Plasmodium falciparum exhibits high translational plasticity during its development in RBCs, yet the regulation at the post-transcriptional level is not well understood. The N6-methyl adenosine (m6A) is an important epigenetic modification primarily present on mRNA that controls the levels of transcripts and efficiency of translation in eukaryotes. Recently, the dynamics of m6A on mRNAs at all three developmental stages of P. falciparum in RBCs have been profiled; however, the proteins that regulate the m6A containing mRNAs in the parasites are unknown.P. falciparum genome encodes two putative YTH (YT521-B Homology) domain-containing proteins, which could potentially bind to m6A containing mRNA. We developed a modified methylated RNA immunoprecipitation (MeRIP) assay using PfYTH2 and find that it binds selectively to m6A containing transcripts. The PfYTH2 has a conserved aromatic amino acid cage that forms the methyl-binding pocket. Through site-directed mutagenesis experiments and molecular dynamics simulations, we show that F98 residue is important for m6A binding on mRNA. Fluorescence depolarization assay confirmed that PfYTH2 binds to methylated RNA oligos with high affinity. Further, MeRIP sequencing data revealed that PfYTH2 has more permissive sequence specificity on target m6A containing mRNA than other known eukaryotic YTH proteins. Taken together, here we identify and characterize PfYTH2 as the major protein that could regulate m6A containing transcripts in P. falciparum.Using sequence analysis, we computationally identified that the Plasmodium spp. lost the canonical m6A-specific demethylases in their genomes, however, the YTH domain-containing proteins seem to be retained. This study presents a possibility that the YTH proteins are involved in post-transcriptional control in P. falciparum, and might orchestrate the translation of mRNA in various developmental stages of P. falciparum. This is perhaps the first characterization of the methyl-reading function of YTH protein in any parasites. P. falciparum strains that have developed resistance to all existing drug therapies, including artemisinin combination therapy [Plasmodium parasites undergo various developmental stages in the human host. Comparative transcriptomic and proteomic studies of different P. falciparum stages have shown that there is a marked delay between mRNA synthesis and protein expression for nearly 30% of the 2584 analyzed genes [P. falciparum are marked by broad changes in transcriptional activity [Plasmodium spp. have distinct regulatory mechanisms, both at the post-transcriptional and translational levels that regulate various developmental stages of the Plasmodium lifecycle. Such unique mechanisms to fine-tune the global gene expression might help P. falciparum to establish successful pathogenesis inside the human host.Malaria is a major vector-borne disease predominantly affecting tropical countries. Of great concern is the emergence of drug-resistant  therapy , 2. Plasactivity . These fP. falciparum [P. falciparum and P. vivax, as compared to other eukaryotes [Epigenetic modifications such as DNA and histone-methylation play a pivotal role in regulating gene expression and safeguarding genome integrity. Nevertheless, the knowledge of the nucleic acid methylome in human malaria parasite is limited. Discovery of reversible adenine methylation on mRNA in many higher eukaryotes has opened new avenues to explore the post-transcriptional gene regulation in eukaryotic pathogens , 8. Intelciparum . In addikaryotes –16.P. falciparum may undergo stringent regulation to facilitate delayed protein synthesis in a stage-dependent manner. The stability and fine-tuning of mRNA processing could happen at two levels: (i) addition of poly (A) tails at 3′ end of RNA and/or (ii) epigenetic modifications of mRNA, particularly N6-adenosine methylation of the matured mRNA. RNA modifications have emerged as critical biological regulators of tRNA, rRNA, and mRNA homeostasis in eukaryotes [P. falciparum tRNA methylation may have a significant role in the regulation of poly-aspartic acid containing proteins [P. falciparum [Presence of only a few transcription factors suggests that parasite might have a unique modes of gene regulation at the RNA level , 18. Thekaryotes . For inskaryotes , whereasproteins . Importalciparum .6-methyl adenosine (m6A) modifications of mRNA and lncRNAs regulate the RNA tertiary structure and recruit the m6A binding proteins [P. falciparum. Recently, m6A has been identified as predominant modification on mRNAs of P. falciparum and that this post-transcriptional modification is dynamically distributed [In higher eukaryotes, Nproteins . m6A is tributed .P. falciparum. We searched YTH-specific homologue in the genome of the parasite and identified two putative proteins in P. falciparum (PfYTH), in concordance with a previous report [P. falciparum during its development in RBCs. We anticipate that the findings presented here will propel further research in elucidating the role(s) of m6A on P. falciparum mRNAs and PfYTH proteins in regulating the development and pathogenicity of the parasite.In this study, we aimed to identify and characterize the potential m6A modification methyl-reader proteins in s report . As PfYTPlasmodium falciparum exhibits a high translational shift in the RBC stages during its development [P. falciparum mRNAs are enriched with m6A modifications, we searched for the m6A reader protein YTH homologs in the genome of P. falciparum. The YTH domain family proteins are known to bind m6A on mRNA in other eukaryotes [P. falciparum genome encoded two YTH domain-containing proteins (PfYTH1 and PfYTH2), as previously reported by Baumgarten et al. [P. falciparum m6A mRNAs. For this, we developed a modified MeRIP assay using PfYTH2 protein and performed methylated RNA immunoprecipitation to identify the m6A specific interaction. ImageJ analyses showed that the quality of the PfYTH2 is nearly 80%, and sufficient to perform in vitro pull-down assays  with default parameters revealed that PfYTH2 forms basic amino acid patches to bind with the negatively charged m6A-containing RNA ligand . To study the effect of the point mutations on RNA binding, we performed atomic-level molecular dynamics simulations of the mutant protein–RNA complexes for 30 ns using Gromacs simulation package [To consolidate the m6A-specific interaction of PfYTH2 protein, we performed MeRIP pull-down assay with PfYTH2 F98A methyl-binding pocket mutant and found a significant loss of interaction with m6A containing mRNA Fig. e, f. To  package , 27. HowWe first assessed the overall potential energy changes of the protein–RNA complex to comprehend the effect of point mutations. Potential energies were calculated for wild type (WT) and mutant protein–RNA complexes over the simulation timescale. Interestingly, protein–RNA complexes with F98A point mutation showed a significant drift of − 122.349 (kJ/mol) over the timescale. This suggests a reduced binding interaction between the mutant PfYTH2 F98A and m6A-mRNA, corroborating well with our modified MeRIP assay, where a significant loss in binding is observed between mutant protein F98A and m6A-containing mRNA . To measure the binding affinity, various concentrations of PfYTH2 proteins were added to the cuvette containing m6A-containing RNA oligos. Fluorescence depolarization measurements revealed that PfYTH2 binds to m6A-containing RNA oligos with high affinity than to unmodified RNA oligos Fig. a, b. TheP. falciparum genome, using Bowtie2 U(C) with guanine or adenine at −1 position to target adenine, purines at −2 and adenine at −3 to be significantly enriched in more than 80% of the TTS reads G(AU)G(A)ads Fig. c. We valads Fig. d. The stPlasmodium spp. has not been studied in detail. The m6A-based regulation of mRNA is achieved through N6-adenosine methyltransferases (writer), adenine demethylases (erasers), and methyl-reader proteins such as the YTH family of proteins. In this study, we have identified m6A-specific methyl-binding protein PfYTH2 in P. falciparum, and characterized its m6A-specific binding and showed that the protein has relaxed sequence specificity to recognize the m6A containing mRNAs.Accumulating evidences suggest that m6A modification on mRNA is a major epigenetic player that regulates the mRNA fate, translation, and stability in diverse eukaryotes –30. The P. falciparum genome encodes only two YTH homology domain proteins [P. falciparum [P. falciparum. Surprisingly, the apicomplexa group of parasites, including P. falciparum contains conserved writer complexes (mRNA N6-adenosine methyltransferase) [P. falciparum. Given these findings, it is tempting to speculate that YTH proteins may have significant functions to regulate differential stage-specific global gene expression patterns in P. falciparum.Multiple YTH domain-containing proteins are encoded by eukaryotic genome; for instance, five YTH domain-containing proteins have been identified in humans . Howeverproteins . Human Yproteins . Similarproteins . These flciparum . Our PfYlciparum   , but doeP. falciparum lead a complex life cycle for multiplication to establish infection in the host(s). This parasite undergoes multiple developmental stages in different hosts and employs stringent gene regulatory mechanisms to achieve the optimal gene expression during various developmental stages. The stage-specific transcriptome and proteome analysis of P. falciparum have shown a delay in mRNA synthesis and its corresponding proteins between various developmental stages [P. falciparum, and showed that the protein has relaxed sequence specificity to recognize the m6A containing mRNAs. This study sheds light on the thus far unknown layer of regulatory processes that is essential for post-transcriptional regulation in various stages of parasite development to fine-tune the stage-specific translation in P. falciparum.Apicomplexa parasites including l stages . This suP. falciparum 3D7 was cultured in the 37 °C incubator with 5% haematocrit in RPMI 1640 (Gibco Cat # 11875093) supplemented with 10% of O+ve plasma and 0.5% albumax II (Gibco Cat # 11021037) and maintained by sub-culturing when sufficient parasitemia was achieved. Growth of P. falciparum was verified by preparing the thin smear followed by Giemsa staining and proceeded to RNA isolation.The blood stage of P. falciparum 3D7. The ORF sequence for each gene was cloned in pGEX6P2  vector using restriction sites BamH1 in the forward primer and Xho1 site in the reverse primer . The quality of the RNA was analyzed on formaldehyde-denatured gel and the RNA stored at − 80 °C for further experiments.The dot blot assay was carried out to detect the presence of methyl adenines in the immunoprecipitated RNA from MeRIP assay. The MeRIP enriched RNA was spotted on positively charged nylon membrane  and RNA was cross-linked using UV cross-linker with 0.1 J for 6 min. Presence of RNA in the membrane was confirmed by Methylene blue staining and the stain was washed using 1X TBST, and the membrane was blocked in 5% skimmed milk overnight. The mouse anti-m6A antibody (Diagenode—Cat # C15200082) was used to probe m6A on RNA and anti-mouse HRP conjugated was used as secondary antibody. The blot was developed using chemiluminescence ECL substrate (Bio-Rad # 1705061).2, 2 mM EDTA, 0.1% Triton, 10% glycerol) for three times and a fourth wash was carried out using MeRIP buffer  followed by the addition of P. falciparum RNA and in vitro transcribed RNA for control experiments. The protein and RNA mixture was incubated overnight at 4 °C with gentle rotation. The bound RNA fractions were eluted with elution buffer containing competitor N6-methyladenosine (Abcam Cat # ab145715). Eluted RNA was purified using Qiagen miRNeasy purification kit (Qiagen Cat # 217004) and used for dot blot experiments. We used PfYTH protein to map the m6A modifications on the RNA of P. falciparum. The RNA was fragmented into ~ 300 bases at 95 °C for 2 min using fragmentation buffer . The fragmented RNAs were subjected to interaction with PfYTH2 and GST protein (control), the complexes were incubated at 4 °C for overnight with rotation. The unbound RNAs were washed with wash buffer and the bound RNA fraction eluted with elution buffer containing N6-methyladenosine (Abcam Cat # ab145715). Eluted RNA was purified using Qiagen miRNeasy purification kit (Qiagen Cat # 217004) and stored at − 80 °C for sequencing.We developed modified MeRIP (methylated RNA immunoprecipitation) to study the PfYTH2 interaction with m6A-containing RNAs. The GST tagged PfYTH2 domain protein (15 µg) was coupled with GST resin. GST protein was used as a control in the parallel reaction. The protein-bound beads were washed with protein interaction buffer  to study the m6A specificity for PfYTH2 in vitro using synthetic RNA oligos. We procured synthetic RNA oligos containing m6A with appropriate flanks and unmodified oligos for control experiments . The RNA oligos were reconstituted in RNAse-free water and the presence of m6A was verified by dot blot assay  for molecular dynamics simulations were computationally designed for modeled PfYTH2 using rosetta backrub method for flexible backbone design described . A total6-methyl adenosine base. The protein–RNA complex was solvated by TIP3P water model in a cubic box with a distance cutoff 10 Å between the edge of the periodic box and surface of the protein–RNA complex.The all-atom molecular dynamics simulations were carried out using GROMACS 2019.3 molecular dynamics simulation package , 27. The+ counter ions followed by energy minimization using steepest-descent algorithm down to a 1000 kJ/mol/nm till the energy gets converged. Before production run, all systems were equilibrated for pressure and temperature by position restraining the protein–RNA for 100 ps using canonical Number of particles (N), Pressure (P) Temperature (T) and Number of particles (N), Volume (V), Temperature (T) ensembles at respective temperatures. The long-range electrostatic interactions were calculated using Particle Mesh Ewald (PME) [The system was neutralized with Nald (PME)  method wld (PME) . All simld (PME) , and ParDNA libraries were prepared with illumine compatible SMARTer Stranded Total RNA-Seq Kit v2 . 400 pg of total RNA was taken for first strand synthesis followed by second strand synthesis by template switching mechanism. This was further processed with addition of Illumina adaptors and barcode as per SMARTer Stranded Total RNA-Seq Kitv2 in final PCR amplification of 16 cycles. The bar code ligated libraries were purified using HighPrep beads (Cat # AC-60050) followed by library quality control check. Illumina-compatible sequencing library was quantified by Qubit fluorometer  and the fragment size distribution was analyzed on Agilent 2200 Tapestation. We got Illumina-compatible sequencing libraries with mean fragment size of ~ 338 bp.P. falciparum 3D7 from PlasmoDB  and mapped the sequencing reads using Bowtie2 v0.9.6 tool with default parameters  cutoff to call significant regions. The enriched peaks with q-value 0.01 obtained using MACS2 and fold enrichment for each peak summit was calculated against random Poisson distribution with local lambda. The MACS tool was used for peak identification, during the peak calling step. The location with the highest fragment pileup is referred as the absolute summit which provides the details about precise binding location. Further, the precise m6A position was used for HOMER annotation for the details of annotated regions. The summit file had only one base (m6A) information identified by MACS which was used as input for HOMER annotation. The HOMER annotation includes peaks in Transcription Start Site , Transcription Termination Site , Exon (Coding), Intronic, or Intergenic. To identify the target motif and annotate transcripts from MeRIP analysis, we used HOMER software. The HOMER helps in motif identification, considering peaks with ≥ onefold change. We identified the enriched motifs in these peak regions. For this, the broad peak sequences for both samples are fetched out and analyzed for possible motifs. With total number of target sequences, the HOMER identified 120 (81.6%) number of target sequences are with the motif. It uses default the find MotifsGenome program and performs de novo motif discovery as well as checks for the enrichment of motifs. Further, the peaks are annotated using reference genome annotation file by annotate peaks program in HOMER tool with default parameter. This program first determines the distance to the nearest TTS and assigns the peak to that gene and subsequently determines the genomic annotation of the region occupied by the center of the peak/region.The generated raw data was checked for the quality using FastQC software and reads were preprocessed to remove the adapter sequences and removal of the low quality bases (< q30) towards 3′-end using TrimGalore. We downloaded the reference genome P. falciparum RNA. The bound RNA from PfYTH2 protein was eluted using elution buffer containing competitor (Abcam Cat # ab145715) for m6A modification. The eluted RNA was used for cDNA synthesis. We performed a cDNA synthesis using Maxima H minus First strand synthesis kit (Thermo Scientific Cat # K1681). Briefly, the eluted RNA from YTH, GST and the input RNA were subjected to DNase digestion with 10× DNase buffer for 2 min at 37 °C in the preheated water bath separately. Then, oligo dT primers, random hexamer and 10 mM dNTPs were added to the DNase treated tubes and reaction volume was made up to 15 µl. The reaction mixtures were mixed gently and incubated at 65 °C for 5 min. Then 5× reverse transcriptase buffer, Maxima H Minus enzyme mix were added and volume of the mixtures were made up to 20 µl. Afterwards, cDNA synthesis for all the reactions were carried out in PCR machine using following conditions, 25 °C for 10 min followed by 50 °C for 15 min, reactions were terminated at 85 °C for 5 min. The qRT-PCR was carried out with freshly synthesized cDNA for 6 transcripts using Sybr Green mix from Thermo Scientific was used for amplification, and in parallel we have used 1% input for qRT-PCR reaction. The enrichment of the transcripts from YTH and GST samples were calculated from the Ct values by converting the input Ct values to 100% and fold enrichment of transcripts was represented to % input.To validate the NGS data of PfYTH2 pull-down samples, we performed MeRIP using PfYTH2 and GST as control for Additional file 1. Additional figures and table.


Candida species can use carboxylic acids, like acetate and lactate, to survive and successfully thrive in different environmental niches. These nonfermentable substrates are frequently the major carbon sources present in certain human body sites, and their efficient uptake by regulated plasma membrane transporters plays a critical role in such nutrient-limited conditions. Here, we cover the physiology and regulation of these proteins and their potential role in Candida virulence.Opportunistic pathogens such as  Candida species can use carboxylic acids, like acetate and lactate, to survive and successfully thrive in different environmental niches. These nonfermentable substrates are frequently the major carbon sources present in certain human body sites, and their efficient uptake by regulated plasma membrane transporters plays a critical role in such nutrient-limited conditions. Here, we cover the physiology and regulation of these proteins and their potential role in Candida virulence. This review also presents an evolutionary analysis of orthologues of the Saccharomyces cerevisiae Jen1 lactate and Ady2 acetate transporters, including a phylogenetic analysis of 101 putative carboxylate transporters in twelve medically relevant Candida species. These proteins are assigned to distinct clades according to their amino acid sequence homology and represent the major carboxylic acid uptake systems in yeast. While Jen transporters belong to the sialate:H+ symporter (SHS) family, the Ady2 homologue members are assigned to the acetate uptake transporter (AceTr) family. Here, we reclassify the later members as ATO (acetate transporter ortholog). The new nomenclature will facilitate the study of these transporters, as