N1-methyl-pseudouridine (m1Ψ), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, we compared the incorporation fidelity of uridine analogs with different RNA polymerases. We demonstrate that m1Ψ is incorporated with higher fidelity than Ψ. The fidelity of nucleotide incorporation differs between RNA polymerases; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel method to improve uridine analog incorporation fidelity during IVT. Our proof-of-concept experiments for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing RNAPs for the generation of modified uridine-containing mRNAs in vitro.In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs—such as pseudouridine (Ψ) and  Despite there being several advantages over conventional protein-based approaches, mRNA-based therapeutics are still in early stages of development. Instability of the synthetic mRNAs and the immune responses generated against these synthetic molecules have been key hurdles in the adaptation of this technology, particularly for therapeutic applications where prolonged expression from the synthetic molecule is desirable and repeated dosing of the drug product is required2. The use of chemically modified bases in synthetic mRNAs is an innovation that has allowed for both an ameliorated immune response to the synthetic molecules and increased protein expression from the mRNA, thereby providing an unprecedented opportunity to use synthetic mRNAs as a novel class of therapeutics for a wide range of indications, including two approved vaccines against COVID-195. It has been shown that the incorporation of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A) and 2-thiouridine (s2U) into synthetic mRNAs results in reduced immune responses and increased protein expression in vivo14. Pseudouridine-modified mRNAs have been shown to result in reduced activation of 2′-5′-oligoadenylate synthetase (OAS), RNA-dependent protein kinase (PKR), and toll-like receptors10. Investigation of ψ derivatives with improved pharmacological properties led to the identification of m1ψ, the current benchmark for synthetic mRNA-based applications. m1ψ is a naturally occurring modification found in 18S rRNA and tRNAs17, and similar to ψ-modified synthetic mRNAs, the presence of m1ψ in synthetic mRNAs has been demonstrated to result in reduced activation of RNA sensors in cells18. Furthermore, the presence of m1ψ in synthetic mRNAs show increased translation efficiency in cell-free extracts, multiple mammalian cell lines, and mouse models20. The exact mechanism of how m1ψ enhances translation is not well understood but it has been demonstrated that presence of m1ψ, alone or in combination with other chemical modifications, can alter ribosome transit time on the modified mRNA, and can increase the mRNA half-life by altering the secondary structure of the synthetic mRNA22.Synthetic messenger RNAs (mRNAs) are a novel modality for vaccines, and they are also being evaluated as a vector for therapeutics1. In contrast to endogenous mRNAs, in which modified nucleotides occur in specific positions in the mRNA24, in synthetic mRNAs, the modified nucleotide is present at almost every position where the naturally occurring nucleobase would be. This complete substitution approach is preferred from a regulatory perspective because it results in less molecule-to-molecule variation in the positions of the modified nucleotides along the synthetic mRNA. The exact implications of incorporating the chemical modifications throughout the body of the mRNA is still under investigation. For attaining the maximum effectiveness from the drug product and to ensure that the expression from the synthetic molecule is optimal, it is critical that the modified nucleotide is incorporated in the right place, be compatible with the functional elements of the mRNA, and does not alter the biological function of the synthetic mRNA. Numerous studies have demonstrated that ssRNAPs can incorporate chemically modified nucleotides into RNA25, but it is unclear whether all ss-RNAPs incorporate the modified nucleotides with comparable fidelity.Current methods to incorporate modified nucleotides in synthetic mRNAs include complete substitution of the standard nucleotide with a chemically modified nucleotide during the process of in vitro transcription (IVT) by single-subunit DNA-dependent RNA polymerases 26. In the previous study, T7 RNAP exhibited higher combined error rates in the synthesis of ψ-, m6A- and 5-hydroxymethylcytidine (hm5C)-modified RNAs, with increased misincorporation of ψ across from dT templated bases. In light of the widespread use of m1ψ for mRNA vaccines, in the current study, we focused on understanding the fidelity of incorporation of m1ψ during in vitro transcription using multiple ssRNAPs. The fidelity with which m1ψ is incorporated during in vitro transcription is a timely question to address because both the SpikeVax (mRNA-1273) and Comirnaty (BNT162b2) SARS-CoV2 mRNA vaccines are synthesized by substituting uridine with m1ψ throughout the body of the mRNA and both have demonstrated efficacy greater than 94%5. We investigated the fidelity of m1ψ incorporation with three commonly used ssRNAPs  and compared it to the fidelity with which uridine and ψ are incorporated. Using four different RNA sequences, including two functional mRNA sequences, we analyzed the effects of sequence context in promoting misincorporation of uridine analogs and identified rA → rU substitution errors as the predominant error when uridine analogs (m1ψ and ψ) are present in the reaction. Finally, based on the nature of the substitution errors observed, we combine sequence optimization of the synthetic mRNA together with an altered RNA synthesis process to reduce the uridine analog incorporation error during in vitro transcription. These proof-of-concept experiments establish a novel method to synthesize mRNAs with improved fidelity of uridine analog incorporation and provide considerations for choosing ssRNAPs for the generation of modified nucleotide-containing mRNAs in vitro.We previously established a Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing-based assay to determine the combined transcription and reverse transcription errors and the effects of RNA modifications27 and Cypridina luciferase mRNA sequence (CLuc mRNA) when in vitro transcription was performed with T7 RNAP  that represent all possible four-base combinations (templates described as DNA-1 and DNA-2 previously 26). The error rates of pooled RNA1 and RNA2 (referred as RNA1/RNA2) in reactions with canonical uridine, using the Sequel I system was observed to be 6.4 ± 0.4 × 10−5 error/base as compared to 5.6 ± 0.8 × 10−5 error/base using the PacBio RSII system  sequencing-based assay , while that in m1ψ-modified RNAs were comparable to unmodified RNAs (7.4 ± 0.7 × 10−5 error/base for m1ψ-containing reactions) , CLuc mRNA as well as BNT162b2 sequences when in vitro transcription reactions are performed with T7 RNAP under standard high-yield reaction conditions. The combined error rates in ψ-modified RNAs were observed to be two-fold higher than unmodified RNAs  and SP6 RNAP (20 mM rNTP). In order to ensure that the differences in combined error observed for T7 RNAP and SP6 RNAP are not due to differences in the rNTP concentrations in the reactions, we performed in vitro transcription reactions with T7 RNAP under low rNTP reaction conditions with either 20 mM or 10 mM rNTP . The total combined error rates as well as the base substitution errors observed in unmodified RNA1/RNA2 when reactions were performed with T7 RNAP under low rNTP (10 or 20 mM) reaction conditions were comparable to that observed with high rNTP (40 mM) reaction conditions .First, as a control, we analyzed the error rates when in vitro transcription reactions were performed under standard high-yield rNTP condition where all the rNTPs are added equally to a final concentration of 40 mM . As observed with T7 RNAP, ψ-modified RNA had the most pronounced (three-fold) reduction in total combined error rate (from 6.0 ± 2.1 × 10−4 errors/base to 1.7 ± 0 × 10−4 errors/base) when the rNTP ratios were altered to match the sequence of the RNA. Furthermore, the reduction in total combined error observed in proportional rNTP reaction conditions is attributable specifically to reduction in the rA → rU substitution errors  and reactions were performed with either equimolar rNTPs or rNTPs matched to the nucleotide sequence. In presence of proportional rNTPs, the total combined error rate in unmodified RNA was reduced to 1.3 ± 0 × 10ors Fig. .Figure 5−5 errors/base, 6.4 ± 0 × 10−5 errors/base and 6.0 ± 0.9 × 10−5 errors/base, respectively  of synthetic RNA duplexes containing either uridine, ψ or m1ψ, have shown that both ψ- or m1ψ-containing duplexes have a higher Tm than uridine-containing duplexes13. Increased base pairing and stacking has been postulated to contribute to the increased Tm observed39. One of the critical features of ψ and m1ψ is the C5-C’1 bond that enables rotation between the sugar moiety and the nucleobase, which, in contrast to canonical uridine, could provide improved base pairing and stacking. As compared to uridine and m1ψ, ψ contains an extra hydrogen bond donor group (N1H) that imparts a universal base character to ψ. Hence, ψ can not only pair with A but can also wobble base-pair with G, U, or C36. On the other hand, m1ψ has a methyl group in the N1-position and therefore does not have the extra hydrogen bond donor and therefore wobble pairing with other nucleotides is not favored. It is plausible that the lower error rates observed in m1ψ-containing RNAs is due to the lack of the extra hydrogen bond donor39. Additionally, it is likely that the methyl group in the N1 position of m1ψ may alter the polarity of the C2 position and make it less favorable to pair with dT in the DNA template. It is also worth noting that stability difference between RNA duplex containing four consecutive ψ’s and m1ψ’s was not distinguishable13. Stability measurements in which a single rψTP or rm1ψTP pairs with DNA template to mimic the errors observed during in vitro transcription will likely be more relevant to distinguish the base-pairing differences we observe in this study. Not much is understood about the pairing of rψ or r m1ψ with DNA, but there is precedence for RNA-RNA interactions in presence of ψ and m1ψ which might help us understand the increased error rates observed in presence of the modified analogs40.Even though the combined error rates observed for the same set of RNA sequences were significantly different between T7 and SP6 RNAPs, there were few similarities. First, the fidelity with which the analogs were incorporated, followed the same trend—uridine > m1ψ > ψ—with m1ψ-modified RNA exhibiting lower total errors and rA → rU/dT → dA substitution than ψ-modified RNA Figs.  and 5. S30. Our data demonstrates that combining uridine depletion of the RNA sequence with altering the rNTP composition of the reaction, reduces the rA → rU substitutions that are introduced during in vitro transcription  and the sequences are available in Supplementary Table 26. For in vitro transcription reactions with SP6 or T3 RNA polymerase, the corresponding promoter sequences were inserted in the DNA templates using Q5 Site-Directed Mutagenesis Kit . DNA templates encoding functional mRNAs, Cypridina luciferase (CLuc) mRNA (1707 nucleotides) and part of BNT162b2/Comirnaty mRNA (4187 nucleotides)30, were synthesized by GenScript Inc.  and introduced into standard high-copy plasmids. DNA templates for uridine-depleted artificial sequences  were synthesized by GenScript and introduced into standard high-copy plasmids. The plasmids were propagated in E. coli  and purified with the Monarch Plasmid Miniprep Kit . T-depleted (13.5% T-content) version of the Cypridina luciferase (CLuc) mRNA template was synthesized by GenScript and introduced into standard high-copy plasmids. The plasmids were propagated in E. coli  and purified with the Monarch Plasmid Miniprep Kit . Plasmids were digested with restriction enzymes to generate linearized templates for in vitro transcription. The linearized plasmids were treated with PreCR Repair Mix  and purified with the Monarch PCR & DNA Cleanup Kit .Generation of the DNA templates for the artificial RNA sequences RNA1 and RNA2  were described earlier For short RNAs (30-nucleotide and 60-nucleotide