the guidance of BOX C/D snoRNAs . A recen’-O-methyltransferase activity was recognized and attenuated in wild-type primary cells and mice, but was pathogenic in the absence of IFN-I signaling  that FBL takes effect. In the VSV entry assay Figure 2MAVS-deficient A549 cells, down-regulation of FBL cannot increase the expression of IFN-I and ISGs, implying that the RNA sensor upstream of MAVS might account for the activation of IFN-I signal in FBL-deficient macrophages. Our results further showed that MDA5 is the RNA sensor responsible for the activation of IFN signal induced by FBL deficiency.In FBL-deficient immune cells, which PRR misidentifies self RNA with reduced Nm modifications as “non-self” RNA to activate the immune signaling pathway still needs further investigations. For RNA-sensing in the cytoplasm, RIG-I monitors the uncapped 5’-ends of RNA molecules . The carOur results reveal that FBL inhibits the expression of IFN-I and ISGs by suppressing the innate immune activation, which promotes virus entry and further viral infection in macrophages. In sum, we propose the following working model of FBL in viral infection and immunity. FBL directly catalyzes the formation of Nm RNA modifications. When FBL is at low expression level, the reduced Nm modification may be recognized as “non-self” RNA by MDA5, which activates innate immune response and promotes the IFN-I expression thus widely increasing the expression of antiviral ISGs, such as IFIT1, OAS2, IFIT3 and so on. Our findings also indicate the possible role of FBL in homeostasis maintenance by preventing autoinflammation. This study may provide a potential target for the control of infectious diseases and autoimmune diseases.https://www.ncbi.nlm.nih.gov/geo/, GSE185660 and https://www.ncbi.nlm.nih.gov/geo/, GSE185661.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by the Animals Care and Use Committees of the Institute of Laboratory Animal Sciences of Chinese Academy of Medical Sciences (ACUC-A01-2021-040).XC designed the experimental approach and supervised the study. PL, YL, RS, LZ, JY, and FL performed experiments. PL, YL, and XC analyzed data and wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China , and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Saccharomyces cerevisiae, a eukaryotic model organism widely used in industrial biotechnology, two different growing conditions are considered for comparison, including the poly-A messenger RNA isolated from yeast cells grown in minimum media under respirofermentative conditions supplemented with glucose (glucose growth conditions) and from cells that had shifted to ethanol as a carbon source (ethanol growth conditions). Library preparation for direct RNA sequencing is shorter than that for direct cDNA sequencing. The sequence characteristics of the two methods were different, such as sequence yields, quality score of reads, read length distribution, and mapped on reference ability of reads. However, differential gene expression analyses derived from the two approaches are comparable. The unique feature of direct RNA sequencing is RNA modification; we found that the RNA modification at the 5′ end of a transcript was underestimated due to the 3′ bias behavior of the direct RNA sequencing. Our comprehensive evaluation from this work could help researchers make informed choices when selecting an appropriate long-read sequencing method for understanding gene functions, pathways, and detailed functional characterization.Direct sequencing of single molecules through nanopores allows for accurate quantification and full-length characterization of native RNA or complementary DNA (cDNA) without amplification. Both nanopore-based native RNA and cDNA approaches involve complex transcriptome procedures at a lower cost. However, there are several differences between the two approaches. In this study, we perform matched native RNA sequencing and cDNA sequencing to enable relevant comparisons and evaluation. Using  The RNA sequencing (RNA-seq) method is now routinely used to explore a collection of all the gene readouts present in a cell or its transcriptome. Transcriptomic changes are a result of biological differences, making RNA-seq an exceptional opportunity to explore global regulatory networks in cells, tissues, organisms, and diseases. The most common RNA-seq methodology currently offered by next-generation sequencing (NGS) platforms  produces hundreds of millions of short-read sequences in the range of 100–600 base pairs. The short-read RNA-seq power provides not only large data output but also high accuracy in base calling. The short-read RNA-seq, therefore, is now a core component of research in nearly all biological fields .Despite dominant position of NGS in transcriptomics, short-read RNA-seq has been poorly suited for