well as the analysis of their relevance for Candida pathogenesis.Opportunistic pathogens such as  Saccharomyces cerevisiae, Jen1 and Ady2 are the main transport systems responsible for the uptake of monocarboxylates at conditions where these substrates can be used as main carbon sources. The aquaglyceroporin channel Fps1 has also been reported to mediate the import of undissociated acetic acid by facilitated diffusion  searches in NCBI (https://blast.ncbi.nlm.nih.gov) and identified all of the orthologues of the functionally validated S. cerevisiae monocarboxylate transporters (Jen1 and Ady2) for a set of medically relevant Candida species. In particular, these two transporters were independently used as BLAST queries in searching the proteome of C. albicans, Candida glabrata, Candida tropicalis, C. parapsilosis, Candida orthopsilosis, Candida dubliniensis, Candida krusei, Candida auris, Candida lusitaniae, Candida haemulonis, Candida guilliermondii, and Candida kefyr. This set includes the most studied and well-characterized Candida species but also infrequent species that are emerging as successful pathogens. Finally, we reclassified and renamed some of these homologues based on recently reported structural and functional data. In particular, we propose the exclusive use of ATO (acetate transporter ortholog) for all of the yeast Ady2 ortholog members of the acetate uptake transporter (AceTr) family to better describe their function as acetate transporters  orthologues were functionally characterized in C. albicans, one encoding a monocarboxylate transporter (CaJen1) for lactate, pyruvate, and propionate , resembles the common topology of other MFS members. Structure-function studies, benefiting from the solved structure of other MFS members, have identified several conserved amino acids that are essential for the binding and cotranslocation of the substrates , 33. WheYarrowia lipolytica Gpr1 protein (S. cerevisiae Ady2 (ScAdy2) and Aspergillus nidulans AcpA in eukarya , the genome of S. cerevisiae harbors two additional homologues, FUN34  and ATO3. These three transporters are highly induced under carbon-limiting conditions and during the stationary phase of glucose grown cells  was also reported to be expressed during meiosis, which is required for the regulation of meiotic plaque components in sporulation . Mutantsormation . The staormation , 45.S. cerevisiae, this volatile alkaline compound is transmitted between yeast colonies as a signal to inhibit growth of the facing parts of both colonies, a signaling process that requires amino acid uptake  family (TCDB 2.A.96)  based on18–10.1128/mBio.00156-20.2TABLE S2Candida species with annotated/former designations and the following suggested designation based on ScAdy2 gene homology. Download Table S2, DOCX file, 0.03 MB.GenBank accession numbers of ScAdy2 homologues present in twelve Copyright © 2020 Alves et al.2020Alves et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Candida species resulted in a phylogenetic tree containing 65 Ato-like proteins organized into two asymmetrical clusters  showed that these proteins only contain 3 and 5 helices, respectively, instead of the expected six detected for this transporter family. The analysis of the genomic sequence following the termination codon of the protein revealed the presence of the homologous absent portion, indicating that the proteins were truncated, likely recently in evolution. As we have no indication if the truncated proteins are fully functional, we excluded these sequences from further analyses.The phylogenetic analysis of ScAdy2/Ato orthologues (hereafter referred as ScAto1) in the selected clusters . Clusterhologues , which ihologues . In partproteins . Howeverproteins  were shoEscherichia coli (ecSatP) and Citrobacter koseri (ckSatP) are so far the only members of the AceTr family whose structures are available  CgDtr1, which is responsible for the export of acetate and involved in weak acid stress resistance in RPMI medium at pH 4.5  transporters belonging to the ATP-binding cassette (ABC) superfamily have been so far associated with the export of carboxylates in acid stress conditions. Since weak acid stresses are common in many host niches, the proliferation of the Ato family in pathogenic species may be a response to these stresses.These results taken together suggest that carboxylic acid transporters are important mediators of host-pathogen interactions by allowing rapid adaptation to different environmental conditions and modulating the virulent properties of tic acid . This setic acid . In factt pH 4.5 . In thisCandida when these nutrients are the main exogenous carbon sources available. Jen and Ato family members, in certain conditions, seem to play critical roles in Candida pathogenesis, as they allow cells to sustain metabolism and survival when thriving in the human host. However, many details are missing regarding the energetics, specificity, and regulation of these transporters. Further research will be needed to determine individual transport properties, potential redundancies, and functional roles of carboxylate transporters in Candida virulence and pathogenesis.The ability to transport carboxylates inside the cell represents an advantage for


The coronavirus (COVID-19), discovered in 2019, has been creating havoc since it first emerged in China and is now spreading worldwide. Its presentation is somewhat similar to influenza. We hereby discuss the salient features of the coronavirus and present the case of a 33-year-old male who was tested positive for COVID-19.  Virus SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) or the 2019 novel coronavirus (2019-nCoV) belong to the broad family of coronaviruses (subgenus Sarbecovirus). Viruses use a nested set of messenger ribonucleic acids (mRNAs) to replicate. There are four genera of coronaviruses: alpha, beta, gamma, and delta. Two of these genera are known to infect humans: alpha coronaviruses (the human coronavirus 229E (HCoV-229E) and human coronavirus NL63 (HCoV-NL63)) and beta coronaviruses (human coronavirus HKU1 (HCoV-HKU1), human coronavirus OC43 (HCoV-OC43), the Middle East respiratory syndrome-related coronavirus (MERS-CoV), and the severe acute respiratory syndrome coronavirus (SARS-CoV)) [The CoV-2 is an enveloped, single-stranded, positive-sense ribonucleic acid (RNA) virus. The virus has a spheric shape, 60 - 140 nm in diameter, surrounded by glycoprotein spikes (S) of 9 - 12 nm of length that gives them a crown-like appearance . About 8The most recent outbreak of the coronavirus (CoV) started in December 2019 in the Wuhan Hubei province in China and has so far affected 31 countries . A largeHCoV spreads via respiratory transmission. Based on available data of SARS and MERS, the Centers for Disease Control and Prevention (CDC) has estimated an incubation period for the COVID-19 to be between two and 14 days . The sev3/uL , a hemoglobin (Hgb) of 13.9 g/dL , a hematocrit (Hct) of 42.9% , and a platelet count of 176 x 103/uL . C-reactive protein was 6.1 mg/dL , complement component (C3) 165 mg/dL , lactic acid 8.7 mg/dL , lactate dehydrogenase (LDH) 746 U/L , and procalcitonin < 0.05 ng/ml . We hereby present the case of a 33-year-old man with a past medical history of alpha thalassemia who came to the emergency department (ED) with generalized weakness, fever, chills, productive cough with scant yellow sputum, myalgia, and arthralgia for 10 days. The patient endorsed a lack of appetite and an episode of non-bilious vomiting with epigastric pain and dysuria. At presentation, the patient denied chest pain, shortness of breath, palpitations, or diarrhea. The patient had not traveled outside of the United States (US) and had no known allergies. Physical examination was remarkable for scattered rhonchi bilaterally. The patient was not found to be in any distress. Heart sounds were normal. The patient was alert and oriented to time, place, and person. Cranial nerves, II-XII, were grossly intact, sensation was intact to touch, and strength was 5/5 throughout the upper and lower extremities bilaterally. In the ED, his vital signs included a temperature of 100.7°F, blood pressure (BP) of 105/54 mmHg, a mean arterial blood pressure of 71 mmHg, heart rate of 70 beats/minute, respiratory rate of 16/minute, and saturation of 97% on room air. Laboratory results were as follows: white blood count (WBC) 3.9 Lx10An echocardiogram (EKG) showed normal left ventricular systolic function. Left ventricular ejection fraction, estimated by two-dimensional 2D) EKG at 60% - 65%. Blood, urine, and sputum cultures were all negative. Chest x-ray showed bilateral ground-glass opacities in the lower lobes Figures -2. An acD EKG at The current understanding regarding transmission of the coronavirus to this day is still being researched. The suspected cause has been researched from the initial source of the outbreak in Wuhan, China. There was an association between the initial patients and a local seafood market which sold live animals. The patients were mostly workers or visitors to that area which has since been closed. However, the transmission of the virus outside of Wuhan has been more linked to person-to-person contact. This was suspected when reports of the virus were being made in places, such as Hubei and the US, where individuals did not have exposure to animal/seafood markets. Transmission by this method has been thought to be due to respiratory droplets from person to person. Close contact with infected individuals (within six feet) through respiratory droplets (coughing/sneezing) landing in the mouths/noses of people nearby or inhaled through the lungs are the suspected mode of transmission of the virus between people. In addition, there are still reported cases of the virus in locations where it is still not known how or where the individuals were exposed -11. ThouCOVID-19 can prove to be deadly in immunocompromised individuals and individuals with comorbidities; otherwise, the illness in healthy people is no more than the flu. Prevention is the key to avoiding crowded places and limiting exposure to others.