long), double-stranded DNA templates were generated by annealing DNA oligonucleotides for template and non-template strands in annealing buffer  by heating at 95 °C for 5 min followed by gradual cooling to room temperature.DNA templates corresponding to three uridine-depleted artificial RNA sequences with base composition of (1) 30.9% A, 33.4% C, 30.1% G, 5.5% U, (2) 29.7% A, 32.0% C, 31.7% G, 6.6% U and (3) 30.2% A, 30.4% C, 27.1% G, 12.3% U were synthesized by GenScript Inc. . Additionally, to evaluate if this approach is viable for a functional mRNA, we also generated a uridine-depleted CLuc mRNA sequence where the uridine content was reduced from 22.8 to 13.5%. The uridine-depletion of the CLuc mRNA sequence was performed without perturbing the amino acid sequence of the encoded protein so that there are no other differences between the two mRNAs, other than the nucleotide content of the sequences . For the uridine-depleted sequences , two sets of reactions were performed—one in which all the rNTPs were present in equimolar ratio and another in which the rNTPs were altered to match the template sequence composition. Following IVT, the DNA template was digested with Turbo DNase  digestion at 37 °C for 30 min and then purified with the Monarch RNA Cleanup Kit  for long RNA (1020 nucleotides to 4187 nucleotides) or with the Oligo Clean-Up and Concentration Kit  for short 30-nt RNA and 60-nt RNA.In vitro transcription reactions were performed according to standard protocols provided with the high-yield in vitro transcription kits , consisting of 40 mM rNTP (pH buffered with sodium phosphate) for T7 RNA polymerase and 20 mM rNTP (pH buffered with Tris) for SP6 RNA polymerase. In vitro transcription with T3 RNA polymerase  was carried out following manufacturer’s instruction. For modified RNAs, UTP was replaced with either pseudouridine-5’-triphosphate  or NLow-yield IVT reaction conditions were carried out with either 20 mM or 10 mM rNTPs  at 37 °C for two hours. DNA template removal and cleanup were performed as described above.For the rUTP optimized T7 RNAP reactions, rNTPs proportional to the template sequence were used . For the rUTP optimized SP6 RNAP reactions, rNTPs proportional to the template sequence were used  rATP, 6.6 mM (33%) rCTP, 6 mM (30%) rGTP and 1.2 mM (6%) rUTP was used for RNA sequence with 5.5% uridine content].In vitro transcription of RNA1/RNA2 with excess rATPs were performed with either 16 mM rATP with 8 mM of other rNTPs or 20 mM rATP with 10 mM of other rNTPs.Eluted RNA samples from the in vitro transcription reactions were diluted based on concentrations measured on a Nanodrop spectrophotometer  and denatured at 70 °C for 2 min and snap-cooled on ice. 250 ng RNA samples were analyzed with RNA 6000 Nano kits  and the integrity and size distribution of the RNA was assessed using mRNA Nano series 2 assay .Purified modified RNA and RNA without any chemical modification were digested with the Nucleoside digestion mix  at 37 °C for 1 h. Base composition analysis was performed by Liquid Chromatography-Mass Spectrometry (LC–MS) using an Agilent 1290 Infinity II UHPLC equipped with G7117A Diode Array Detector and 6135XT MS Detector, on a Waters Xselect HSS T3 XP column  with the gradient mobile phase consisting of methanol and 10 mM ammonium acetate buffer (pH 4.5).https://www.enovatia.com/our-products/promass/?gclid=EAIaIQobChMIoZS6xKDn-AIViNrICh3peATrEAAYASAAEgLRNPD_BwE).Purified 30-nucleotide and 60-nucleotide RNA with different uridine modifications were analyzed at Novatia, LLC using on-line desalting, flow injection electrospray ionization on an LTQ-XL ion trap mass spectrometer (Thermo Fisher Scientific) and analyzed with ProMass Deconvolution software from Novatia, LLC 26. For the sequencing on the Sequel platform, about 1.5 µg cDNA was treated with NEBNext End Repair Module  at room temperature for 5 min, followed by purification with Monarch PCR & DNA Cleanup Kit . The end-repaired cDNA was ligated with 2 µL barcoded adaptor  with T4 DNA Ligase  in 50 µL reaction volume at room temperature for 1 h, followed by purification with Monarch PCR & DNA Cleanup Kit . The un-ligated adaptor and cDNA were digested with E. coli Exonuclease III  and Exonuclease VII  in 1X standard Taq buffer at 37 °C for 1 h, followed by cleaning up with Monarch PCR & DNA Cleanup Kit . The ligated DNA was repaired with PreCR Repair Mix  at 37 °C for 30 min. The libraries were purified with 0.6X volume of AMPure PB beads  and pooled for sequencing runs. SMRT Link was used to generate the protocol for primer annealing, polymerase binding [Sequel Binding Kit 3.0 ], cleanup and final loading to SMRT Cells LR and sequencing using Sequel system.The library preparation for sequencing on RSII system was performed as described before26. In short, high-accuracy consensus sequences were built for the first and second strand for each sequenced double-stranded DNA. The consensus sequences were aligned to the reference sequence and base substitutions, deletions, and insertions were determined. The first strand error rates were determined by comparing the first strand consensus sequences to the reference sequence (RNA strand), and mutations were required to be present in both strands. The average error and standard deviation of total errors were then calculated by combining the data from the templates for each reaction condition: for RNA1 and RNA2, one measurement was performed and for other RNA sequences, two independent repeats were performed. Substitution error profiles are represented separately for each RNA sequence. The relative fold change was calculated for each substitution as (M − U)/U, where M is the substitution rate on modified RNA (m1ψ or ψ) and U is the substitution rate on unmodified RNA. For comparing total combined error rates and for estimating the statistical significances, p-values were calculated using unpaired t-tests from the measurements of unmodified and modified RNAs, and those under equal and proportional rNTP conditions. The local context of base substitutions was extracted, and sequence logos were built using WebLogo software41. Also, the frequency of mutations at each position was plotted to examine distribution of errors along the reference sequence.Analysis of sequencing data was performed as previously describedSupplementary Information.


Although noncoding RNAs (ncRNAs) have been shown to regulate maladaptive neuroadaptations that drive compulsive drug use, ncRNA-targeting therapeutics for substance use disorder (SUD) have yet to be clinically tested. Recent advances in RNA-based drugs have improved many therapeutic issues related to immune response, specificity, and delivery, leading to multiple successful clinical trials for other diseases. As the need for safe and effective treatments for SUD continues to grow, novel nucleic acid-based therapeutics represent an appealing approach to target ncRNA mechanisms in SUD. Here, we review ncRNA processes implicated in SUD, discuss recent therapeutic approaches for targeting ncRNAs, and highlight potential opportunities and challenges of ncRNA-targeting therapeutics for SUD. Substance use disorder (SUD) continues to be a worldwide public health crisis . AlthougAs only 1%–2% of the human genome encodes for protein , 4 and mIn animal models of SUD, several ncRNAs have been shown to play functional roles in drug-seeking behaviors , and in In humans and other primates, ncRNA expansion has fostered the intricate regulatory network required for brain evolution and cognitive advancement . ncRNAs MicroRNAs are a class of small noncoding RNAs with a highly conserved single-stranded sequence of approximately 22 nucleotides . InitialBDNF, CREB, MeCP2, CaMKIIa)  . In partCaMKIIa) . In addiCaMKIIa) , 85–94. While there have been no clinical trials using miR-targeting therapeutics in SUD patients, several miRs have been explored functionally in preclinical SUD models . For exaLong noncoding RNAs (lncRNAs) are a diverse class of RNA molecules that are greater than 200 nucleotides in length and are generally classified based on their genomic location or function  . Many lnMIAT, NEAT1, MALAT1, and MEG3 lncRNAs was observed in the NAc of heroin-using subjects, and MIAT, MALAT1, MEG3, and EMX2OS upregulation was observed in the NAc of cocaine-using subjects. These well-studied lncRNAs contribute to various cellular processes, including GABA neuron neurogenesis, synapse formation, and cAMP signaling  are a class of lncRNAs that have also been implicated in SUD . NATs arof abuse , 114. Thstration , and in toxicity . Thus, wCircular RNAs (circRNAs) are single-stranded noncoding RNA molecules produced from pre-mRNAs by a non-canonical splicing process called back-splicing, resulting in covalently closed RNA loops. Approximately 20% of mammalian genes express circRNAs, and while these ncRNAs are present in various organs, their enriched expression in the brain makes them an appealing target for the treatment of neuropsychiatric disorders , 118. ciIn several recent papers, a role for circRNAs in SUD has been explored. For example, RNA-sequencing analysis of post-mortem human NAc samples identified several circRNA–miR interactions that were associated with alcohol dependence , and in Due to significant improvements in safety, selectivity and delivery, RNA-based pharmaceuticals have received considerable attention and 14 RNA-based drugs have received FDA or EMA approval since 2015. See reference  for a covia steric block or RNA degradation (Antisense oligonucleotides (ASOs) are small, synthetic single-stranded nucleic acid molecules that hybridize with the target RNA to alter splicing or translation radation . The smaradation . Indeed,radation . Also, uradation .NCT05127226) after successful in vitro and in vivo investigations  have been successfully targeted using ASOs in other preclinical disease models  to improve stability, cellular uptake, and in vivo delivery  have been targeted with miR mimics (In preclinical studies, researchers have demonstrated the effectiveness of antimiRs in animal models of alcohol , 164–166R mimics –170. AltR mimics –175. To Emerging research indicates that the three-dimensional structure of RNA, which creates well-defined recognition sites and motifs, can be selectively targeted with small molecules . Other tGas5 (let-7 family, miRs with a known link to SUD, are suppressed by RNA-binding proteins called LIN28. Recently, Wang et al. successfully identified six small molecule disruptors of LIN28 and subsequently let-7 suppression (Despite the aforementioned challenges, several SMIRNAs have been identified and validated in preclinical studies –189, andGas5 , a lncRNGas5 . Finallypression . Togethevia intravenous, intrathecal, or intranasal routes of administration (Despite several advances, treating CNS diseases with nucleic acids-based platforms remains a major challenge due to the blood-brain barrier. Comprised of tight junctions between brain capillary endothelial cells, the blood-brain barrier prevents large molecule therapeutics from entering the brain parenchyma. To circumvent this issue, researchers have developed several RNA delivery systems that are capable of entering the brain stration , 196–200via more feasible routes of administration. For example, intrathecal injection of an AAV that expresses an artificial miR resulted in robust gene silencing with no observed side effects in nonhuman primates (via intrathecal or intravenous routes of administration (In preclinical studies, viral vectors are widely used to transfer nucleic acids to brain cells with high efficiency . The mosprimates . Using tprimates . In animprimates . Severalstration –215 and stration . In summNanoparticle-mediated delivery of ncRNA therapeutics is a promising approach for the treatment of SUD . Nanoparvia systemic administration remains an ongoing challenge, miR mimic and antimiR encapsulated nanoparticles have successfully targeted the brain in multiple CNS disease models following systemic administration (Although nanoparticle-mediated brain delivery stration –233. Forstration . A more stration , 235. Instration –238, trastration , angiopestration , 241, anstration . Thus, aThe lipid bilayer is a billion-year-old barrier that prevents large, charged molecules like RNAs from entering the cell. In addition to this barrier, there are other formidable obstacles that protect cells from RNAs including, RNases, the innate immune system, and for neurons, the blood-brain barrier . Despitevia less invasive routes of administration, a hypothesis that merits further exploration.An additional strategy to move the field forward is to repurpose or test clinical-stage nucleic acid-based therapeutics that may also have relevance to SUD. For example, several companies have developed miR mimics or antimiR that target miRs linked to SUD , 258–260BCL2 mRNA, showed promise in preclinical experiments but lacked efficacy in multiple clinical trials (Although many promising opportunities are listed above, multiple clinical trials using RNA-based treatments have been withdrawn due to severe side effects or limited efficacy , 264. Thl trials , 269. Ful trials –272. On l trials . Thus, gDosing is another major issue that needs to be addressed in ncRNA-targeting therapeutics, as many ncRNA studies have used supraphysiological concentrations that may lead to unpredictable side effects , 274. Fo