transcriptome assembly, novel splice isoform discovery, and novel gene detection. Because most eukaryotic messenger RNA (mRNA) transcripts are 1–2 kb in length , no mattThe rise of long-read technologies now opens the possibility to overcome those limitations and biases. Long-read RNA-seq captures a full-length transcript within a single read, thereby allowing accurate transcript annotation and enabling a comprehensive view of the transcriptome. Sequencing prokaryotic transcriptomes using the long-read technology reveals complex operon structures, which provide an important resource for functional annotation . CurrentSaccharomyces cerevisiae strain, CEN.PK113-7D, a yeast strain that is used extensively in academic and industrial research. We determined transcriptomic profiling under two different growth conditions (diauxic growth). Approximately 70% of the reads corresponded to full-length transcripts. Some full-length transcripts over 5 kb were also detected and mapped. In addition, identification of polyadenylated non-coding RNAs  is allowed using this sequencing protocol  protocol was subsequently released by ONT. The latter protocol is also PCR-free and carries out large complex whole-genome analysis at lower cost. However, there are several differences between the two approaches; notably, RNA and DNA sequencing speeds are different  following the manufacturer’s protocol.The details of cell growth and RNA purification were previously described . In brieIn this work, we started from aliquoted poly-A RNA used in our previous work . BrieflyFor dRNA-seq, the SQK-RNA001 Kit was used (ONT), and Superscript IV Reverse Transcriptase (ThermoFisher) was applied for the RNA stabilization step by formation of DNA–RNA hybrids through reverse transcription. After this, the motor protein was attached specifically to the RNA strands . Each liThe ONT raw data (.fast5 files) generated by MinKnow software, version 1.7.14 (ONT), were converted to basecalled fastq files using the local-based software Guppy, version 3.4.5 (ONT). This step automatically classifies failed and passed reads based on a specific cut-off for mean quality scores of 7, and only reads of >200 bases were included. The ONT reads (in standard fastq format) were aligned to the yeast S288c version R64 reference sequences, downloaded from SGD database, using Minimap2  to generThe dRNA reads were converted to DNA sequences, and reverse complement sequences of dcDNA reads were generated before alignments. For analysis of mapping results of yeast, we used SAMtools, version 1.6 , to invep-values of individual transcripts between the two compared growth conditions. We considered that the gene that has adjusted p-value < 0.001 was differentially expressed. Consequently, functional gene enrichment analysis based on Gene Ontology (GO) annotation was performed using the Platform for Integrative Analysis of Omics data (PIANO) package  software  with twoin silico approach provides an advantage over other SHAPE prediction tools, which require manually curated reference RNA structures. We implemented an in-house python script using libraries from the ShaKer tool for training a SHAPE model and to predict RNA structure and accessibility. A general model of SHAPE reactivity was trained on an experimentally determined SHAPE dataset provided in the ShaKer repository. Then, the predicted SHAPE model was used to support the prediction of structure and accessibility on each nucleotide of a given RNA sequence using RNAplfold  prediction using the graph kernel (ShaKer) tool . This inNAplfold . The scoNAplfold .The relative location of considered loci with reference to gene position was compared using Bedtools version 2 .The parameters and commands used are summarized in To compare dcDNA-seq vs. dRNA-seq, the poly-A mRNA isolated from yeast cells grown in minimum media supplemented with glucose and from cells that had switched to ethanol as a carbon source were aliquoted and used as the input of the two sequencing strategies to rescued batch effect. For each condition, three biological replicates were analyzed. The sequencing workflow of the two sequencing strategies is summarized in The differences in read characteristics obtained from dcDNA-seq and dRNA-seq for the two transcriptomes are summarized in p-value > 0.05) between dcDNA and dRNA sequences. Furthermore, the read quality score distribution of total reads differed between dcDNA and dRNA reads  by aligning both dcDNA and dRNA reads onto a reference genome. As presented in NA reads , with hiNA reads . This inNA reads .r = 0.96 on average, range: 0.94–0.98), while on average, r = 0.92 (range: 0.90–0.94) was obtained when dcDNA and dRNA sequences were compared for the same growth conduction. We recently demonstrated that the negative binomial statistic is a