RPPH1 RNA, is overexpressed and accumulates in mitochondria of senescent fibroblasts, possibly modulated by the RNA-binding protein AUF1. In addition, RPPH1 RNA overexpression promotes spontaneous replicative cellular senescence in proliferating fibroblasts. Using MS2 aptamer-based RNA affinity purification strategy, we identified putative target mRNAs of RPPH1 RNA and revealed that partial complementarity of RPPH1 RNA to its target mRNAs prevents those mRNAs decay in proliferating fibroblasts. Altogether, our results demonstrate the role of mitochondrial noncoding RNA in the regulation of mRNA stability and cellular senescence.Post-transcriptional gene regulation is an important step in the regulation of eukaryotic gene expression. Subcellular compartmentalization of RNA species plays a crucial role in the control of mRNA turnover, spatial restriction of protein synthesis, and the formation of macromolecular complexes. Although long noncoding RNAs (lncRNAs) are one of the key regulators of post-transcriptional gene expression, it is not heavily studied whether localization of lncRNAs in subcellular organelles has functional consequences. Here, we report on mitochondrial lncRNAs whose expression fluctuates in the process of cellular senescence. One of the mitochondrial lncRNAs,  Eukaryotic gene expression is tightly regulated in transcription and post-transcriptional steps ,2. Post-Even though amounts of total transcripts in cells may determine efficiency of protein output, additional factors also contribute critically in protein production . For exaRPPH1 RNA (Ribonuclease P RNA component H1) . 1–2 μg of plasmids that expressed AUF1 isoforms, RPPH1, or RMRP were transfected using Lipofectamine 2000 according to the manufacturer’s protocol. Transfected cells were generally analyzed 48 h later.Human fibroblasts WI-38  and IDH4  were cultured in DMEM  supplemented with 10% (v/v) Nonidet P-40, 1 mM EDTA, and 0.1% (w/v) SDS). Protein samples were separated on 10% SDS-polyacrylamide (SDS-PAGE) gels, and transferred onto Nitrocellulose membranes . The membranes were incubated with a specific primary antibody in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T) and 5% nonfat dry milk at 4 °C for overnight. After three washes with TBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature, immunoblots were visualized using Amersham ECL Prime Western Blotting Detection Reagent . The following antibodies were purchased from Millipore : anti-AUF1, Anti-HuR, anti-beta-Actin, anti-GAPDH, and anti-ATP5A were purchased from Santa Cruz Biotechnology .Western blot analysis was performed as reported previously . Briefly2 and 0.5% NP-40 for 10 min on ice and centrifuged at 10,000× g for 15 min at 4 °C. The lysates were incubated with 1 µg of antibodies against AUF1, HuR , or with control IgG  conjugated with protein A-Sepharose beads for 1 h at 4 °C. After the beads were washed five times with NT2 buffer composed of 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 0.05% NP-40, the beads were incubated with 20 units of RNase-free DNase I for 15 min at 37 °C. Then the beads were further incubated with 0.1% SDS/0.5 mg/mL Proteinase K for 15 min at 55 °C. The beads-associated RNA was extracted with acidic phenol and further examined by reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) analysis using the primers listed (RPPH1 RNA was used to construct plasmid pRPPH1-MS2 which expresses chimeric RNA (RPPH1-MS2 hairpins). WI-38 cells were co-transfected with pRPPH1-MS2 and pMS2-GST which expresses a fusion protein that contained a glutathione-S-transferase domain fused to a domain that recognizes MS2 hairpins. Forty-eight hours later, cells were lysed with the NT2 buffer aforementioned, then mRNAs associated with the chimeric RNA were pulled down by using glutathione (GSH)-coated beads. Following extraction of RNA from the beads, we employed RT-qPCR analysis to confirm the biochemical interaction between RPPH1 RNA and target candidate mRNAs observed from the BLAST-based search. Normalization of RIP results was carried out by quantifying in parallel the relative levels of GAPDH mRNA. These abundant RNAs are nonspecific contaminants present in the IP components .Immunoprecipitation (IP) of endogenous RNP complexes  from whole-cell extracts was performed as described previously . Brieflys listed . MS2 pulGAPDH mRNA, MT-RNR2 RNA, and 18S rRNA. All oligonucleotides are displayed in Total RNA of cells was isolated by Trizol  according to the manufacturer’s protocol. Acidic phenol  was used to extract RNA for RIP analysis. Isolated RNA was reverse transcribed using random hexamers and reverse transcriptase . After RT, cDNAs were assessed via qPCR analysis with SYBR green master mix , gene-specific primers listed , and an g for 10 min at 4 °C. The supernatant was used for the cytosolic fraction. The pellets were centrifuged at 12,000× g for 15 min at 4 °C, supernatant was removed, pellets were washed with Reagent C, centrifuged at 12,000× g for 5 min at 4 °C to get mitochondrial fraction. Subcellular fractionation was verified by western blot analysis.Cytosolic and mitochondrial fractions were collected as described in Manufacturer’s Protocol . Briefly, cells were lysed with Reagent A for 2 min on ice, Reagent B for 5 min on ice with vortexing every minute, and Reagent C, then the resulting lysates were centrifuged at 700× http://blast.ncbi.nlm.nih.gov/) to identify local regions of sequence similarity between RPPH1 RNA and MLC1, CCR7, HEXA, and GAPDH mRNA (NM_002046.3). The similarity regions with E-Value < 2, Identity > 75%, Nucleotide Match > 13 matching to the reverse complementary sequence of RPPH1 RNA were selected as and considered as possible interaction regions through base-paring between RPPH1 RNA and each mRNA. We used BLAST (WI-38 cells were cultured from population doubling level (PDL) 15 to reach PDL 55 after splitting the cells into half upon confluency. IDH4 cells, in which the expression of SV40 large-T antigen is regulated by a steroid-inducible promoter, were cultured in the presence of 1 mM Dexamethasone (Dex) to suppress cellular senescence and promoted proliferation. Suppression of SV40 large-T antigen in IDH4 to induce senescence was mediated by Dex removal from the IDH4 culture media . Cellular senescence was examined 7 days later after transfection. Senescence-associated β-galactosidase activity was assessed by senescence β-galactosidase staining kit  according to the manufacturer’s instructions. The percentages of G1, S and G2/M cells was determined by standard fluorescence-activated cell sorting analysis.MLC1 and CCR7 mRNAs interacting with RPPH1 , and ligated into the digested pmiR-GLO vector. Positive colonies were confirmed by PCR, and the construction of pmiR-GLO-MLC1 and pmiR-GLO-CCR7 vectors were verified by sequencing. Generated luciferase reporter plasmids were co-transfected with pRL-null vector using Fugene HD transfection reagent  according to the manufacturer’s protocol. Transfected cells were generally analyzed 48 h later. Luciferase activities were measured using a DualGlo Luciferase Assay Kit  according to the manufacturer’s protocol.Luciferase reporter constructs bearing the putative binding sites in th RPPH1  were clot test. Results were considered statistically significant at ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.Quantitative data are presented as the mean ± SD, unless otherwise indicated. Differences between means were evaluated using Student’s unpaired


Saccharomyces cerevisiae Pols I and II using a series of quantitative in vitro transcription assays. We find that Pol I single-nucleotide and multinucleotide addition rate constants are faster than those of Pol II. Pol I elongation complexes are less stable than Pol II elongation complexes, and Pol I is more error prone than Pol II. Collectively, these data show that the enzymatic properties of the Pols have diverged over the course of evolution, optimizing these enzymes for their unique cellular responsibilities.Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult because studies are often conducted under disparate experimental conditions and techniques. Here, we directly compare and reveal functional differences between  In contrast to prokaryotic cells, which express a single RNA polymerase, eukaryotes express at least three nuclear RNA polymerases  , 2, 3. SSaccharomyces cerevisiae is organized into a single genetic locus consisting of approximately 150 tandem 9.1 kb repeats on chromosome XII. Pol I synthesizes a single transcript from each repeat, producing the 35 S pre-RNA that is cotranscriptionally and posttranscriptionally modified to form the 25 S, 18 S, and 5.8 S rRNAs. Together with the 5 S rRNA (synthesized by Pol III), these RNAs form the backbone of the eukaryotic ribosome . The rDNA in ribosome . Despiteribosome , 12.Pol II is localized in the nucleus and is responsible for transcribing approximately 6000 protein-coding genes, far more loci than for either Pol I or Pol III . The avePol III is localized in the nucleus and is responsible for a diverse set of noncoding RNAs including tRNAs, 5 S rRNA, U6 snRNA, and several microRNAs and snoRNAs . The aveThe differences between the Pols in subunit composition, localization, transcript demand, regulatory mechanisms, and RNA products illustrate the different selective pressures that have been exerted on these enzymes during eukaryotic evolution , 26.By expressing three unique nuclear RNA polymerases, eukaryotic cells gain the capacity for more complex control of gene expression. For example, unique transcription factors can regulate the expression of individual genes by modulating the activity and environmental response of a specific polymerase without influencing the other two polymerases . We hypoin vitro transcription assays to compare several intrinsic enzymatic properties of Pols I and II using a fully purified system. Pols I and II exhibit divergent properties in every assay performed. Nucleotide addition catalyzed by Pol II was found to be slower compared with Pol I. However, Pol II displays higher transcription elongation complex (EC) stability and much lower misincorporation activity. Together, all of these findings support the hypothesis that unique selective pressures have driven the divergence of DNA-dependent RNA polymerases, resulting in enzymes whose transcription elongation properties are suited to their cellular roles.To test this idea, we utilized quantitative in vitro transcription assay, previously developed to describe Pol I . ECs were radiolabeled by incubation with α-32P-CTP and the cofactor Mg2+ (A). ECs with a radiolabeled 10-mer RNA were rapidly mixed with the next cognate nucleotide, ATP, and stopped at fixed reaction times (0.005–10 s) using a chemical quench-flow (B).To quantitatively compare the constants that govern nucleotide addition catalyzed by both Pol I and Pol II, we employed a promoter-independent be Pol I . The EC A strand A. ECs wetor Mg2+ A. ECs winch-flow B.Figure Time courses of Pols I and II single-nucleotide addition were collected in triplicate at a saturating concentration of ATP (1 mM). Samples were run on polyacrylamide gels, exposed to phosphorimager screens, and analyzed.32P-CTP nascent RNA generated during the labeling step), an 11-mer (the 10-mer extended by AMP addition), and because of Pol I’s intrinsic nuclease activity, a GC and a CA dimer (A). The GC dimer was produced from the 10-mer RNA during labeling, and the CA dimer resulted from cleavage of 11-mer RNA during the nucleotide incorporation reaction (A).For Pol I, we observed bands on the gels consistent with a 10-mer (the α-CA dimer A. The GCreaction A.