DNA information can be copied into mRNA  and proteins can be subsequently synthesized using the information in mRNA as a template . Approximately 4000 RNA-binding proteins (RBPs) in the cells coordinately regulate these multiple processes between transcription and translation. It has been recently recognized that some of the RBPs have abnormal expression and/or function, leading to the initiation or maintenance of malignant disorders including sarcomas, which is the general term for a broad group of malignancies that begin in the bones and soft tissue. Unfortunately, there are currently very few effective treatments for many types of sarcomas in advanced stages. Therefore, we need to understand more deeply how sarcomas develop in our body and how they are efficiently eradicated by therapeutic intervention. Studies on the disease mechanisms in terms of RBPs will provide us with the opportunity to have a better understanding of the sarcoma pathogenesis.RNA-binding proteins (RBPs) are proteins that physically and functionally bind to RNA to regulate the RNA metabolism such as alternative splicing, polyadenylation, transport, maintenance of stability, localization, and translation. There is accumulating evidence that dysregulated RBPs play an essential role in the pathogenesis of malignant tumors including a variety of types of sarcomas. On the other hand, prognosis of patients with sarcoma, especially with sarcoma in advanced stages, is very poor, and almost no effective standard treatment has been established for most of types of sarcomas so far, highlighting the urgent need for identifying novel therapeutic targets based on the deep understanding of pathogenesis. Therefore, defining the network of interactions between RBPs and disease-related RNA targets will contribute to a better understanding of sarcomagenesis and identification of a novel therapeutic target for sarcomas. RNA-binding proteins (RBPs) are involved in multiple steps of RNA metabolism including RNA splicing, polyadenylation, transport, maintenance of RNA stability and degradation, intracellular localization, and translation control. There is an increasing amount of evidence that alterations in the expression or activity of RBPs can lead to a variety of disorders including cancers and sarcomas. Defining the network of interactions between RBPs and disease-associated RNA targets will contribute to a better understanding of tumor pathology and a development of novel therapeutic agents.RBPs generally have conserved domains and common sequences that interact with RNA and other proteins to form ribonucleoprotein (RNP) complexes. RBPs determine the fate of target RNAs by recruiting a variety of proteins and other cellular components . RBPs weRecent intensive genomic sequencing studies in malignant disorders have revealed recurrent mutations and aberrant activation and deactivation of RBPs due to both genetic and nongenetic factors, suggesting essential roles of RBPs in the initiation and progression of tumors. In addition, advanced technologies assessing RNA–protein interactions have identified more than 4000 proteins as potential RBPs. These advances associated with RBPs and RNA have greatly enhanced basic and translational research on how these RBPs contribute to tumorigenesis.+ hematopoietic stem and progenitor cells [In fact, it is now clear that RBPs are dysregulated in several types of cancer, thereby affecting the expression and function of oncoproteins and/or tumor suppressor proteins ,8. Clinior cells . A domaior cells ,13.On the basis of recent advances in the understanding of RBPs in the pathogenesis of malignant tumors, we provide a concise review of RBPs covering their functional roles in RNA metabolism and dysregulation in sarcomas.RBPs are involved in almost all layers of post-transcriptional regulation (RNA metabolism) . RBPs esAlternative splicing (AS) plays an important role in gene regulation, through which pre-mRNA transcripts are processed to produce multiple mRNA variants with different stability and protein-coding potential. AS perturbations are frequently observed across cancers. RBP is one of the molecular determinants of AS, and the disturbance of RBP network activity has a causal relationship with cancer development ,16. FuncPolyadenylation is a key process for the generation of mature RNA transcripts. Selective polyadenylation occurs within the 3′ UTR of mRNA and produces 3′ UTR of varying length by 3′-terminal cleavage and polyadenylation (CPA). Each transcript of mature mRNA contains a polyadenylate tail that determines its stability. RBPs involved in polyadenylation include U1 small nuclear ribonucleic protein (U1 snRNP), cleavage and polyadenylation-specific factor 1 (CPSF1), embryonic lethal abnormal vision (ELAV) L1/human antigen R (HuR), and poly(A) RNA-binding protein , and the ZFP36 ring finger protein (ZFP36/TTP) . CPEB fa7G cap, and 3′ poly (A) tail [RNA stability is associated with its nucleotide sequence, modification, 5′ m(A) tail . These d(A) tail . More sp(A) tail .7GpppN). The 5′ cap is important for promoting splicing of the first intron, exporting mRNA to the cytoplasm, and allowing translation of the mRNA. Removal of the cap (“decapping”) results in silencing of mRNA expression. The decapping enzyme DCPS is characterized as a pyrophosphatase that hydrolyzes the 5′ m7Gppp and m7Gpp cap structure generated following 3′-to-5′ and 5′-to-3′ decay. Messenger RNAs containing an AU-rich element (ARE) in the 3′ UTR are rapidly degraded in the cytoplasm. ARE-mediated decay is initiated by deadenylation, which is followed by the 3′-to-5′ decay through a complex of exonucleases (termed exosome). In this process, DCPS hydrolyzes the remaining cap [7Gpp and monophosphorylated RNA, followed by the DCPS-mediated hydrolysis of m7Gpp to release m7Gp [Several RBPs such as the mRNA decapping enzyme scavenger (DCPS), CUGBP Elav-like family member 2 (CELF2), insulin-like growth factor 2 mRNA protein (IGF2BP) family, HuR, QKI-5, RBMS3, and TARBP2 play important roles in cancer biology . For exaning cap . On the ase m7Gp . TherefoAnother layer of regulation on the stability, as well as the translation, of mRNA is achieved by RBP-mediated control on intracellular localization . SubcellThe translation process consists of the following three steps: initiation, extension, and termination. Controls on these processes are crucial for cancer development and progression both globally and in specific mRNAs which promote cancer biology, such as cell survival, transformation, metastasis, and stemness. Multiple factors including 43S ribosome initiation complex, cap-dependent mRNA translation, and cap-independent mRNA translation are involved in carcinogenesis. Dysregulations in these translational processes in malignant disorders are beyond the scope of this review and are excellently summarized elsewhere ,49,50.No-coding RNAs (ncRNAs) are commonly expressed RNAs in human cells that lack protein-coding ability. In the research results of FANTOM and ENCODE, two large genome projects, 80% of the human genome has transcriptional activity, while only 2% of the human genome codes for proteins. The noncoding regions of the human genome (98%) are primarily responsible for regulating gene expression ,52. RBPsThe RBP LIN28 is one of the four factors sufficient to reprogram human somatic cells into induced pluripotent stem cells, which upregulates or inhibits the maturation of different members of the let-7 microRNA family in many cancer cells . LIN28A CircRNAs are covalently closed RNA molecules, usually derived from precursor mRNA (pre-mRNA) through reverse splicing events, where the downstream 5′ splicing donor is reversely spliced to the upstream 3′ splicing receptor . Recent https://depmap.org/portal/ (accessed on 1 June 2022)) and list the top-ranked RBPs , inhibit RNA decay, and enhance the expression of oncogenes such as MYC and LIN28 in multiple types of cancers [Some other well-known RBPs also contribute to the Ewing sarcoma pathogenesis. The LIN28 paralog is canonically known to regulate gene expression through let-7 miRNA family biogenesis. Keskin et al. discovered that LIN28B is highly expressed in a subset of Ewing sarcoma . LIN28B  cancers . Thus, IRhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and rarely affects adults. Pathologically, RMS is a high-grade mesenchymal tumor showing aberrant myogenic differentiation. The 5 year survival rates are approximately 60% in children and 30% in adults . A multiTargeting the insulin growth factor (IGF) pathway is considered an attractive therapeutic target for RMS ,79. RMS According to the DepMap database, Quaking (QKI) was identified as a candidate of the therapeutic target of RMS . AlthougSynovial sarcomas account for 8–10% of all soft-tissue sarcoma cases and most commonly affect the AYA generation younger than 30 years old. Clinical prognosis of adult synovial sarcoma remains poor; the expected 5 year survival is 50–60% despite intensive multimodality treatment . SynoviaTP53 (95%), RB (29%), ATRX (52%), and DLG2 (52%) [Osteosarcoma is the most common bone-derived malignant tumor, affecting mostly adolescents. The clinical prognosis is dramatically improved by multimodality therapy; long-term survival is obtained in >60% of patients with localized disease. However, limited progress has been achieved over the last four decades. Histologically, osteosarcoma is characterized by an abundant osteomatrix synthesized by malignant cells and classified as osteoblastic, chondroblast, or fibroblastic according to the predominant differentiation pattern . Recent G2 (52%) .The role of RBPs in the osteosarcoma phenotype is poorly understood. A few RBPs have been reported as potentially relevant to clinical prognosis and cell proliferation. SUB1/PUC4, a coactivator of RNA polymerase II, was identified as a potential targetable vulnerability of osteosarcoma in the DepMap database . The molAlthough our understanding of the genomics, molecular biology, and therapeutic implications of altered RBPs in a variety of cancers and sarcomas has been greatly improved by the recent elegant studies using cutting-edge techniques such as single-cell RNA/DNA sequence and CRISPR/Cas9 mediated screens, standard treatment strategies targeting sarcomas in advanced stages have been only partly established, which underscores an urgent need for the development of novel therapeutics for sarcomas. In addition, the full contribution of aberrant RBPs to sarcomagenesis has not yet been elucidated. Interestingly, driver fusion genes in sarcomas favor RBPs with LCDs such as EWS/FLI1 and TAF15/NR4A3 . AlthougThere remain several unsolved problems in terms of the pathogenesis of sarcomas and the development of therapeutic avenues for sarcomas with functionally aberrant RBPs. On the other hand, the identification of RBM39 as a dependency molecule in AML, for example, is a promising result as RBM39 is directly targeted by sulfonamides, providing a unique example of translation from a basic RBP screen to the development of pharmacological intervention against malignancies.In summary, this review highlights the functional and pathological roles of RBPs in the initiation and maintenance of sarcomas and other malignant disorders. We are just beginning to understand how aberrant RBPs contribute to tumorigenesis and how we could therapeutically target aberrant RBPs in tumors, indicating the need for further studies.