valid approach to analyze dRNA-seq data  were compared by scatter plots, and a correlation matrix was constructed . Within seq data ; here, wp-values  and the p-values . Even thp-values . The prep-values  did not We performed the RNA modification profiling using ELIGOS software by comparing the ESB of dRNA sequences with rBEM. Based on the three biological replicates of yeast grown on glucose and ethanol, we identified 134,980 sites of putative RNA modification with glucose and 192,240 sites with ethanol using the cut-off of odd ratios ≥ 3 and adjPval < 1e-50. The distribution of the identified sites on the individual transcripts is shown in ACC1), which is the rate-limiting step enzyme of fatty acid biosynthesis. We further investigated the transcript in detail through the Integrative Genomics Viewer (IGV) browser  growth and 3,817 sites differential sites for oxidative (ethanol) growth. The higher number of differential sites in the glucose-limited condition is along the line with the study of  100 see . Zooming 100 see  of yeastACS1 and ACS2. The main gene ACS1 was transcriptionally upregulated in ethanol , fatty acid biosynthesis , and a gene involved in glycogen–trehalose homeostasis (TPS1). These results indicate that there exists a complex association between posttranscriptional modifications and metabolic reprogramming.Genes under regulation to switch from glycolysis to ethanol utilization produce a very important metabolite acetyl-CoA, which as acetyl-CoA synthase has two isozymes, To study transcriptomes without application bias using ONT, we can perform either native or cDNA sequencing. Library preparations of native RNA sequencing has fewer steps, enabling a rapid characterization of RNA molecules as demonstrated in many studies . HoweverThe differential gene expression, which is a key result to study transcriptomes, derived from dRNA-seq and dcDNA-seq, is quite consistent at both the gene level and functional analysis level. Therefore, either method can be used to identify key transcriptionally regulated transcripts. Native RNA sequences provide opportunities to study posttranscriptional regulation, such as RNA methylations , leadingThe RNA ribonucleotide modifications are known to be critical regulators of a wide range of biologically relevant processes. One of the unique advantages of dRNA-seq, which we have looked into here, is the ability to detect RNA ribonucleotide modifications directly, which cannot be accomplished by dcDNA-seq. However, the reads generated by dRNA-seq could contain higher error rates that are derived from modified bases. The summary of comparison between the two library preparation approaches is shown in In summary, our study showed the advantages and disadvantages of using dRNA-seq or dcDNA-seq to study transcriptomes in yeast. This will be useful information for research studies to select a method for transcriptional characterization in various research interests.


Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible. Yellow fever virus (YFV) is the etiological agent of yellow fever (YF), a hemorrhagic disease endemic to sub-Saharan Africa and tropical regions of South America. Due to the threat YF poses to public health, a single case of constitutes an outbreak, making accurate diagnosis paramount. Viremia during YFV infection is variable and may be quite low depending on disease severity and the timing of sample collection. In addition, viral RNA is highly liable, especially in the hot and humid conditions that accompany YFV transmission season. To reduce RNA degradation, clinical samples are transported to diagnostic laboratories via cold chain. As cold chain is not always reliable or available, the capacity of certain regions to perform the molecular diagnosis of yellow fever is stunted, leading to a delay in diagnosis and outbreak management. Here we optimized a protocol for the incorporation of Whatman FTA cards into the YF molecular diagnostic with the goal of producing a cold chain-independent viral RNA stabilization method. These cards are compact, easy to use and were shown to inactivate YFV and stabilize YFV RNA at high temperature and humidity. The implementation of this protocol in endemic regions could aid in the rapid and accurate diagnosis of YF. Yellow fever virus (YFV) is a flavivirus endemic to 47 countries in sub-Saharan Africa, Central America, and South America. The virus is responsible for periodic outbreaks of yellow fever (YF) disease, a hemorrhagic disease with a case fatality rate of 30–60% in severe cases . Each yeDiagnosing YF can be difficult as patients present with non-specific, flu-like symptoms that can be confused with leptospirosis, malaria, and other viral hemorrhagic fevers during differential diagnosis. As such, laboratory diagnosis is required to confirm