(1)FracB). These data were fit to a sum of two exponentials to describe single-nucleotide addition, which is governed by rate constants kobs,fast and kobs,cleavage (Equation B). This strategy, whereby single-nucleotide addition data are fit to a sum of two exponentials, has been successfully used previously to describe Pol I’s nucleotide incorporation mechanism (Pol I single-nucleotide addition time courses displayed a rise in the fraction of 11-mer RNA and subsequent decrease because of RNA cleavage B. These Equation B. This sechanism .C). Pol II lacks intrinsic nuclease activity; thus, this was expected.Conversely, for Pol II, we observed only two RNA species, a 10-mer and 11-mer RNA, but no cleavage products C. Pol IID). Weighted fits were performed using both one exponential  s-1, whereas the RNA cleavage rate constant, kobs,cleavage, was much slower at (0.19 ± 0.01) s-1. Pol II single-nucleotide addition was described by two rate constants, determined to be (38 ± 9) s-1 and (4 ± 0.6) s-1 for kobs,fast and kobs,slow, respectively. Pols I and II exhibit dramatically different nucleotide addition kinetics under these conditions. Furthermore, only one rate constant is required to describe nucleotide addition by Pol I . From these time courses, we measured the appearance and disappearance of each RNA intermediate over time. To analyze and compare individual incorporation events by Pols I and II, a minimal kinetic model was developed using Scheme 1 and Scheme 2 for Pols I and II, respectively (C–D). The additional kinetic parameter, kobs,10, was included to describe the intrinsic cleavage activity of Pol I conferred by its A12.2 subunit . Experimental data sets consisted of nine time courses describing the abundance of each RNA intermediate (11-mer through 19-mer) were fit simultaneously. The mean and standard deviation between replicates were calculated for each of the parameters of Schemes 1 and 2 . The average kobs for Pol I was measured to be (53 ± 4) s-1, whereas the average kobs for Pol II was (8.6 ± 0.1) s-1. These values were consistent with the trend observed in Time courses for Pols I and II were collected using the chemical quench-flow in the presence of 1 mM ATP and 1 mM GTP in buffer A , A–B. Frectively , C–D. Th subunit , 32. Thi subunit , and TFI subunit , and the 1 and 2 . Plots oobs values that ranged from (23–200) s-1, compared with (5.5–17.1) s-1 for Pol II . Radiolabeled ECs were mixed with RNase A in a destabilizing salt concentration (1 M KCl), and reactions were carried out with the reaction time spanning from 0 s to 40 min for Pol I and 0 s to 48 h for Pol II. (A). As intact ECs protected the 10-mer RNA from cleavage, accumulation of cleaved RNA indicated EC collapse over time.We previously developed an RNase protection assay for Pol I that quantifies the stability of stalled ECs on a DNA:RNA hybrid . ECs weron assay A. Radiol Pol II. A. As intB). The observation that Pol II EC collapse was dramatically right-shifted compared with Pol I indicates that Pol II forms a much more stable EC (C). The difference between the EC stabilities of Pols I and II suggests that they have diverged over time to suit their individual roles. It is known that Pol II transcribes nucleosome-bound templates, whereas the rDNA template for Pol I is thought to be nucleosome-free or loosely bound by histones. Perhaps this difference in template provided a selective pressure toward a more stable EC for Pol II.We found that almost all Pol I ECs collapsed within 40 min, whereas Pol II required 48 h to release the DNA B. The obtable EC C. The di32P-CTP, or a noncognate nucleotide, α-32P-ATP (A). The RNAs generated from either correct incorporation or misincorporation are compared with measure relative misincorporation by Pols I and II.Nucleotide addition by Pol I was significantly faster than by Pol II. It has been suggested that faster incorporation may lead to reduced fidelity , 36. Thu-32P-ATP A. The RN32P-CTP to yield a 10-mer RNA and a small population of those 10-mers were cleaved and resolved as dimers (B). Quantification of total 10-mer and dimer species intensity resulted in a value reflective of number of active ECs in the reaction. Upon incubation of the noncognate α-32P-ATP, Pol I misincorporated, extending to a 10-mer, and subsequently cleaved two nucleotides from the 3’ end of the RNA, resulting in a dimer species (B). The intensity of 10-mer and dimer species resulting from misincorporation was summed, and the summation was divided by the intensity of the abundance of RNA produced by correct incorporation of CTP. This procedure yields a relative misincorporation value of the number of misincorporation events per active EC.For the CTP control, Pol I incorporated α-s dimers B. Quanti species B. The in32P-ATP Pol I ECs produced up to 0.30 ± 0.06 misincorporation events per active EC (C). Misincorporation was clearly evident in the accumulation of the dimer species, but the rate of misincorporation remains low (∼0.005 events per EC per minute). It is important to note that this value is also not reflective of misincorporation events by Pol I in the presence of all NTPs. Rather, this value is indicative of the maximal relative misincorporation per Pol I EC over a long incubation when only a noncognate nucleotide is provided.Over a 60 min incubation with α-ctive EC C. MisincB). Pol II incorporated the correct nucleotide, α-32P-CTP, but misincorporation during incubation with α-32P-ATP was almost undetectable (B and D). Relative misincorporation was quantified as described for Pol I but only summing the amount of 10-mer for correct and incorrect incorporation events. Pol II produced only 0.003 ± 0.002 misincorporation events per active EC during 60 min incubation with noncognate NTP (D). These data demonstrate that the fidelity of nucleotide addition is substantially different for Pols I and II.For Pol II, correct and incorrect incorporation are only indicated by the presence of a 10-mer RNA species, because Pol II lacks intrinsic cleavage activity B. Pol IItectable B and D. nate NTP D. These A). The Pol I and II time courses were fit identically as for It is possible that these studies revealed overall faster nucleotide incorporation rate constants for Pol I based on a reaction condition, buffer A, that was optimized for Pol I . To testFig. 6-1, whereas the slowest was observed in both buffer B and buffer D, ∼30 s-1 . Contrary to what we observed with Pol I, where nucleotide addition is described only by kobs,fast, Pol II nucleotide addition is governed by both kobs,fast and kobs,slow. Pol II single-nucleotide rate constants, kobs,fast and kobs,slow, were within error across buffers A, B, and C . The lower pH in buffer D reduced the rate constants for nucleotide addition by both enzymes at every position in the template, and the amplitude of the reduction was approximately the same at all positions (B). These data support our previous conclusion that Pol I is faster than Pol II and demonstrate that nucleotide addition by both enzymes is sensitive to pH.The slowest step of single-nucleotide addition by Pol I was consistently and substantially faster than Pol II’s slowest step across buffers A–D, ranging from ∼50 to ∼8 fold faster than kfer used  and 3. Pfer used A. The loositions B. These This study examined several key enzymatic properties of both Pols I and II, revealing substantial divergence between these two enzymes in every assay performed. We suggest that these properties arose under selective pressure that favored the optimization transcription elongation. In other words, the biochemical characteristics of each polymerase have diverged to meet the unique demands of their separate roles within the cell. Pol I is faster at single-nucleotide and multinucleotide addition, more prone to EC collapse, has a higher misincorporation activity and is more sensitive to changes in reaction conditions than Pol II. Showing that in the absence of Pol-specific transacting factors, each polymerase displayed fundamentally unique enzymatic properties.obs,fast alone  . As mentst alone , ΔA12.2 the data . Notablybona fide cleavage factors by Pols I and III, A12.2 and C11, respectively, led to structurally distinct Pols with a substantial functional impact on misincorporation, backtrack recovery, elongation rate, and reinitiation  shared by Bacteria, Archaea, and Eukarya . Bacteriitiation , 44, 45.in vivo, then each engaged Pol I would yield more rRNA over time. This outcome would appear to be beneficial, because during periods of rapid growth, there is robust demand for new ribosome synthesis. However, there would be extensive functional consequences of this enhanced rRNA synthesis rate. Because rRNA synthesis is tightly coupled to rRNA processing  nt/s versus (24.6 ± 2.6) nt/s (versus (8.6 ± 0.1) nt/s  nt/s . Here, w.1) nt/s . The var.1) nt/s .Pol I has emerged as a validated anticancer target , 73, 74.BMH-21 is an example of a compound identified for its repressive effects on rRNA synthesis and derivatives of BMH-21 are in preclinical development as a cancer therapeutic . BMH-21 The yeast strain for Pol I purification was described previously . For Pol4)2SO4, 50 mM Tris-SO4 pH 7.8, 10 mM MgCl2, 10 μM ZnCl2, 10% glycerol). Supernatant was loaded on five 5 ml Ni columns  following ultracentrifugation . Ni columns were washed in KCl low imidazole buffer  and subsequently eluted directly onto a heparin column  attached downstream with KCl high imidazole buffer (same as KCl low imidazole but with 250 mM imidazole). Ni columns were removed, and heparin column was washed into low salt KCl buffer without imidazole  and eluted with high salt KCl buffer (same as low KCl buffer but 2 M KCl) into a 50 ml conical tube. Eluate was run over desalting columns  to change to MonoQ suitable buffers . Flow-through was collected and manually loaded onto a MonoQ column . Fractionation was performed using a 0 to 100% buffer B gradient at a flow rate of 0.25 ml/min over 100 min. Pol fractions identity and purity were confirmed with Coomassie-stained SDS PAGE gels and Western blots.Yeast strains expressing tagged Pols I and II were grown at the Bioexpression and Fermentation Facility, Department of Biochemistry and Molecular Biology at University of Georgia. The Pols were purified from approximately 150 g of wet cell mass. The cells were washed and lysed using a cell disrupter in breakage buffer  are achieved by different length loops of the quench-flow. Additional HCl and neutralization buffer is added to each quenched reaction to yield equal volumes of each time point and to neutralize the HCl. An aliquot of the reaction mixture is transferred to the RNA loading dye and subsequently boiled and loaded on a 28% polyacrylamide denaturing urea gel with 1X TBE running buffer.Single-nucleotide addition assays utilized to investigate Pols I and II were first described in the supplemental of a previously published manuscript . Buffer EC stability assays for Pols I and II were executed essentially as described previously . ECs aret and DNAnt strand in buffer A. The EC mix is split, one to measure correct incorporation, the other to measure misincorporation. The EC mix aliquots was either incubated with α-32P-CTP and Mg2+, to observe correct incorporation, or α-32P-ATP and Mg2+, to observe misincorporation. Time points were collected and quenched in RNA loading dye. Samples were boiled and ran on a 28% polyacrylamide denaturing urea gel with 1X TBE for the top (anode) buffer reservoir and 1X TBE with 1 M NaOAc in the bottom (cathode) buffer reservoir to resolve dimer species. To control for variable radioactive intensity of the α-32P-CTP and α-32P-ATP stocks, a series of dilutions were spotted on Whatman paper, and a ratio of intensity was calculated.Misincorporation assay for Pol I and II was described previously . ECs forMultinucleotide addition assays were set up identical to the single-nucleotide addition assays previously described , with thMulti-start Evolutionary Nonlinear OpTimizeR, is a custom-built hybrid nonlinear least squares (NLLS) genetic algorithm which is useful in optimizing parameters describing a wide variety of biochemical problems. Traditional NLLS methods exhibit an initial guess dependence which can result in optimization routines stalling in a local solution. The genetic algorithm component of MENOTR addresses this issue by starting a variety of starting points and subsequently performing NLLS on partially refined initial guesses. MENOTR also has the capabilities to perform Monte Carlo and grid search error analysis. An original manuscript is currently being written to outline MENOTR’s capabilities. MENTOR was used throughout this manuscript to optimize parameters describing Pols I and Pol II transcription kinetics.MENOTR, KaleidaGraph  was also used in this manuscript to perform parameter optimization. KaleidaGraph has the benefit of being a quick and easy method to perform simple parameter optimization problems.To optimize a set of parameters in a given scheme, MENOTR requires the user to input the set of ordinary differential equations (ODEs) describing each species in the scheme. The set describing Scheme 1 are 1.1-1.10, and the set describing Scheme 2 are 2.1-2.10. MENOTR used the sets of ODEs in the built in MATLAB function ODE23tb to numerically simulate a set of time courses for each species. The time courses are then separated based on their respective RNA species component and summed. The reason for this is because some intermediates are not able to be separated in this experimental strategy. MENOTR then optimizes the parameters and reports the set of parameters that describe the data best.All data described are contained within the manuscript.The authors declare that they have no conflicts of interest with the contents of this article.