Background: Neuroendocrine carcinoma of the cervix (NECC) is an aggressive and rare type of cervical cancer. The five-year overall survival is low at 30% and there is no standardized therapy based on controlled trials for this type of tumour. Most are locally advanced or metastasized at the time of the diagnosis. Extracellular vesicles (EVs) could be a carrier of viral DNA/RNA, given their vital role in cellular communication. The content of EV derived from NECC cells has not been investigated due to the lack of cell line, and it is not known whether they contain human papillomaviruses (HPV) DNA/RNA or not. Methods: The presence of viral E7 DNA/RNA in EVs purified from a culture of a recently established NECC cell line, GUMC-395, was evaluated by using droplet digital polymerase chain reaction (ddPCR). These EVs were characterized using nanoparticle tracking analysis (NTA) for size distribution, transmission electron microscopy (TEM) for morphology, Western blot for CD63, and bioanalyser for RNA quantity and quality. Results: HPV16 viral-RNA, but not DNA, was detected in EVs from GUMC-395 using ddPCR. NTA identified EVs with a mean diameter of 105.0 nm, TEM confirmed normal morphological shape and size, and Western blot analysis confirmed the presence of EV-associated proteins CD63. The EVs were found to be enriched with small RNAs using a bioanalyser. Conclusions: HPV16 RNA is found in EVs from a neuroendocrine cervical cancer and could be involved in the pathogenesis of the disease and used as a diagnostic biomarker. High-risk human papillomaviruses (hrHPV) are proven carcinogens in the oropharynx, penis, cervix, vagina, vulva, and anus . Cervica2 at 37 °C [g for 10 min then filtered using 0.22 nm filter. The resulting supernatant was used to isolate EVs using ExoQuick reagent  following the manufacturer’s protocol and resuspended in 50 µL of PBS.GUMC-395 cells (p3) were cultured in a collagen type I-coated flask using F medium supplemented with 10% fetal bovine serum, 0.125 ng/mL epidermal growth factor , 25 ng/mL hydrocortisone , 5 μg/mL insulin (Sigma-Aldrich), 0.1 nM cholera toxin (Sigma-Aldrich), in addition to 10 μM Y-27632 (Sigma-Aldrich). HFK cells (p4) were grown in serum-free medium . All cells were maintained in a humidified incubator with 5% COat 37 °C ,16. Dneaat 37 °C . Primer Using ZetaView PMX-420 Analyser  NTA measurements were performed. The simultaneously built-in lasers, with an excitation wavelength of 405 nm and 640 nm, were used for fluorescence detection, whereas the 488 nm laser was used for all scatter applications. Analysis of videos that were taken at 30 frames per second for concentration and size using the ZetaView software version 8.05.10  with anti-bleach technology.The EV pellet was resuspended in PBS/2% paraformaldehyde, then 2 µL were plated on carbon-coated grids . After allowing for adsorption, the grids were transferred to 1.5% glutaraldehyde for 10 min after being washed many times in PBS. Then, the grids were transferred to a uranyl-oxalate solution for 5 min. They were dried and observed at 80 kV in a transmission electron microscope equipped with CCD camera for image acquisition .Proteins were isolated and Western blot analysis was performed as previously described , with anThe extraction of total RNA from GUMC-395 and EVs using an miRNeasy Kit  was conducted as described previously . The intFor the HPV16 detection assay, the QX200 Droplet Generator  was used to generate the droplets as per the manufacturer’s directions. Reactions were partitioned into a median of approximately 15,000 droplets per well using the QX200 droplet generator. All reactions were performed on a QX200 ddPCR system  in technical duplicates. The HPV16 E7 Tagman assay used was forward primer 5′-(CAAGTGTGACTCTACGCTTCGG)-3′, (reverse primer) 5′-( GTGGCCCATTAACAGGTCTTCCAA)-3′and probe FAM-5′- TGCGTACAAAGCACACACGTAGACATTCGT -3′- MGBNFQ.Two independent assay measurements of total DNA and RNA concentration were made for the HPV E7 assays. Each assay measurement comprised data from four replicates of ddPCR wells for E7 and two replicates for the no-template control.GUMC-395 cells were grown on a collagen-coated flask in the presence of Y-27632 A and werTotal RNA was extracted from GUMC-395 cells and GUMC-395 cells derived from EVs and were analyzed using the RNA 6000 Pico kit and the small RNA kit from Agilent bioanalyser. While there were ribosomal RNAs in GUMC-395 cells  irrespective of the integration status in the cell . FurtherOur study is limited by the limited number of cell lines generated (one cell line); however, rare cancers such as neuroendocrine cervical cancer are very difficult to obtain. Additionally, blood samples are lacking to test whether ddPCR is sensitive enough to detect the viral RNA in biological fluid.Our results confirm the presence of HPV RNA in EV with a potential clinical utility in the form of valid tests for the detection of circulating tumour HPV RNA, therefore providing a tool for diagnosing HPV-neuroendocrine cancer. Specifically, since such cancer shows false negative results on the Pap smear, it can be challenging to diagnose. Droplet digital PCR is an efficient technique for viral RNA quantification and detection and, therefore, can be used for neuroendocrine cervical cancer diagnosis.


Cutaneous malignancies, including basal cell carcinoma, cutaneous squamous cell carcinoma, and cutaneous melanoma, are common human tumors. The incidence of cutaneous malignancies is increasing worldwide, and the leading cause of death is malignant invasion and metastasis. The molecular biology of oncogenes has drawn researchers’ attention because of the potential for targeted therapies. Noncoding RNAs, including microRNAs, long noncoding RNAs, and circular RNAs, have been studied extensively in recent years. This review summarizes the aspects of noncoding RNAs related to the metastasis mechanism of skin malignancies. Continuous research may facilitate the identification of new therapeutic targets and help elucidate the mechanism of tumor metastasis, thus providing new opportunities to improve the survival rate of patients with skin malignancies. BCC andNoncoding RNAs (ncRNAs) include microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) . NcRNAs MiRNAs are essential ncRNAs that regulate protein biosynthesis by modulating transcription . The synLncRNAs are defined as non-protein coding RNAs with transcripts longer than 200 bp , and thein vivo because they do not have 5′ caps or 3′ Poly A tails, which makes them highly resistant to the nucleic acid exonuclease (RNAse)  . Current (RNAse) . The RBPNcRNAs play an essential role in the occurrence and development of skin cancers. This review summarizes the noncoding RNA aspects related to the metastasis mechanism of skin malignancies. The information may help identify new therapeutic targets related to the mechanism of tumor metastasis and provide new opportunities to improve the survival rate and quality of life of patients with skin malignancies..Sand et al.  identifiVarious miRNAs are upregulated in CSCC and function as oncogenes to promote the malignant behavior of CSCC . MiR-365Decreased expression of various miRNAs results in CSCC suppression. MiR-20a is downregulated in CSCC tissues and inhibits cell proliferation and metastasis by targeting LIM domain kinase 1 . DecreasIn summary, miRNAs play an essential role in the development and progression of CMM, promoting or inhibiting malignant progression by regulating cell proliferation, invasion, metastasis, drug resistance, the immune microenvironment, cell cycle progression, and apoptosis. Detailed information is provided in in vitro and in vivo  and ncRNA-associated nanoparticles have been the subject of clinical trials . The miR


Diabetic nephropathy (DN) is an increasing threat to human health. The impact of hyperglycemia or its metabolites, advanced glycation end-products (AGEs), on glomerular endothelial cells (GECs) and their pathophysiologic mechanisms are not well explored. Our results reveal that AGEs increased the expression and secretion of the KIT ligand (KITLG) in GECs. Both AGEs and KITLG promoted endothelial-to-mesenchymal transition (EndoMT) in GECs and further increased the permeability of GECs through the AKT/extracellular-signal-regulated kinase pathway. Inhibition of KITLG’s effects by imatinib prevented AGE-medicated EndoMT in GECs, supporting the belief that KITLG is a critical factor for GEC injury. We found higher KITLG levels in the GECs and urine of db/db mice compared with db/m mice, and urinary KITLG levels were positively correlated with the urinary albumin-to-creatinine ratio (ACR). Furthermore, type 2 diabetic patients had higher urinary KITLG levels than normal individuals, as well as urinary KITLG levels that were positively correlated with urinary ACR and negatively correlated with the estimated glomerular filtration rate. KITLG plays a pathogenic role in GEC injury in DN and might act as a biomarker of DN progression. Approximately 463 million adults have diabetes mellitus (DM) worldwide, and its prevalence has doubled in the past 20 years . DM remaKidney injury caused by DM primarily involves the glomerulus. In the early stage of DN, the histological features include mesangial matrix expansion and glomerular basement membrane thickening . DM-indu−/− mice [The KIT ligand (KITLG), also named the stem cell factor, is expressed in human endothelial cells . KITLG h−/− mice . A recen−/− mice , suggestTo investigate the influence of HG on the permeability of GECs, we assessed the effect of HG and its metabolites, AGEs, on the leakage of GECs. A transendothelial permeability assay was used to examine the permeability of human GECs (HGECs) treated with normal glucose (NG), HG, bovine serum albumin (BSA), or AGE–BSA, and we found that AGEs, not HG, increased the vascular permeability of HGECs A,B. AccoSince KITLG may play a potential role in DN ,29, we iEndoMT has been indicated as one of the major pathophysiologic mechanisms of DN ; thus, wAccording to the positive role of KITLG in AGE-induced glomerular endothelial injury in DN, we used imatinib, as a KITLG inhibitor, to further examine whether the suppression of KITLG rescued the glomerular endothelial injury induced by AGEs. Firstly, imatinib did not change the viability of HGECs A, indicaSince KITLG contributed to glomerular endothelial injury in vitro model of DN, we next examined the expression of KITLG in vivo model of DN. Immunohistochemistry (IHC) staining revealed that the expression of KITLG and N-cadherin at the protein level were increased in the GECs of kidney sections of diabetic db/db mice compared with those of non-diabetic db/m mice A,B. We fWe enrolled 16 normal individuals and 56 type 2 DM patients in our study  and measOur study investigates the signal pathway and novel biomarkers of glomerular endothelium injury in the DN microenvironment. Instead of HG, AGEs increased the expression and secretion of KITLG in HGECs, and KITLG promoted EndoMT in HGECs and further increased the permeability of HGECs through AKT/ERK1/2 activation. Conversely, suppression of KITLG’s effects by imatinib ameliorated AGE-mediated