YF. Both serological methods  and/or detection of YFV-specific RNA are included in the WHO algorithm for laboratory diagnosis of YF . AlthougAlthough YF molecular diagnostic assays can differentiate between WT and rare adverse events associated with 17D vaccination , samplesWhatman FTA cards have been shown to be effective in the molecular diagnosis of multiple viral and bacterial diseases –24. The 10 plaque forming units (pfu)/ml] and WT strain Asibi (titer: 6 log10 pfu/mL) used in this study were obtained from the CDC Arboviral Disease Branch, Arbovirus Reference Collection. Experiments involving 17D-204 virus were performed according to biosafety level 2 requirements and experiments involving Asibi virus were performed according to biosafety level 3 safety requirements. Serial dilution of virus was performed in PBS  or flavivirus-negative human serum .The vaccine sub-strain 17D-204 [titer: 7 logContrived specimens were spotted onto FTA micro cards , allowed to dry and RNA extracted from discs punched from the card according to the manufacturers protocol . In order to minimize cross-contamination, cards were also pre-punched with a sterilized, 6 mm hole punch prior to inoculation. Punches were inoculated with 10 μL of contrived specimen and allowed to dry for one hour at room temperature in the biosafety cabinet. Individual punches were then placed into 1.5 mL tubes and stored as described until RNA extraction was performed using the QIAmp viral RNA extraction kit .To extract RNA, individual FTA punches were placed directly into 140 μL of PBS, thoroughly vortexed, added to 560 μL buffer AVL and allowed to incubate for 10 minutes at room temperature. When pooling RNA from two punches, individual punches were placed into 70 μL of PBS and vortexed. The extracts in PBS were then combined  and added to 560 μL of AVL. The remaining steps of the QIAmp protocol were then followed as previously described . Final eViral RNA was detected using the Quantitect probe RT-PCR kit  with YFall primers as previously described  in a tot10 pfu/ml), allowed to dry, and were rehydrated in 100 μL sterile water. The resulting FTA extract was used in two assays to confirm inactivation. First, 100 μL of FTA extract was inoculated onto Vero cells and allowed to incubate for 1 hour at room temperature after which DMEM media was added, and the flask placed at 37°C/5% CO2 and allowed to incubate for one week. Cells were monitored for cytopathic effect (CPE) which occurs with vaccine strains of YFV. After one week, 100 μL of media from the week one flask was transferred onto fresh Vero cells. This process was repeated for three weeks. Secondly, the FTA extract was used in a viral plaque assay. Ten-fold serial dilutions  of FTA extract were prepared in Bovine albumin-1 media (BA-1) and used to perform a plaque assay as previously described [Confirmation of viral inactivation was performed using institutionally approved protocols. The vaccine strain, 17D-204 virus was used as a surrogate for all YFVs in order to reduce biosafety concerns. FTA punches were inoculated with 10 μl of 17D-204 virus , 6 log10 pfu/mL , 5 log10 pfu/mL , 4 log10 pfu/mL (100 pfu/punch), 3 log10 pfu/mL (10 pfu/punch) and 2 log10 pfu/mL (1 pfu/punch). The ability of FTA cards to stabilize YFV RNA at an elevated temperature was measured by holding FTA punches with variable titers of YFV at 37°C for two weeks and comparing RNA positivity to control punches held at room temperature (~25°C) for the same amount of time. Samples were testing in triplicate and experiments were conducted in duplicate, leading to n = 6 for all groups. Punches in the 37°C group were placed directly into heat blocks after inoculation to ensure that any effect of temperature on the drying process was captured in the experiment. In experiments utilizing a single punch, RNA was extracted from one punch daily for seven days and at 14 days post-inoculation. For studies using pooled RNA from two punches, only 10 pfu/punch and 1 pfu/punch were utilized. Once a punch no longer tested positive for YFV RNA, punches from that dilution were removed from collection on subsequent days.For stability experiments, 17D-204 was diluted in PBS or human, flavivirus-negative serum and inoculated onto punches at concentrations of 7 logLaboratories in YF-endemic areas are often subject to high humidity. To assay the effect of humidity on the utility of the FTA cards, a two-part experiment was conducted using the one punch protocol described above. The first part sought to determine whether exposure of cards to high humidity post inoculation affected their ability to stabilize YF RNA. FTA cards were inoculated with 10 pfu/punch or 1 pfu/punch of 17–204 virus and incubated at high humidity (80–85%). RNA was extracted from one punch daily for seven