Circular RNAs (circRNAs), which are single-stranded closed-loop RNA molecules lacking terminal 5′ caps and 3′ poly(A) tails, are attracting increasing scientific attention for their crucial regulatory roles in the occurrence and development of various diseases. With the rapid development of high-throughput sequencing technologies, increasing numbers of differentially expressed circRNAs have been identified in bladder cancer (BCa) via exploration of the expression profiles of BCa and normal tissues and cell lines. CircRNAs are critically involved in BCa biological behaviours, including cell proliferation, tumour growth suppression, cell cycle arrest, apoptosis, invasion, migration, metastasis, angiogenesis, and cisplatin chemoresistance. Most of the studied circRNAs in BCa regulate cancer biological behaviours via miRNA sponging regulatory mechanisms. CircRNAs have been reported to be significantly associated with many clinicopathologic characteristics of BCa, including tumour size, grade, differentiation, and stage; lymph node metastasis; tumour numbers; distant metastasis; invasion; and recurrence. Moreover, circRNA expression levels can be used to predict BCa patients’ survival parameters, such as overall survival (OS), disease-free survival (DFS), and progression-free survival (PFS). The abundance, conservation, stability, specificity and detectability of circRNAs render them potential diagnostic and prognostic biomarkers for BCa. Additionally, circRNAs play crucial regulatory roles upstream of various signalling pathways related to BCa carcinogenesis and progression, reflecting their potential as therapeutic targets for BCa. Herein, we briefly summarize the expression profiles, biological functions and mechanisms of circRNAs and the potential clinical applications of these molecules for BCa diagnosis, prognosis, and targeted therapy.The online version contains supplementary material available at 10.1186/s12943-020-01300-8. Circular RNAs (circRNAs) are single-stranded closed-loop RNA molecules without terminal 5′ caps and 3′ poly(A) tails . AlthougBladder cancer (BCa) is the most common malignant tumour of the urinary system, and its incidence is increasing worldwide . BCa is An increasing number of studies have shown that differential expression of circRNAs is associated with the carcinogenesis and progression of BCa. In this review, we summarize the expression profiles, biological functions and mechanisms, and clinical significance of BCa-related circRNAs.CircRNAs are produced from pre-mRNAs and are thought to be the result of exon-skipping events. Although the specific mechanism of circRNA biogenesis is still unidentified, two widely accepted models of circRNA circularization can explain the back-splicing processes known as lariat-driven circularization and intron pairing-driven circularization . In the CircRNAs were initially regarded as functionless by-products of aberrant RNA splicing , 3. WithTo date, genome-wide annotation of circRNAs, experimental validation of circRNAs, and overexpression/suppression of circRNAs are the main approaches to explore the functional implications of circRNAs. First, ribo-RNA-seq profiles rRNA-depleted total RNAs, including both poly(A) (linear) and nonpoly(A) (circular) RNAs. In addition, p(A)- RNA-seq profiles only non-poly(A) RNA. Ribo-RNA-seq or p(A)-RNA-seq combined with RNase R, which digests linear RNAs and preserves circRNAs, is more suitable for biochemical enrichment detection of circRNAs . In addiA full review was performed using Web of Science to search for reports with the key words (“circular RNA” or “circRNA”) and  published over the past 10 years (January 2009–March 2020). Research regarding the discovery and characterization of circRNAs has increased annually, while protein-coding gene (mRNA) discovery research has remained stable   and micrTo better study the biological functions and applications of circRNAs, numerous circRNA-associated public databases have recently been developed to facilitate circRNA analyses. These online databases are currently used for circRNA identification, prediction, localization, characterization and functional analysis and provide tools for investigating the interactions of circRNAs with targets. For example, CircBase contains circRNA information from different species . CircRNAWith the rapid development of high-throughput sequencing technologies, a large number of novel dysregulated circRNAs have been identified in BCa cell lines and tissues, most of which are differentially expressed between BCa tissues and adjacent normal tissues, indicating the important roles of these circRNAs in BCa development and progression. Primary expression profiles obtained via ribosomal RNA-depleted RNA-seq and circRNA microarrays have been widely employed for the discovery of novel circRNAs , 59, 62.P-value threshold, and an FDR < 0.05 was recommended. CircRNAs  have been reported to be significantly differentially expressed [For microarray or RNA-seq data analyses, paired t tests were performed to analyse significant differences. The false discovery rate (FDR) was applied to determine the xpressed , 64. Forxpressed –83. No uIn 2011, Hanahan and Weinberg proposed ten hallmarks of cancer that result in the progressive conversion of normal cells into cancerous cells . Here, wRecently, several oncogenic and antioncogenic circRNAs have been discovered to regulate cell proliferation, tumour growth suppression, cell cycle arrest, apoptosis, invasion, migration, metastasis, angiogenesis, and cisplatin chemoresistance in BCa cells Fig. .Table 2Tumour cells can sustain active proliferative states via activation of cell proliferation signalling pathways . The PI3Apart from sustaining proliferative signals, cancer cells continuously evade the growth-suppressive effects of the tumour suppressor pathway . TP63 isAdditionally, dysregulation of cell cycle regulators contributes to limitless tumour cell growth and proliferation . C-MYC aApoptosis, autophagy, and necrosis are major mechanisms leading to controlled cell death that are strictly controlled by tumour cells . Tumour Invasion, migration and metastasis of tumour cells into lymphatic and blood vessels for dissemination into the circulation eventually results in tumour colonization of distant organs . MMP9, aIncreasing evidence has revealed that circRNAs can act as metastasis activators or suppressors in BCa. The Wnt/β-catenin signalling pathway is highly involved in tumour metastasis , 146. HaAngiogenesis, the process by which rapidly growing malignant tissues are continuously supplied with nutrients and oxygen and cleared of metabolic wastes, is essential for tumour growth and progression. Without angiogenesis, tumours are unlikely to grow beyond a size of 100–200 μm . VasculaAlthough BCa is relatively sensitive to chemotherapy, decreasing cisplatin chemoresistance is a crucial therapeutic strategy for MIBC , 163. RACircRNAs perform regulatory roles mainly by acting as miRNA sponges , interacThe high incidence and mortality of BCa reflect the need for strategies to improve its early diagnosis, prognosis, and effective treatment. CircRNAs show considerable potential for use as diagnostic and prognostic biomarkers in BCa. First, circRNAs, as unique endogenous noncoding RNAs, are highly conserved and broadly expressed in various tissues, including human BCa and normal bladder tissues , 64, 179CircRNAs have been reported to be significantly associated with many clinicopathologic characteristics in BCa, including tumour size, grade, differentiation, and stage; lymph node metastasis (LNM); tumour numbers; distant metastasis (DM); invasion; and recurrence. Li et al. observed that circ_0018069 is significantly downregulated in BCa tissues and in T24 and Biu-87 cells and that circ_0018069 levels are correlated with tumour grade, tumour stage, and muscular invasion depth in the context of BCa . FurtherThe clinical value of circRNAs as diagnostic biomarkers has been explored in many studies. The area under the receiver operating characteristic (ROC) curve (AUC) of circ_0018069 for BCa diagnosis is 0.709, and the sensitivity and specificity are 97.6 and 46.3%, respectively . The AUCCircRNA levels can also be used to predict patient survival parameters, such as overall survival (OS), disease-free survival (DFS), and progression-free survival (PFS). To further analyse the prognostic value of circRNAs in BCa, we collected information from studies reporting survival information and evaluated the associations between circRNA expression levels and OS, DFS, and PFS. Fourteen upregulated circRNAs were reported to predict poor OS , 187, whOver the past 10 years, the importance of elucidating circRNA biology to our understanding of tumorigenesis has become evident. As outlined in this review, considerable evidence indicates that circRNAs play key roles in BCa. To date, fifty-five circRNAs among hundreds of aberrantly expressed circRNAs have been identified to be specifically associated with BCa. Notably, BCa-related circRNAs have been discovered to regulate cancer-related biological behaviours via ceRNA regulatory mechanisms. Existing reports feature methodologies and study designs that others can use for further investigation of circRNAs of interest. CircRNAs have been reported to be significantly associated with many clinicopathologic characteristics of BCa and with BCa patient survival parameters, and the abundance, conservation, stability, specificity and detectability of circRNAs render them potential diagnostic and prognostic biomarkers for BCa. Additionally, circRNAs play crucial regulatory roles upstream of various signalling pathways related to BCa carcinogenesis and progression, reflecting their potential as therapeutic targets for BCa.Some limitations of previous research on circRNAs in BCa should be noted. First, the biogenesis of circRNAs and the regulatory mechanisms involved in circularization remain vague. More research is needed to help us understand the circRNA circularization processes in depth. Second, no unified standards are available to determine thresholds for circRNA detection. Third, previous studies on circRNAs in BCa lacked circRNAs with BCa specificity. More circRNAs with relative bladder cancer specificity may be further characterized in future studies. Fourth, almost all reported circRNAs in BCa exert functions via miRNA sponge mechanisms. The other three classical mechanisms, including sponging of RBPs, regulation of transcription and translation into peptides or proteins, have rarely been studied in BCa. Fifth, all circRNAs reported in BCa are currently in the basic research stage. Further investigation of circRNAs as diagnostic biomarkers, prognostic biomarkers, or targeted therapy for BCa in well-designed multicentre cohort studies is necessary.Additional file 1: Table S1. Database for circRNA research