EndoMT and increased the permeability of HGECs. Moreover, elevated levels of urinary KITLG/Cr were noted in vivo model of DN and type 2 DM patients compared with normal groups. Urinary KITLG/Cr levels were positively correlated with the severity of albuminuria as a glomerular injury marker. These findings reveal that KITLG could be an indicator of injury in the glomerular endothelium of DN . This st3K-AKT and Ras-MAPK-ERK [DN is characterized by abnormalities in the function and structure of glomeruli. Glomerular endothelial injury and dysfunction play a central role in the development and progression of DN . PreviouMAPK-ERK . These pMAPK-ERK . AberranMAPK-ERK , non-smaMAPK-ERK , glioblaMAPK-ERK , and thyMAPK-ERK . Our finOne of our novel findings showed that imatinib significantly mitigated AGE-induced EndoMT and further impaired the permeability of HGECs by suppressing KITLG-mediated phosphorylation of AKT and ERK1/2. Imatinib, a tyrosine kinase inhibitor, is used for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors. Interestingly, previous studies have indicated that imatinib can regress type 2 DM  and atteSodium–glucose cotransporter 2 (SGLT2) inhibitors exert a renoprotective effect by halting the progression of DN . MoreoveHGECs  were cultured in Endothelial Cell Medium  plus 5% fetal bovine serum (FBS), according to the manufacturer’s suggestions. Cells were treated with NG (5.5 mM), HG (25 mM), BSA , AGE–BSA , KITLG  or imatinib  for the indicated times.The transendothelial permeability assay was performed using an In Vitro Vascular Permeability Assay kit . HGECs grown to confluence on collagen-coated inserts were exposed to NG, HG, BSA, AGE–BSA, NC, or KITLG with or without imatinib for 48 h. After treatment, FITC-labelled dextran was added to the top of the cell monolayer for 20 min, and then FITC–dextran was measured across the HGECs’ monolayer to the bottom wells by relative fluorescence excitation at 485 nm and emission at 530 nm using a fluorescence plate reader.−ΔΔCt method. The primers  used are listed in Oligo (dT) primer and reverse transcriptase  were utilized to prepare the cDNA after total RNA extraction of cells. The SYBR Green system of QuantStudio3  was used to analyze quantitative RNA. The PCR reaction was carried out with the following temperature profile: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. The relative expression levels of the mRNA in cells were normalized to interval control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expression was assessed using the 2The supernatants of HGECs treated with NG, HG, BSA, or AGE–BSA for 48 h were collected. All urine samples of humans and mice were aliquoted and stored in a −80 °C freezer. The KITLG levels of the supernatants of GECs and the urine of humans and mice were measured using ELISA .https://imagej.net/WelcomeUSA accessed on 15 January 2022).HGECs were lysed in Radio-Immunoprecipitation Assay (RIPA) lysis buffer . Equal amounts of protein were subjected to 9% SDS-PAGE for electrophoresis, followed by transfer onto a polyvinylidene difluoride (PVDF) membrane, which was blocked with 5% blocking buffer and sequentially immunoblotted with each primary antibody overnight at 4 °C. The membrane was washed with tris-buffered saline (TBS)/Tween-20 (0.2%) and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody. The corresponding bands were detected using a chemiluminescent HRP substrate kit . The chemiluminescence signal was captured using Proteinsimple + Fluorchem Q . Antibodies against N-cadherin , vimentin , and E-cadherin  were obtained from BD Biosciences  and those against GAPDH  were obtained from EMD Millipore . Antibodies against phospho-AKT , AKT , phospho-extracellular-signal-regulated kinase1/2 (phosphor-ERK1/2) , and ERK1/2  were obtained from Cell Signaling technology . The densitometry of the bands was analyzed using Image J software  , and db/db mice  were obtained from the National Laboratory Animal Center in Taiwan. Urine and kidney samples were collected at the 12th week of age. The kidneys were fixed in 4% paraformaldehyde for IHC staining. All animal experiments in this study were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by Kaohsiung Medical University and Use Committee.https://imagej.net/WelcomeUSA accessed on 15 January 2022).The kidney tissue sections were fixed in 4% paraformaldehyde for IHC. KITLG antibody  and CD34 antibody, as glomerular endothelial cell markers , were used in IHC. The densitometry of KITLG was analyzed using Image J software  . eGFR was calculated using the equation of the 4-variable Modification of Diet in Renal Disease Study [Fifty-six type 2 DM patients with eGFR ≥30 mL/min/1.73 mse Study . ConcentLevels of urinary albumin, as a marker of glomerular injury , were ast-test or ANOVA, followed by a post hoc test adjusted with a Tukey correction, as appropriate. The associations among continuous variables were examined by Spearman correlation. Statistical analyses were conducted using SPSS version 22.0 for Windows  and Graph Pad Prism 9.2.0 . Statistical significance was set at a two-sided p-value of <0.05.The continuous variables were expressed as the mean ± S.E.M or median , as appropriate. The categorical variables were expressed as percentages. Differences in the distribution of categorical variables were tested using the chi-square test. The significance of differences in continuous variables between the groups was tested using Student’s This study demonstrates the role of KITLG in HGECs within a DN microenvironment, which results in EndoMT in HGECs and enhances the permeability of HGECs. KITLG could be a clinical biomarker of kidney injury in type 2 DM patients, and might represent a potential therapeutic target for DN.


Pepino mosaic virus (PepMV) is a potexvirus of the family Alphaflexiviridae within the order of Tymovirales that threatens tomato production worldwide. PepMV possesses a positive-strand RNA genome with a 5′-methylguanosine cap and a 3′-polyA tail. Previously, using partially-purified viral RNA polymerase important secondary structures within the 3′-untranslated region (UTR) of PepMV RNA were identified. Here we show that an RNA pseudoknot can be formed in the 3′-UTR that includes part of the polyA tail. Using protoplasts, we demonstrate that the pseudoknot is required for replication of PepMV RNA. Mutational analysis and native gel electrophoresis further show that the pseudoknot is stabilized by UAU base triples, as is the human telomerase RNA pseudoknot. The presence of a pseudoknot in several other members of the Alpha- and Betaflexiviridae is supported by covariance analysis and native gel electrophoresis of other potexvirus, capillovirus and trichovirus RNAs. The ubiquitous presence of the pseudoknot in viruses of the Betaflexiviridae, suggests that the pseudoknot is a typical trait of the Betaflexiviridae that may have been adopted by many potexviruses during evolution. Tymovirales are currently subdivided into five families: Alpha-, Beta-, Gamma- and Deltaflexiviridae, and Tymoviridae1. All members possess a single plus-stranded RNA genome of 6–9 kilobases (kb) that encodes an RNA-dependent RNA polymerase (RdRP), a coat protein (CP), and one or more proteins involved in cell-to-cell movement of the virus. RdRP is translated from the genomic RNA, CP is translated from a subgenomic (sg) RNA, and the movement proteins are expressed from a gene that overlaps with the RdRP gene or from additional sgRNAs. Members of the Tymoviridae are distinct in that they form icosahedral-shaped particles and possess a tRNA-like structure (TLS) at the 3′-end of the RNA, which is usually charged with valine. Alpha-, Beta-, Gamma- and Deltaflexiviridae are characterized by flexuous, filamentous-shaped particles that are 470–1000 nm in length and 12–13 nm in diameter. Their genomes lack a TLS but have a 3′-polyA tail, which for Bamboo mosaic potexvirus (BaMV) has been shown to fold into a pseudoknot structure. So far, a pseudoknot structure has not been identified in other potexviruses or any other member of the Alpha-, Beta-, Gamma- and Deltaflexiviridae.Plant viruses belonging to the order Pepino mosaic virus (PepMV) is a mechanically-transmitted potexvirus (Alphaflexiviridae) with an approximately 6.4 kb genome with a 5′-methylguanosine cap and a 3′-polyA tail2. The genome contains five open reading frames, coding for an RdRP, three proteins involved in virus movement, and CP, flanked by 5′- and 3′- untranslated regions (UTRs) of ~ 86 and ~ 64 nucleotides (nts), respectively3. In vivo and in vitro experiments have shown that three 3′-co-terminal sgRNAs are produced that express CP and the other proteins5.8. In the case of the type member Potato virus X (PVX) 3′-UTR, the RNA folds into three stem loops . hp1 is not required for minus-strand synthesis but hp2, which harbours a U-rich loop, is essential for plus– and minus-strand synthesis9. The BaMV 3′-UTR folds into a cloverleaf-like structure of four hairpins, followed by a pseudoknot that incorporates approximately 13 adenosines of the polyA tail7. The PepMV 3′-UTR folds into three stem-loop structures  but an analogous structure had not been detected nor proposed for any related potexviruses. Alignment of potexvirus RNA sequences, however, shows that a stem equivalent to stem S1 of the BaMV pseudoknot can be formed in the majority of potexviruses, including three unclassified potexviruses . For BaMV it was shown that the 3′-proximal Us (highlighted in yellow) can base-pair with 6 As of the polyA tail (highlighted in yellow), thereby forming stem S2. In the potexvirus RNAs shown here, 2–7 Us are available for base-pairing with As from the polyA tail. The conserved 5′-proximal U-rich motif (highlighted in green) can in principle also base-pair with As from the polyA tail but this would lead to an unusual and rare pseudoknot structure wherein the polyA tail has to span both S1 and S2. Since we found that the 5′-proximal Us are involved in base triples (see below) we did not consider this alternative pseudoknot conformation.e.g. PepMV and PVX), and those for which L2 is composed of As from the polyA tail (e.g. BaMV). A few viruses have one (Allium virus X and Strawberry mild yellow edge virus), two (Yam virus X) or three Us (Hydrangea ringspot virus) that separate S1 from the polyA tail. While the role of this additional structure remains unknown and may be host-related, its presence is probably not required for the synthesis of minus- or plus-strand RNA, as was reported for PVX8. Also, replication of PepMV RNA in vitro and in protoplasts is not affected by removal of this hairpin10.Potexvirus pseudoknots can thus be grouped into those that have additional sequences capable of forming a stem-loop structure within the loop L2 of the pseudoknot  led to barely detectable accumulation of gRNA and sgRNAs , whereas translation of the transcript incorporating 6 As was only twofold less than the wt RNA with 25 As  is important for replication in vivo. Disruption of the middle GC bp to a CC mismatch (pep2) greatly reduced replication. , although a loss or change of putative tertiary interactions could also contribute to a lower accumulation. Note