days. Humidity readings were taken at each time point using a humidity and temperature pen (Fisher Brand) to ensure no large fluctuations in humidity occurred due to opening and closing of the incubator door. The second evaluation sought to determine whether exposure of cards to high humidity prior to inoculation affected their ability to stabilize YF RNA. FTA cards were incubated at 37°C and high humidity (80–85%) for one, two, and three days prior to inoculation cards were removed from the incubator, immediately punched, and inoculated with 10 pfu/mL or 1 pfu/mL of 17–204 virus. Punches were returned immediately to the incubator so that drying would occur at high humidity.Finally, the effect of desiccating cards that were previously exposed to humid conditions was assayed to establish if dry FTA cards were required to stabilize RNA after inoculation. FTA cards were placed in an incubator at 37°C for three days at 75–80% humidity, then were transferred into plastic bags containing one or two silica 1-gram gel desiccation packets  and sealed. A card placed in a bag with no desiccation packets was also included as a control at each timepoint. Bags were returned to the incubator for one, two or seven days prior to inoculation when FTA cards were punched and inoculated with 10 pfu/punch or 1 pfu/punch of 17D-204 virus. The punches were returned immediately to the bag containing the specified number of desiccant packets and placed into the incubator. RNA was extracted from punches and assayed for YFV RNA positivity every day for seven days. The experiment was repeated with a different brand of desiccation packet  to compare the efficacy of 1-gram desiccation packets.t value reached the cut-off threshold of 37. The goodness of fit for these regressions was calculated using R2 values. For high titer samples, the mean Ct value never reached 37 before the end of the time course, so the time to detection loss was extrapolated based on the linear regression model. Significant differences in LOD and time to negative result were determined by calculating the likelihood that the 95% confidence intervals of Ct value 37 for two experiments (95% CI) would overlap as described previously [t of 37 was extrapolated.Limit of detection (LOD) and time to negative result were calculated by linear regression modeling, estimating when the mean Ceviously . In caseThe vaccine strain of YFV, 17D-204, was confirmed to be inactivated after storage on FTA cards. No CPE was observed in Vero cells inoculated with 17D-204 FTA punch extract after three blind passages of 1 week each. Additionally, no plaques were observed after a plaque assay was performed on 17D-204 FTA punch extract.10 pfu/mL). These Ct values were compared to a standard extraction (140 μL of sample directly extracted) of both 17D-204 and Asibi. Although the mean Ct values were lower when RNA was extracted from an FTA punch, both strain 17D-204  and strain Asibi  were readily detected from FTA punches whereas the PBS control was negative for YFV RNA   .t values when sample was applied to the whole card and punched later  and when sample was applied to pre-punched cards   was shown to be significantly lower  than the LOD of one FTA punch ; however, there was no significant difference  in the LOD of RNA extracted from one FTA punch compared to 10 μL of virus extracted directly  (2 = 0.98) when compared to extracting RNA from one punch. This trend continued when RNA was extracted from three and four punches  . To dete = 0.97) . Increas punches .2 = 0.96) was significantly lower  than the LOD of an FTA punch inoculated with 10 μL virus diluted in human serum , but the LOD of an FTA punch inoculated with 10 μL virus diluted in serum was not significantly different  than a FTA punch inoculated with 10 μL virus diluted in PBS  as the diluent. As was observed in the experiment utilizing PBS as the diluent, the LOD of 140 μL of virus diluted in human serum , 10,000 pfu/punch (R2 = 0.80), 1,000 pfu/punch (R2 = 0.64) and 100 pfu/punch (R2 = 0.82) could be detected for over two weeks (2 = 0.88) and 1 pfu/punch for 3.5 days (R2 = 0.74) (2 = 0.78) and 10,000 pfu/punch (R2 = 0.73) were still detectable for more than 14 days; however, the 1,000 pfu/punch sample (R2 = 0.64) was detectable for 13.7 days (2 = 0.70), 10 pfu/punch  and 1 pfu/punch  samples decreased at 37°C  . At 37°C3.7 days . Similar at 37°C . Using 9To test if RNA extraction from multiple FTA punches improved the sensitivity of YFV molecular detection at high temperatures, RNA was pooled from two punches at 10 pfu/punch and 1 pfu/punch. It was shown that extracting RNA from two punches significantly increased the number of days RNA could be detected .t values when compared to dry cards incubated at low humidity . To test if these humidity conditions positively