The amidites were used to introduce ‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic 19F–13C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic 13C–19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted /[5‐19F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial 19F–13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The ‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.We present the access to [5‐ 19F and13C in RNA—a perfect match. An aromatic 19F–13C isotope labeling protocol is introduced for uridines and cytidines in RNA up to 60 nucleotides. The favorable TROSY properties of this spin pair will be useful to probe large RNAs by NMR spectroscopy. The fluorine nucleus offers a 100 % natural abundance, a high sensitivity, a larger chemical shift dispersion than protons, and is biorthogonal making it an ideal candidate for in vivo NMR applications. Thus, it is not surprising that several 19F‐NMR spectroscopic studies to probe structure/function of nucleic acids can be found in literature. Fluorinated nucleotides, such as sugar fluorinated residues,bacillus subtilis.19F stable isotope labeling scheme to yield a 19F–13C‐spin topology, such as a ‐labeling pattern in pyrimidines, would be desirable to evade resonance overlap issues in larger RNAs by adding a second 13C dimension. The application of 19F‐NMR to study high molecular weight systems is also hampered by the fast transverse relaxation by the chemical shift anisotropy (CSA) mechanism. In a recent work this issue was addressed by introducing aromatic 19F–13C‐spin topologies in proteins and DNA, in which the cancellation of the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms lead to a very favorable NMR spectroscopic behavior in a TROSY type experiment.19F, 5‐13C]‐uridine (U) and ‐cytidine (C) phosphoramidites for solid phase RNA synthesis. We demonstrate the favorable spectroscopic behavior of the ‐pyrimidines in two model RNAs: the 61 nt human hepatitis B virus encapsidation signal epsilon (hHBV ϵ) and the 59 nt pre‐microRNA 21.13C/19F labeling by solid phase RNA synthesis is shown and used for a resonance assignment protocol based on /[5‐19F]‐uridine labeling schemes. For the 61 nt hHBV ϵ RNA we observed a favorable TROSY effect and compared the performance of ‐U labels with ‐U labels. The novel stable isotope (SI) labeling scheme will facilitate NMR studies of large RNAs and high molecular weight RNA protein complexes as it was recently demonstrated for high molecular weight proteins systems.13C5 labels we synthesized the ‐uridine and ‐cytidine building blocks 7 and 11  RNA 13 with 61 nts (ca. 20 kD)  21 after processing by the microprocessor enzyme complex and carries three ‐cytidine  and two ‐uridine  labels. Further, the apical stem loop 15 a with the same labeling pattern was produced to probe the modular composition of this pre‐miRNA 21  group, followed by the 5′‐tritylation reaction to give compound 6. Finally, the ‐uridine 3′‐phosphoramidite 7 was obtained. Noteworthy, we found it crucial to substitute triethylamine by pyridine as the additive for the eluents used in the silica chromatography to get reproducible recovery rates after column chromatography. Phosphoramidite 7 could be obtained in a six‐step synthesis with an overall yield of 8 %. Using the protected 5 the ‐cytidine 3′‐phosphoramidite 11 could be obtained by transforming the uridine nucleoside into the respective cytidine derivative 8 followed by an exocyclic amino selective acetylation step yielding 9. The final steps included the removal of the transient 5′‐, 3′‐hydroxy protection, followed by the 5′‐tritylation to give compound 10. Finally, the ‐cytidine 3′‐phosphoramidite 11 was obtained via the phosphitylation reaction with a yield of 70 %. We observed a 30 % proton back‐exchange of the deuterium at position 6 during the uridine to cytidine transformation using aqueous ammonia in acetonitrile at room temperature. Thus, we decided to produce only building block 11, as we presume that during the strongly alkaline deprotection at elevated temperatures in the oligonucleotide synthesis a mixture of ‐cytidine will be obtained. Both amidites were then used in the solid phase synthesis approach to label RNAs up to 60 nts. A short discussion on the aspects of chemical solid phase synthesis of ‐pyrimidine modified RNAs is given in the supporting information.Our initial efforts focused on the production of the 5‐1F, 5‐13C‐19F NMR assignment data for an in vitro transcribed [5‐19F]‐uridine labeled version is available.19F, 5‐13C]‐counterparts , that is, with four sequences 15 resonances, with five sequences 31 resonances can be assigned. The sequence design for RNA 13 is described . The five RNAs were labeled with 25 % diluted ‐Us at the respective positions, whereas all other uridine residues were replaced by ‐Us . We observed the fold dependent 19F chemical shifts for all Us and the so far not reported shift signature for G–U wobble base pairs at −163 ppm could be confirmed for all four G–U base pairs. Based on the 19F shift of U49 the A13–U49 base pair is weak and forms only transiently leaving U49 mainly single stranded. An overlay of all spectra with all assignments can be found in the supporting information .We then turned to the development of an efficient resonance assignment procedure for 19F, 1C‐labeled19F, 5‐13C]‐cytidine labeling to address structural and dynamic features of RNA. We picked the SAM VI riboswitch as the target and labeled the single cytidine C26 with 19F/13C  G7–G27 base pair and four stacked A28, A30, A31and G32 residues and by a U8 that recognizes the SAM‐adenine base. First, the ligand binding was monitored by observing the imino proton region of 1H‐NMR spectra. The binding of SAM was nicely reflected, and several additional canonical and non‐canonical base pairing interactions were formed upon the complex formation . This could be either an effect of the fluorine modification, but given the minimally invasive nature, we think that this could represent a functional feature of the riboswitch. In‐line with NMR and fluorescence spectroscopy results on riboswitches conformational dynamics is a key feature in the ligand recognition process.In the next step we exemplified the suitability of ‐C labels was not feasible due to back exchange of the deuterium to a proton under alkaline conditions. As a consequence, the ‐C labels experience scalar coupling interactions between the F5/C5 and the H6 leading to broadened resonances compared to the corresponding position 6 deuterated U. This is illustrated for U31 and C32 in RNA 15‐1 . We, thus, recommend for larger RNAs to mainly use the ‐U labeling protocol, especially if proton decoupling is not feasible with the available NMR hardware as in our case.As the final example the larger non‐coding pre‐miRNA 21 19F, 5‐13C]‐cytidine and a ‐uridine phosphoramidite suitable to modify RNAs with 19F/13C‐spin topologies. A regioselective fluorination of [5‐13C]‐uracil using Selectfluor is the key step to give ‐uracil. The nucleobase is fused to a sugar unit under silyl‐Hilbert‐Johnson conditions. Optionally, the proton H6 can be deuterated at this stage to minimize the scalar coupling network of the fluorine and to give sharper lines with a higher signal to noise ratio. The deuteration at position 6 can only be retained in the uridine building block, whereas for the cytidine we observed a re‐protonation at position 6 during the U to C transformation. We further anticipate that this re‐protonation would also occur during the alkaline deprotection step after the RNA solid phase synthesis leading to a mixture of C6‐deuterated and ‐protonated ‐cytidines. Thus, we decided to only synthesize the ‐cytidine phosphoramdite 11. Standard transformations allowed the synthesis of the phosphoramidites 7 and 11. We found it crucial to use pyridine instead of triethylamine as eluent additive for silica column chromatography to obtain reproducible yields. This is attributed to the higher acidity of the H3 imino proton in [5‐19F]‐modified U, which can react with triethylamine to give the ammonium salt. Once the RNA phosphoramidites 7 and 11 were available RNAs with sizes between 30 and 60 nts were synthesized. First, the 30 nt HIV TAR‐2 with six uridines and the 61 nt hHBV ϵ RNA with eighteen uridines were synthesized and six and five ‐U labels were incorporated in each RNA, respectively. We obtained high quality 19F–13C TROSY spectra within a short time on a Prodigy TCI probe with the proton coil tuned to the 19F resonance frequency for 0.5 mm RNA samples. The additional 13C dimension allowed to resolve the resonance overlap, which was earlier observed for U23, U25 and U31 in the 1D‐19F spectrum.12 did not show a pronounced TROSY effect. Thus, the TROSY properties of the FC‐U labels were probed in the 61 nt hHBV ϵ RNA with ≈20 kD. Here, we observed a more pronounced TROSY effect manifested in the 19F spectrum without 13C decoupling. We qualitatively compared a 13C‐detected out‐and‐stay TROSY experiment with a 1H‐13C TROSY experiment of a ‐uridine labeled 61 nt hHBV ϵ RNA. Both stable isotope labeling schemes gave well resolved correlation peaks at 25 °C but at 0 °C, where the slowed down molecular mobility leads to a higher molecular weight like behavior, the superior spectral properties of the 19F/13C spin pair were manifested. All five ‐U labels could be observed, in contrast only two of five 1H‐13C uridine resonances were found . We also assigned all eighteen 19F–13C uridine resonances of the 61 nt hHBV ϵ RNA. The resonance assignment confirmed the fold specific 19F chemical shifts in [5‐19F]‐uridines.19F chemical shifts not only allows to discriminate single stranded and A–U base paired uridines, but we also found a distinct chemical shift signature for G⋅U wobble pairs.We present an access to a ‐C label into the SAM VI aptamer and could follow the ligand binding process and get insights into the recognition process. We observed a minor 19F‐resonance indicating that the binding competent state is already present in the apo RNA supporting a conformational selection