that a CG pair as in pep3 is rarely found as the penultimate bp in stem 1 of potexviruses  and of other noncoding RNAs15. In these studies, UAU base triples could be functionally replaced with isosteric CGC triples. To investigate the presence of UAU base triples in PepMV we disrupted three potential UAU triples identified from homology with the human telomerase pseudoknot, by replacing Us with Cs . Replication of pep5 was moderately reduced, while replication of pep7 and pep9 was severely reduced  which corresponds to the PepMV pseudoknot from which hp1 has been removed, potentially three UAU base triples can be formed, whereas in PepB two U-to-C changes disrupt one base triple, and in PepC, in addition to these two U-to-C changes, an A-to-G change in the polyA tail is introduced to create a CGC base triple. Figure  nts whic8. At pH 8 PvxA migrated faster than PvxB which has two U to C changes that disrupt the UAU triple, and PvxC with the CGC triple , and the trichovirus Apple chlorotic leaf spot virus (AclsvA) which has an A at the junction between the stems and 2 nts upstream of the U-stretch , whereas replication was strongly reduced. On the other hand, 9 As were sufficient to obtain wt levels of RNA replication in vivo. This contrasts with the results of previous in vitro experiments using partially purified PepMV replicase that showed no detectable minus strand synthesis with polyA tails of 10 or 15 As5. This discrepancy may be due to the addition of As in vivo by a cellular polymerase, acting in competition with the degradation of templates with short polyA tails. Folding of the 3′- terminus into a pseudoknot with 6, 10 or 15 As may protect against this degradation in vivo and allow further extension of the polyA tail to produce a replicable template.To date, the only member of the 18. Pillai-Nair et al.8 achieved successful infection of protoplasts using PVX transcripts with 17 As that also contained the 4 additional nts. In any case, as 3 As form part of hp1, the remaining 14 As are sufficient to form the proposed pseudoknot for PVX.For BaMV, no replication in vivo was detected with a polyA tail of 10 A or less. Since in BaMV 4 As are necessary for the formation of stem S1 of the pseudoknot, the remaining 6 As are apparently not sufficient to make up stem S2 and loop L2. The minimum length of the polyA tail for replication of another well-studied potexvirus, PVX has not been definitively determined: 8 As were reported to yield very low replication but the transcript also incorporated 4 additional nts derived from a restriction siteMutational analysis and native gel electrophoresis also indicated that the PepMV pseudoknot is stabilized by a number of UAU base triples, as their disruption reduced replication whereas their substitution with isosteric CGC base triples preserved replication. We observed that not all triples are equally important. The triple closest to stem S1 can be disrupted by CAC (pep5) or replaced by CGC (pep6) without much effect on replication. On native gels, among PepMV RNA oligonucleotides lacking hp1, mutation of the triple closest to S1 was sufficient to change the migration and structure of the pseudoknot, and alter the NMR spectrum. Whether hp1 exerts a stabilizing effect on the pseudoknot and possibly mitigates the effect of destabilizing mutations remains to be investigated.19. Other possible reasons for the lower levels of replication are: (i) the PepMV triples are in a different register and so were here inadvertently introduced at the wrong position, thereby forcing the structure into a conformation less favorable for replication. At present, we cannot rule out this possibility, but the fact that constructs with CGC triples replicated better than their corresponding CAC mismatches at least indicates that the PepMV pseudoknot is stabilized by UAU triples. Elucidation of the 3D structure of the PepMV pseudoknot will help to determine the exact nature of these triples and also the extent of the homology with the human telomerase pseudoknot, or with other pseudoknots stabilized by UAU triples20. (ii) The interruption of the polyA tail by one or more G may adversely affect translation leading to decrease in viral protein levels and, indirectly, to less synthesis of gRNA and sgRNAs. Currently, no data exist to support this hypothesis. (iv) The CGC triple may interfere with the formation of an alternate hairpin structure which in the wt is stabilized by UU base pairs, but in the mutants these are replaced by CC or CU pairs. This hairpin structure may be the binding site for a host factor such as that shown by Sriskanda et al. to bind to the U-rich sequence in L1 of PVX9, although it cannot be excluded that this factor recognizes the UAU triples, as mutation of these Us would also disrupt this interaction.CGC triples did not, however, fully restore replication. One reason could be that CGC triples require protonation of one of the cytosines which at neutral pH occurs slowly but can be accelerated by neighbouring UAU triples.8 found U−40, U−41 and U−42, that we predict to be involved in the formation of the UAU base triples, to be largely protected from modification by CMCT. The 4 Us involved in the proposed AU bps in stem S2, however, were more accessible to CMCT but still less so than other Us in hairpin loops in the 3′-UTR of PVX RNA. Interestingly, U−15 to U−17 in loop L1 of the BaMV pseudoknot are protected from RNAse A cleavage but are a substrate for the dsRNA specific RNAse V19 which supports the presence of UAU triples in the BaMV pseudoknot as well. Other data that support the presence of a pseudoknot in PVX come from a study using BAMV-PVX hybrids21 showing that the PVX 3-’UTR can partially substitute for the potex hexamer and pseudoknot domains of BaMV in in vitro minus-strand synthesis. Future research should demonstrate the requirement of the pseudoknot for the replication of PVX.Based on the structural resemblance to the PepMV pseudoknot, and data from native gel electrophoresis, we also propose a 3′-terminal pseudoknot involving the polyA tail for PVX. Although previous structure-probing data on the PVX 3′-UTR did not show clear evidence of the U-rich motifs being involved in base pairing, Pillai-Nair et al22 which we have tentatively assigned here to group 1 Plantago asiatica mosaic virus, Tulip virus X and Cassava common mosaic virus [CasCMV]); group 2 Lily virus X, Phaius virus X and Mint virus X ; group 3 (Clover yellow mosaic virus [ClYMV] and Tamus red mosaic virus [TaRMV]); group 4 (Foxtail mosaic virus [FxMV] and Turtle grass virus X [TGVX]); group 5 . The 3′-ends of group twofold into a so-called pseudotriloop hairpin which functions as a recognition element for Brome mosaic virus replicase23 but is also found at the 3′-ends of some allexiviruses and the botrexvirus Botrytis virus X . Presumably, during evolution these potexviruses exchanged the 3′ pseudoknot with the 3′ stem-loop of plant viruses from a nearby family. Such events have also been documented for other plant viruses25 and avian caliciviruses26.The pseudoknot can be found in 34 classified and three unclassified potexviruses but is absent in 13 classified potexviruses. These 13 viruses cluster together in five subclades based on similarities in their replicase and coat protein genesActinidia virus, Alstroemeria virus X, etc. Group 4 viruses FxMV and TGVX form a hairpin of 6–7 bp with a loop sequence AAUGCACA that shows strong homology with the loop sequence of the pseudoknot of Zygocactus virus X, Cactus virus X, Schlumbergera virus X, Pitaya virus  both form a stem-loop structure with a similar loop sequence that is not found in other viruses . Cassava viruses (group 5) also form a stem-loop structure with loop sequences that, apart from a CAG sequence, have little in common. Interestingly, the loop sequence of CasMV (ACAGUUUA) is very similar to loop L1 of the pseudoknot of other potexviruses like PepMV, rus Fig. . This isMandarivirus, of the Alphaflexiviridae, but not in the genera Allexivirus, Botrexvirus, Lolavirus, Platypuvirus, and Sclerodarnavirus of the Alphaflexiviridae. The only allexivirus sequence that was found to harbour a pseudoknot is the unclassified allexivirus Garlic yellow virus, but this virus is closely related to Allium carlavirus A, an unclassified carlavirus. In the related Betaflexiviridae however, the pseudoknot is widespread: it is found in 11 genera, and is only not present in the genus Vitivirus. The 3′-ends of vitivirus RNAs can be folded into a hairpin that in many cases is stabilized by base pairs involving the polyA tail  similar to allexiviruses. Thus the pseudoknot seems to be a typical trait of the Betaflexiviridae that was somehow adopted by many potexviruses during evolution. It will be of considerable interest to find out whether the pseudoknot also plays an important role in the replication of viruses belonging to the Betaflexiviridae.This type of pseudoknot involving the polyA tail can be found in one other genus, DpnI prior to purification of the generated products by agarose gel electrophoresis and used as templates for in vitro transcription using the mMESSAGE mMACHINE T7 RNA kit (Ambion). The 5′-methylguanosine capped RNA transcripts produced were purified as described in the Ambion manual, and assayed by spectrophotometer before use in downstream experiments.DNA fragments representing differently-sized sequences of the PepMV plus-strand genome were amplified from the pTOPO-T7 PepXL6 DNA template. All PCRs were carried out using LA Taq DNA polymerase (TAKARA), the template DNA was restricted with 5 N. benthamiana mesophyll protoplasts in the presence of PEG 4000, before incubation for 24 h at 25 °C under constant light. Protoplasts were harvested and total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. For subsequent northern blot analysis, 5 µg of each total RNA was resolved, transferred to charged nylon membranes and probed with a Dig-labelled (-) single stranded RNA probe corresponding to the PepMV CP gene coding sequence.Full length PepMV cDNA templates were amplified using a T7pep forward oligonucleotide primer and one of a set of 3′-co-terminal reverse oligonucleotide primers  and luciferase activity in 50 µl samples was measured using a GloMax multi system (Promega).The RLuc-PepMV-3UTR plasmid was obtained by insertion of a DNA fragment covering the PepMV 3′UTR  into a 2. Gels were run in TAEM buffer at ~ 85 V and 12 mA for ~ 4 h in a cold room. For acidic PAGE the pH was adjusted by the addition of acetic acid to all buffers. RNA was visualized using EtBr or Stains-All (Sigma Aldrich).RNA oligonucleotides were purchased from Merck (Sigma-Aldrich) at 0.05 nmole scale/desalted. 100–200 pmol of each RNA were loaded onto polyacrylamide gels containing 12% or 16% acrylamide:bisacrylamide (19:1), Tris (40 mM), acetate (20 mM), EDTA (1 mM) pH 8.3, with 1.5 mM MgAc1H NMR experiments were recorded at 278° Kelvin on a Bruker 600 MHz spectrometer equipped with a cryoprobe using Watergate suppression. RNA oligonucleotides were dissolved in 10 mM Na2HPO4/NaH2PO4 buffer (pH 6.7), 0.5 mM MgSO4 and 10% D2O to a final concentration of ~ 0.25 mM.Supplementary Information.