influenced RNA stability on the FTA cards, cards were inoculated with 10 pfu/punch and 1 pfu/punch of YFV and placed into a humidity chamber (80–85%) for one week. Control samples were left in the biosafety cabinet at ambient humidity for one week. Cards incubated at high humidity showed no difference in the time limit for detection of positive Chumidity . Despitehumidity . As the humidity . There whumidity .In an attempt to reduce the loss of RNA yield at high humidity, pre-humidified cards were incubated with desiccation packets for one, two and seven days prior to inoculation with low titer YFV Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Figt value 37 indicates the cut-off for YFV RNA positivity and is based on cut-offs used in the molecular diagnosis of YFV in the field.WT YFV strain Asibi and YFV vaccine strain 17D-204 were spotted onto FTA cards and allowed to dry for one hour. RNA was extracted from the cards and assayed using YFall qRT-PCR primers. RNA was also extracted directly from the Asibi and 17D-204 isolates using 140 μL of sample directly into lysis buffer. PBS was spotted onto FTA cards as a negative control. The dotted line at C(TIF)Click here for additional data file.S2 Figt value 37 indicates the cut-off for YFV RNA positivity and is based on cut-offs used in the molecular diagnosis of YFV in the field. Dilutions were performed in PBS. Points and error bars represent the average of two experiments and data was fit using a linear regression.YFV 17D-204 virus was inoculated onto FTA cards (130 μL sample over entire card) or onto FTA card punches (10 μL/ punch) and allowed to dry completely. RNA was then extracted from punches made from the whole FTA card or the pre-punched samples. The dotted line at C(TIF)Click here for additional data file.S3 FigMade using BioRender.(PNG)Click here for additional data file.


AcBBX5, a BBX family gene with two conserved B-box domains, was identified from pineapple. Subcellular localization analysis showed that AcBBX5 was located in the nucleus. Transactivation analysis indicated that AcBBX5 had no significant toxic effects on the yeast system and presented transcriptional activation activity in yeast. Overexpression of AcBBX5 delayed flowering time and enlarged flower morphology in Arabidopsis. Meanwhile, the expression levels of AtFT, AtSOC1, AtFUL and AtSEP3 were decreased, and the transcription levels of AtFLC and AtSVP were increased in AcBBX5-overexpressing Arabidopsis, which might lead to delayed flowering of transgenic plants. Furthermore, transcriptome data and QRT-PCR results showed that AcBBX5 was expressed in all floral organs, with the high expression levels in stamens, ovaries and petals. Yeast one-hybrid and dual luciferase assay results showed that AcBBX5 bound to AcFT promoter and inhibited AcFT gene expression. In conclusion, AcBBX5 was involved in flower bud differentiation and floral organ development, which provides an important reference for studying the functions of BBX and the molecular regulation of flower.Flowering is an important factor to ensure the success of plant reproduction, and reasonable flowering time is crucial to the crop yield. BBX transcription factors can regulate several growth and development processes. However, there is little research on whether BBX is involved in flower formation and floral organ development of pineapple. In this study,  Flowering, the symbol of vegetative development to reproductive development is an important factor to ensure the success of plant reproduction . The traBBX transcription factors have a wide range of functions and play important roles in flower initiation, light morphogenesis, anthocyanin synthesis and abiotic stress tolerance  genes receive signals from the photoperiodic regulatory center CO under long day conditions, which drives the transition from vegetative to reproductive growth in Arabidopsis (FT-induced flowering by affecting TERMINAL FLOWER1 (TFL1) expression under short-day conditions  were treated with 30 mL 200 mg/L ethephon to induce flowering, and the control group with the same amount of water instead. The flower organs including petals, ovary, stamens, sepals and styles were collected separately in pineapple. All samples were performed with three biological replications and immediately frozen in liquid nitrogen and then stored at −80 °C until further use.AcActin was used as an internal reference gene of pineapple. The reaction conditions for RT-qPCR consisted of predegeneration (95°C for 2 min), circular reaction  and Dissociation curve . All experiments incorporated three biological samples and three technical replicates. The relative expression levels of each gene were evaluated by using the 2−ΔΔCt method  according to the manufacturer’s instructions. After detecting the concentration and