mechanism. In the final RNA target—the 59 nt pre‐miRNA 21—‐U and ‐C labeling was combined. Both labels gave satisfactory results but in larger RNAs the deuteration of position 6 in the U‐building block gave sharper linewidths and a better signal to noise ratio, as in our NMR hardware setup proton decoupling cannot be accomplished.We incorporated a single ‐U and ‐C labels can be used to quickly screen folding states, ligand binding events and structural and dynamics features of RNA not only under in vitro but also under in vivo conditions facilitated by fluorine's bioorthogonality. Altogether, we anticipate that the stable isotope labeling Scheme introduced in this work is highly attractive for in vivo NMR applications of RNAs in combination with dynamic nuclear polarization to boost sensitivity.To sum up, the novel [6‐D, 5‐The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The majority of the established RNA isolation protocols for use with primary pancreatic tissue involve perfusion of RNA stabilizing reagent into the pancreatic tissue to protect RNA integrity before extraction. However, the additional time needed for this procedure can actually lead to further RNA degradation. We optimized a protocol suitable for high quality RNA isolation from mouse pancreatic tumors that is a simple, fast, and inexpensive modification of existing methods, combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction. Through this procedure, the mean RNA Integrity Number value obtained for RNA isolated from pancreatic tumors was 9.0, and was reproducibly suitable for RNAseq and qPCR.  Specifications tablePancreatic cancer remains among the most lethal cancers 260/A280 absorbance ratio using a spectrophotometer, where a ratio of ~2.0 represents sufficient RNA purity for qPCR 260/A280 ratio of 2.1 can be substantially degraded. Hence, for sequencing purposes, RNA quality and intactness is measured via capillary electrophoresis (CE) and can be quantified using an RNA Integrity Number (RIN) value which ranges from 1 to 10, with 10 representing the highest quality RNA RNA extraction is commonly performed in many labs on a daily basis to quantify mRNA levels by using quantitative Polymerase Chain Reaction (qPCR). For qPCR procedures, RNA quality is usually estimated by measuring the A260/A280 ratio The guanidinium thiocyanate-chloroform extraction method is the most commonly used method for the extraction of RNA from cells and tissues •4-month old NSG mice bearing orthotopic pancreatic tumor allografts (IACUC approved)•Liquid nitrogen•Guanidinium thiocyanate •Chloroform (Pharmaco)•100% ethanol•RNaseZap Wipes (Ambion)•DNase/RNase free pipette tips (VWR)•Nuclease-free water (Ambion)•RNeasy mini-plus kit (QIAGEN)•RNase-Free DNase set (QIAGEN)•RNA stabilizing agent •Chemical hood•Mortar and pestle•Surgical equipment•Bioanalyzer 2100 (Agilent)•Nanodrop 1000 Spectrophotometer (Thermo Scientific)•Synergy H1 Hybrid Plate reader (BioTek)Step 1: Isolation and freezing of Pancreatic tumors1.1.The use of animals for the study was approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.1.2.5 cells of a mouse pancreatic adenocarcinoma (PDAC) cell line Four-month-old NSG mice were injected with 8 × 101.3.Prior to the necropsy and removal of the pancreas, decontaminate the entire workspace and surgical equipment with 70% ethanol and RNase Zap Wipes.1.4.NSG mice bearing pancreatic tumors were anesthetized using isoflurane and euthanized by cervical dislocation. After euthanizing, the mice were fixed to the dissecting table with the ventral side facing upwards. Sterilize the abdominal area using 70% ethanol, and make an incision in the genital area, cutting open longitudinally to expose the abdomen. Move the bowels towards the left side to get better visualization of the pancreas. As pancreatic tumors are evident, unlike the normal pancreas, make an incision at the common bile duct and isolate the pancreas quickly using scissors and forceps 1.5If only a part of the whole pancreatic tumor is needed for RNA isolation, cut the tumor while soaked in RNA stabilizing agent in a petri-dish on ice, then transfer it to a vial/Eppendorf tube and drop in liquid nitrogen to flash freeze the tumor tissue. Transfer the flash frozen tumor vials to −80 °C until extraction.1.6.Tip: Be as quick as possible and try to maintain cold conditions as much as possible.1.7.Tip: Mince the tumor or pancreas into small pieces while soaked in RNA stabilizing agent, as this facilitates later steps. Mincing the tumors improves penetration of RNA stabilizing agent and retains integrity of RNA in the tissues.1.8.Tip on use of RNA stabilizing agent if preferred for long-term storage: Soak the tissue in RNA stabilizing agent before extraction and store as recommended by the manufacturer specifications. We have not compared RNA integrity using RNA stabilizing agent vs. simple snap freezing in liquid nitrogen for the longer-term storage of pancreatic tumors. For next day extraction, snap freezing in liquid nitrogen works effectively and gives the best results.Step 2: Extraction of RNA2.1.Before performing extraction, clean the mortar and pestle using RNaseZap Wipes.2.2.Place pestle in mortar and add liquid nitrogen to cool both, and place TRIzol solution on ice or use it immediately after removing from refrigerator.2.3.Tare the weighing machine using an empty weighing boat and transfer the pancreas/tumor tissue from the flash frozen vial into a weighing boat and record the weight of the tissue. Now quickly transfer the tissue to the mortar, and powder the tissue while maintaining liquid nitrogen in the mortar to keep everything cold and facilitate crushing of the tissue into a frozen powder. Do not allow the tissue to thaw.2.4.. We recommend up to 1 ml of TRIzol per 100 mg of tumor tissue for best yields. Carry out trituration in a chemical hood to avoid hazardous health effects of the TRIzol.After the evaporation of the liquid nitrogen, add TRIzol in 500µl aliquots to the powdered tissue, with 500 µl for every 50 mg of pancreas/tumor tissue, and continue trituration as shown in Fig. 12.5.Transfer the TRIzol triturate, after it is liquefied, to the DNase/RNase free Eppendorf tube, and incubate for 5 mins at room temperature. It is common to observe solidification of the triturate which liquifies upon on continuing further trituration for a longer time.2.6.Add 200 µl chloroform per ml of TRIzol lysate and shake vigorously for 15 s. Incubate the tube for 2–3 mins at room temperature before centrifuging in a pre-cooled centrifuge (4 °C) at 11,600 rcf for 15 mins.2.7.Centrifugation results in separation of the two phases (chloroform aids in separation of the aqueous phase and lower organic phase) in the Eppendorf tube. Now, carefully transfer the upper clear supernatant layer without agitation to a fresh Eppendorf tube and mix with 0.5x volume of 100% ethanol to precipitate nucleic acids .2.8.Tip: Try not to disrupt the bottom solvent layer, as this will result in blocking the RNA spin column and decreases the efficiency of the method.Step 3: Purification and Isolation of RNA3.1.After mixing with ethanol, transfer the mixture to the QIAGEN RNeasy mini spin column and centrifuge at 9600 rcf for 30 s.3.2.Wash the column using 350 µl RW1 wash buffer from the RNeasy mini extraction kit.3.3.Add 40 µl of freshly prepared DNase I solution (Thermo Fisher) and incubate the column for 15 min at RT, after which, wash the column with RW1 wash buffer for the second time and centrifuge at 9600 rcf for 30 s. This eliminates DNA contamination of the extracted RNA.3.4.Discard the flow through, and wash the column with 500 µl RPE buffer supplied in the kit two times at 9600 rcf for 30 s.3.5.Finally, spin the column at 16,000 rcf for 2 min to remove any remaining RPE buffer from column. Make sure you remove the RPE buffer completely for better results.3.6.Add 30–40 µl of RNase free water to the column and incubate the column for 1 min at room temperature.3.7.Centrifuge at 9600 rcf for 30 s to collect the pure RNA.3.8.Tip: Avoid including tissue pieces in the solution that might result in blocking of the column and reduce column efficiency.3.9.Tip: As the binding affinity of the RNase easy spin columns is limited, we recommend dividing the total extract into equal volumes and using multiple columns to avoid clogging of the columns and combine the eluates after isolation to restore sufficient volume.3.10.Tip: Avoid repeated elution, as most of the RNA obtained by repeated elution is not high enough quality for sequencing purposes.Step 4: Quantification of RNA4.1.The concentration of RNA is measured using a Nanodrop  by loading 2 µl of the sample.4.2.260/A280 value obtained in the Nanodrop gives an estimate of concentration and any protein contamination.The concentration and the absorbance ratio A4.3.However, 260/280 value is not a determinant of intact RNA, so make sure you have sufficient RNA for the quality check and sequencing steps.Step 5: Quality Check5.1.Check the integrity of the RNA using a Bioanalyzer 2100 instrument (Agilent), following the standard protocol for running total eukaryotic RNA on a pico chip, to determine the RIN of the sample .Fig. 2Ex5.2.The extracted RNA is stored at −80 °C till use for sequencing.5.3.Tip: Run the RNA samples on an agarose gel as described 5.4.Tip: To avoid multiple freeze thaws, divide the RNA isolate into multiple aliquots and store at −80 °C. Use each aliquot once.5.5.Tip: Use dry ice to transfer the samples for quality check or sequencing to protect RNA integrity.Step 6: Storage6.1.The extracted RNA is stored at −80 °C till use.6.2Tip: It is advisable to use the RNA as quickly as possible for sequencing purposes to avoid any possible degradation.Although numerous methods have been published which describe the extraction of high-quality RNA from pancreas or pancreatic tumors, our method is simple, reliable and reproducible, resulting in extraction of high-quality RNA (mean RIN = 9.0) from mouse pancreatic tumors that is especially well-suited for NGS analyses. Incorporation of these simple tips will help greatly in improving the quality of RNA and make pancreatic-derived RNA more consistently usable for sequencing purposes, saving money and avoiding bias in the sequencing results. With further adaptation, we envision this technique being applicable to the isolation of high-quality RNA from human pancreatic normal and tumor specimens as well.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.