Mutations in Dbp6 core motifs involved in ATP binding and ATP hydrolysis are lethal and impair Dbp6 ATPase activity but increase its RNA binding and RNA annealing activities. These data suggest that correct regulation of these activities is important for Dbp6 function in vivo. Using in vivo cross-linking (CRAC) experiments, we show that Dbp6 interacts with 25S rRNA sequences located in the 5′ domain I and in the peptidyl transferase center (PTC), and also crosslinks to snoRNAs hybridizing to the immature PTC. We propose that the ATPase and RNA clamping/annealing activities of Dbp6 modulate interactions of snoRNAs with the immature PTC and/or contribute directly to the folding of this region.Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA–RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle. We demonstrate that  Saccharomyces cerevisiae, the vast majority of RNA helicases acting on pre-ribosomal particles are essential for growth  consisting of a small and a large subunit, respectively composed of the 18S ribosomal RNA (rRNA) and the 5S, 5.8S and 25S/28S  rRNAs associated with ribosomal proteins (RPs). Ribosomes are ribozymes as their catalytic activity is mediated by specific rRNA domains. For these domains to be functional, the rRNAs must be correctly folded and compacted. One of the key aspects of ribosome biogenesis is, therefore, to convert the nascent rRNA precursor into highly organized and precisely folded mature rRNA molecules thus generating a functional architecture competent for translation. In yeast, production of the two ribosomal subunits is the result of successive maturation steps of a long nascent precursor transcript (35S pre-rRNA), encompassing the sequences of the 18S, 5.8S and 25S rRNAs . Maturatr growth ,8. They r growth . It is ain vitro  as well as snoRNAs that guide nucleotide modifications in the PTC region. Taken together, we propose that Dbp6 ATPase and RNA annealing activities contribute to remodeling events in the PTC region during the early stages of pre-60S particle assembly.The early steps of large ribosomal subunit synthesis involve numerous RNA helicases. Indeed, no less than seven RNA helicases, namely Mak5, Has1, Prp43, Dbp3, Dbp7, Dbp9 and Dbp6 are required for production of the earliest pre-60S particles ,29–32. TDBP6 ORF PCR cassette downstream of the polymerase T7 promoter and a His tag (His6) by the Ligation Independent Cloning (LIC) method using oligonucleotides listed in DBP6 mutants were generated by reverse PCR with In-Fusion® HD Cloning Plus (Takara/Clontech) using primers listed in Escherichia coli Stellar cells (Takara), and plasmids from clones into Rosetta λDE3 strain to produce pET-MII/MI/MIa/MIc or ΔN plasmids.pET-Dbp6 was obtained by cloning into a modified pET-15b (Novagen) plasmid in the NdeI and BamHI restriction sites a GAL1-10 promoter has been transformed with plasmids pHA113/WT/MII/MI/MIa/MIc or ΔN. These plasmids were obtained by cloning into the pHA113 plasmid  has been obtained by transforming the GAL::HA-DBP6 strain with a plasmid derived from pRS415  supplemented with either 2% galactose or 2% glucose or YNB (1.7 g/l) supplemented with ammonium sulphate (5 g/l) and 2% galactose or 2% glucose.in vitro assays) and S3 (northern blot probes). When necessary, the oligonucleotides were radiolabeled using T4 PNK enzyme (Promega) and 25 μCi [γ-32P]-ATP (PerkinElmer) per probe.RNA/DNA oligonucleotides are listed in E. coli Rosetta λDE3 bacteria transformed with plasmids pET-Dbp6/MII/MI/MIa/MIc or ΔN, were grown at 37°C in 2× YT medium supplemented with 100 μg/ml ampicillin and 25 μg/ml chloramphenicol to an OD600nm ∼0.5. Isopropyl-β-d-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.5 mM and culturing was continued overnight (∼15 h) at 18°C. The cells were harvested by centrifugation, washed with 1× PBS and then dry frozen at –80°C. Bacteria were thawed, resuspended in lysis buffer , 0.5 mM phenylmethylsulfonyl fluoride (PMSF) supplemented with EDTA-free protease inhibitors (Roche)) and lysozyme was added to a final concentration of 150 μg/ml. The cell suspension was sonicated on ice and then subjected to affinity chromatography using a HisTrap FF column . His-tagged proteins were eluted with a linear gradient of imidazole. The eluted fractions containing His-tagged proteins were pooled and the proteins of interest were purified by size exclusion chromatography with a Superdex 200 gel filtration column  in GF-ATPase buffer . The integrity of purified His-tagged proteins was checked by PAGE followed by Coomassie blue staining and proteins were stored at –80°C. The identity of the purified polypeptides (WT and mutant) of the size expected for Dbp6 was further checked by mass spectrometry. In vitro assays were repeated with different batches of purified proteins.Immunoprecipitation experiments were performed as described before . Protein32P-labeled oligonucleotide probes using Rapid-hyb buffer . Radioactive membranes were exposed to Phosphorimaging screens and revealed using a Typhoon Tryo . The signals corresponding to the pre-rRNAs were quantified using the ImageQuant software (Molecular Dynamics). Sequences of the oligonucleotides used as probes in this study are listed in Yeast total RNAs were extracted as described . In all Proteins were separated by electrophoresis on 8% or 10% 29:1 polyacrylamide gels in 1× TGS buffer  and transferred onto nitrocellulose membranes using the Bio-Rad TransBlot system. Membranes were saturated by 30 min incubation in TBS-T buffer , containing 5% (w/v) powder milk. Specific proteins were detected by incubation in TBS-T with the following antibodies: anti-HA-HRP  diluted 1000-fold; anti-Dbp6 polyclonal antibodies diluted 10 000-fold ; anti-PG2, 10 mM KCl, 0.02 mM DTT, 10 mg/ml BSA) for 15 min at 20°C, purified recombinant Dbp6 (1.5 or 0.8 μM) was placed in drops on an open petri dish and cross-linked by exposure at 1 cm to a UV312 nm lamp (UVItec Limited) for 6 min, on ice. Samples were then diluted twice in H2O and proteins were separated by SDS-PAGE on 6% (37.5:1) gels. Proteins were analyzed either by western blot or silver staining .Following incubation in 5 μl of RB 1× reaction buffer  and cold ATP  were added to the reaction.ATP cross-linking experiments were performed following the same procedure as described above (‘protein-protein cross-linking’), except that 0.2 μM [α-32P]-ATP (PerkinElmer) with or without 2 μM of ss58-mer oligonucleotide RNA. The reaction mix was incubated at 30°C and 1 μl aliquots taken from the mix at the appropriate time points were loaded on a Polygram CEL 300PEI/UV254 (Macherey-Nagel™) chromatography plate. Chromatography was performed in 0.75 M KPO4 buffer. The radioactive plate was dried and signals corresponding to [α-32P]-ATP and its hydrolysis product [α-32P]-ADP were revealed by Phosphorimager  and quantified using the ImageQuant software (Molecular Dynamics).Dbp6 proteins (0.4–1 μM) were incubated in a reaction mix containing RB 1× (see above), 100 μM cold ATP and 0.6 μCi/μl  under the accession code GSE200692. Other data supporting the findings of this study are available from the corresponding authors upon reasonable request.The CRAC datasets and their analyses for Dbp6-HTP and the wild-type yeast control are deposited in Gene Expression Omnibus (GEO) database [gkac1196_Supplemental_FileClick here for additional data file.