quality of RNA by NanoDrop™ One/OneC Spectrophotometer , reverse transcription was carried out through the Revert Aid First-Strand cDNA Synthesis Kit . RT-qPCR was performed with ChamQ Universal SYBR qPCR Master Mix  on LightCycler 480 II . t method .http://pineapple.zhangjisenlab.cn/pineapple/html/index.html) (The AcBBX5 protein sequence of pineapple was obtained from pineapple Genome Database (ex.html) . The proex.html) . The phyex.html) .AcBBX5 was introduced into the pCAMBIA2300-GFP vector digested with Kpn I and Xba I restriction enzymes to generate the construct 35S::AcBBX5-GFP. After sequencing correctly, it was transformed into Agrobacterium tumefaciens strain GV3101. The epidermal cell transformation of tobacco leaves was injected with the A. tumefaciens carrying out with the recombinant vector. After incubation in the dark for 12 h at 25 °C, the tobaccos were transferred to normal growth for 24–36 h. The fluorescence signal was observed by a confocal scanning microscope Ax-io-Imager_LSM-800  under excitation of 488 nm.The coding sequence of AcBBX5. Nde I and Sal I were selected as restriction sites, and the coding sequences of AcBBX5 were inserted into the bait vector pGBKT7 by homologous recombination. Following the manufacturer’s protocol, the recombinant vectors were transferred into AH109 strain yeast and cultured on SD/-Trp medium at 30 °C for 2–3 days. Single colonies were selected for amplification culture and then transferred to SD/-Trp and SD/-Trp/-His/-Ade solid media for further culture. Three days later, X-α -Gal was added to observe whether the colony was blue.A yeast assay system was used to examine the transcriptional activity of Arabidopsis thaliana (ecotype Colombia) grown under conditions of 21 ± 1°C  and used for heterologous transformation in this study. pCAMBIA2300-GFP-AcBBX5 vectors  were used to transform into Arabidopsis via the floral dip method , 24.53% (CsaBBX14), 24.27% (OsBBX16), 22.93% (PbBBX18), and 25.60% (SlBBX22), respectively  by PCR amplification and QRT-PCR to study their characteristics  condition, the control plants with about 18 rosette leaves began to blossom about 18 days after transplanting. The overexpressed OE-3, OE-9 and OE-13 bloomed at about 58, 50 and 29 days after transplanting, and the number of rosette leaves was about 37, 25 and 23, respectively . Compared with the control plants, the expression levels of FT, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), FRUITFULL (FUL) and SEPALLATA3 (SEP3) genes promoting flower formation in Arabidopsis were inhibited in overexpressed lines, while the expression levels of FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) genes negatively regulating flower formation were significantly increased in transgenic Arabidopsis plants. These results further confirmed that AcBBX5 repressed flower formation in Arabidopsis by regulating the expression of other floral genes and this might suggest the possibility that AcBBX5 represses flowering in Pineapple.In order to better understand the molecular mechanism of  QRT-PCR Figure 5In addition to delay flowering, florets were also larger in AcBBX5 transgenic lines compared to control lines at full flowering Figure 6AcBBX5 in pineapple flower formation, yeast one-hybrid assay was performed to determine whether AcBBX5 could bind to the promoter of AcFT. PB42AD-AcBBX5 and Placzi-proAcFT vectors were combined to transform yeast strain EGY48. PB42AD/Placzi- proAcFT and Placzi/PB42AD-AcBBX5 were used as negative control. The transformed yeast showed blue color in SD/Trp-Ura- medium containing X-Gal, but the negative control did not. This result indicated that AcBBX5 could bind to the promoter of AcFT (AcFT) and effector plasmid (35S:AcBBX5) were constructed and a dual luciferase (LUC) assay was performed in N. benthamiana leaf cells to examine whether AcBBX5 could activate the AcFT promoter. As expected, in the LUC reporter assays, the fluorescence signal was significantly attenuated in sites infected by AcBBX5 and AcFT promoters compared with the negative control, and the activity of the promoter expressed by the LUC/REN ratio was significantly reduced relative to the control  of the CmFTL1 promoter  and photoperiodic flowering pathway genes   . OsBBX14FT, FLC, CO and SOC1 genes. In addition to AtBBX24  were inhibited in Arabidopsis transgenic lines, while negative regulators of flower formation (FLC and SVP) were higher than control lines , the National Key R&D Program of China (2019YFD1001105 and 2018YFD1000504), the Natural Science Foundation of Hainan Province (321RC467 and 322MS013), the major science and technology project of Hainan Province (ZDKJ2021014), and the Scientific Research Start-up Fund Project of Hainan University (KYQD-ZR-20090).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.