Among these inhibitors, we discovered a new mRNA metabolic response in which the addition of antimycin A, an inhibitor of mitochondrial respiratory-chain complex III (complex III), resulted in a marked accumulation of poly(A)+ RNA near the nuclear speckles. Furthermore, dihydroorotate dehydrogenase (DHODH) inhibitors, a rate-limiting enzyme in the intracellular de novo pyrimidine synthesis reaction that specifically exchanges electrons with complex III, also caused a remarkable accumulation of nuclear poly(A)+ RNA adjacent to the nuclear speckles, which was abolished by extracellular uridine supply, indicating that the depletion of intracellular pyrimidine affects poly(A)+ RNA metabolism. Further analysis revealed that ataxia telangiectasia mutated (ATM), a serine and threonine kinase and a master regulator of DNA double-strand break (DSB) and nucleolar stress, is required for this poly(A)+ RNA nuclear accumulation phenomenon. This study reports new insights into novel aspects of nuclear poly(A)+ RNA metabolism, especially the relationship between mitochondrial respiratory-chain functions, pyrimidine metabolism, and nuclear RNA metabolism.Intracellular and intercellular signalling networks play an essential role in optimizing cellular homoeostasis and are thought to be partly reflected in nuclear mRNA dynamics. However, the regulation of nuclear mRNA dynamics by intracellular and intercellular signals remains largely unexplored, and research tools are lacking. Through an original screening based on the mRNA metabolic mechanism, we discovered that eight well-known inhibitors cause significant nuclear poly(A) In eukaryotes, mRNA is first transcribed as a precursor mRNA and undergoes several processings, such as 5′ capping, splicing, and 3′ polyadenylation. These processes are mediated by several ribonucleoproteins and are tightly regulated to ensure that correctly matured mRNAs are facilitated to be exported from the nucleus. However, many aspects of this sophisticated regulation of mRNA processing remain unelucidated . RecentlVarious external signals are altered by specific molecules in the cell and transmitted into the cell, called the signal transduction pathway, and influences the transmission of information in and out of the cell, optimizing cellular responses and regulating cell proliferation and differentiation . Some of+ RNA metabolism  . Additionally, antimycin A increased the nucleolar localization of coilin, suggesting the occurrence of nucleolar stress . Th. Th+ RNAreatment , indicat+ RNA accumulation is induced in response to a decrease in intracellular pyrimidine concentration. Therefore, we investigated whether the nuclear accumulation of poly(A)+ RNAs can be observed by inhibiting purine synthesis. To investigate this, we added methotrexate, an inhibitor of folate metabolism and a typical inducer of purine synthesis inhibition , and 1-mM 4-(trifluoromethyl) benzamidoxime (4-TFMBAO) and heated at 80°C for 4 min. The reaction was stopped by cooling on ice, and the fluorescence intensity was measured using SYNERGY H1MF ; excitation and emission wavelengths were 340 and 460 nm, respectively.DHODH activity was measured using a modified protocol according to Yin et al. . BrieflyClick here for additional data file.


Seminal vesicles play an important role in the male reproductive system, producing seminal fluid and thus adequate environment for sperm. However, mechanisms underlying secretory functions of the seminal vesicles’ epithelium have not been defined yet. The aim of the present study was to characterize expression and immunolocalization of selected membrane transporters and carriers in the seminal vesicles. The study included biopsy specimens collected from non-affected parts of seminal vesicles from 53 patients of Caucasian origin subjected for prostatectomy. RT-PCR was used to define expression of 15 genes coding for ABC-family and 37 genes encoding 37 SLC-family transporters/carriers. Immunohistochemistry was used to define localization of 6 transporters. In the seminal vesicles, the following membrane transporters and carriers were defined: ABCA1, ABCB1, ABCB5, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCG2, SLC01C1, SLC02B1, SLC04A1, SLC04C1, SLC10A1, SLC15A1, SLC15A2, SLC16A1, SLC16A3, SLC19A1, SLC22A1, SLC22A3, SLC22A11, SLC22A18, SLC22A4, SLC22A5, SLC28A1, SLC2A9, SLC33A1, SLC47A1, SLC47A2, SLC51A, SLC51B, SLC7A5, SLC7A6. Age-dependent expression was evidenced for ABCB1, ABCG2, SLC04C1, SLC15A1, SLC16A1, SLC22A11, SLC22A18, SLC47A1 and SLC47A2. ABCG2, P-gp, MRP1, MRP3, MCT1 and LAT1 were localized in the apical membrane and P-gp in the basolateral membrane of the seminal vesicle epithelium. The expression of the membrane transporters and carriers in the seminal vesicle epithelium confirms its secretory and barrier functions. Seminal vesicles constitute an integral part of the male reproductive system, producing seminal fluid and thus adequate environment for sperm. The fluid is responsible for nutrition, normal sperm cells motility, semen liquidity, stabilization of sperm cells chromatin, as well as immune modulation of the female reproductive system. All of the processes are detrimental for preservation of male fertility, and dysfunction of the seminal vesicles is associated with male infertility ,2,3.The epithelium of the seminal vesicles not only forms a mechanical barrier protecting sperm but also provides active defense mechanisms (transporters and carriers) as well as produces seminal fluid. In the latter ions and electrolytes , low molecular weight substances , structural and transport proteins , hormones , enzymes  and enzyme protein inhibitors (sperm motility inhibitor) can be found ,5. NormaABCB1), multidrug-resistance-associated proteins (MRPs/ABCCs), and breast-cancer-resistance protein (BCRP/ABCG2). These transporters are considered as key regulators of intracellular accumulation of lipids, sterols, drugs and toxins. More than 400 transport proteins of SLC superfamily have been identified and are allocated to over 60 families. The classification of SLC transporter superfamily is based exclusively on biological function and not on sequence homology. Members of the same SLC transporter family demonstrate an at least 20% protein sequence identity, but share similar substrates. The SLC family mainly includes organic anion-transporting polypeptides (OATPs/SLCOs), organic anion transporters (OATs/SLC22As), organic cation transporters (OCTs/SLC22As), organic cation and carnitine transporters (OCTNs/SLC22As), peptide transporters (PEPTs/SLC15As) and multidrug and toxin extrusions (MATEs/SLC47As). Most of SLC transporters function as influx carriers, mediating movement of solutes, either by passive diffusion along concentration gradient, by cotransport (acting as facilitated transporters), or counter-transport against concentration gradient using the concentration gradient created by another solute (acting as secondary active transporters). A class of MATEs transporters provide efflux activity. SLC proteins transport a wide array of molecules, including carbohydrates, amino acids, vitamins, nucleotides, metals, inorganic ions, organic anions, oligopeptides and drugs  software. Shapiro–Wilk normality test was used to examine the distribution of data. Statistical significance of differences in mRNA expression and age were determined by either with Mann–Whitney U test or Student T-test and data are presented as median [interquartile range] or mean ± SD depending on distribution. For age-dependent differences in the expression levels, 53 patients were divided into two subgroups: group 1: <60 years old (n = 32), group 2: ≥60 years old (n = 21). Percent contribution of the analyzed transporters was calculated using 2−ΔCT values. Statistically significant differences  were assumed at p < 0.05.mRNA expression data are presented as mean ± standard deviation (SD). Statistical analysis was performed using STATISTICA PL, ver. 13.1 [StatSoft, Inc., Tulsa, OK, USA, 2016, STATISTICA-data analysis software system, version 13.1, ABCA1, ABCB1, ABCB5, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCG2 and for SLC superfamily: SLC01C1, SLC02B1, SLC04A1, SLC04C1, SLC10A1, SLC15A1, SLC15A2, SLC16A1, SLC16A3, SLC19A1, SLC22A1, SLC22A3, SLC22A11, SLC22A18, SLC22A4, SLC22A5, SLC28A1, SLC2A9, SLC33A1, SLC47A1, SLC47A2, SLC51A, SLC51B, SLC7A5, SLC7A6 , for ABC transporter superfamily: , SLC7A6 .−ΔCT values, sum of all analyzed transporters): ABCC3 (14.76%), SLC2A9 (12.65%), SLC7A5 (11.81%), ABCG2 (11.18%), ABCC1 (6.30%), ABCB6 (4.80%), ABCC5 (4.57%), SLC15A2 (4.50%), SLC33A1 (4.43%), SLC7A6 (3.25%), SLC16A1 (2.98%), SLC22A5 (2.77%), ABCB1 (2.45%), ABCA1 (1.89%), SLC04C1 (1.71%), ABCC4 (1.64%), SLC04A1 (1.47%), SLC16A3 (1.40%), SLC19A1 (1.15%), SLC02B1 (0.96%), SLC22A3 (0.81%), SLC22A18 (0.62%), SLC47A1 (0.54%), SLC22A4 (0.41%), ABCC6 (0.28%), SLC15A1 (0.16%), SLC22A1 (0.11%), SLC51B, ABCB5 (0.10%), ABCC2 (0.07%), SLC22A11, SLC47A2, SLC51A (0.03%), SLC01C1, SLC28A1, SLC10A1 (0.01%).The percent contributions of the analyzed transporters in the seminal vesicles were defined as follows  and over 60 (67.8 ± 4.9) years of age, were evidenced for  SLC47A2 . All othABCG2), MDR1/P-gp (ABCB1), MRP1 (ABCC1), MRP3 (ABCC3), MCT1 (SLC16A1) and LAT1 (SLC7A1) in human seminal vesicle cells. The staining revealed luminal localization of ABCG2, MDR1/P-gp, MRP1, MRP3, MCT1 and LAT1. The basolateral immunolocalization was defined for MDR1/P-gp  revealed the presence of the transporter proteins of ABCG2 , membrane transporters also contribute to the mechanical barrier of tight junctions complementing the structure by active barrier functions .ABCA1, ABCB1, ABCB5, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC6, ABCG2—and 25 SLC carriers and transporters—SLC01C1, SLC02B1, SLC04A1, SLC04C1, SLC10A1, SLC15A1, SLC15A2, SLC16A1, SLC16A3, SLC19A1, SLC22A1, SLC22A3, SLC22A11, SLC22A18, SLC22A4, SLC22A5, SLC28A1, SLC2A9, SLC33A1, SLC47A1, SLC47A2, SLC51A, SLC51B, SLC7A5, SLC7A6. The most abundant transporters and carriers were ABCC3, SLC2A9, SLC7A5, ABCG2, ABCC1, ABCB6, ABCC5, SLC15A2, SLC33A1, SLC7A6 and SLC16A1. The antibody immunolocalization evidenced luminal localization of ABCG2, MDR1/P-gp, MRP1, MRP3, MCT1 and LAT1 as well as the basolateral immunolocalization of MDR1/P-gp in the seminal vesicles’ epithelia.The present study, for the first time, demonstrated relevant mRNA expression of 11 ABC transporters—ABCC3). MRP3 belongs to the subfamily of multiple drug resistance (MDR) proteins, but its exact biological characteristics is not entirely defined. This transporter has been shown to play a significant role in cell resistance to anticancer drugs  [Among the transporters examined, the highest level of expression was demonstrated for the gene encoding MRP3 (oposide) . In hepaoposide) . The roloposide)  and in doposide) .ABCC1) is a transporter highly expressed in the epithelial cells of seminal vesicles, which protein immunolocalization in the apical membrane was evidenced. As with MRP3, it belongs to MDR proteins, and was found to translocate neutral or positively charged, hydrophobic and amphipathic compounds  as well as organic anions . MRP1 has perhaps the widest variety of substrates of all the human ABC transporters [MRP1 (sporters . The trasporters . Moreovesporters . The rolABCG2) is the next ABC transporter localized in the apical membrane of the epithelial cells in seminal vesicles. Initially discovered as drug resistance transporter in cancer cells, breast cancer resistance protein (BCRP) is now defined in many cell types and tissues, especially those providing barrier functions  [BCRP (y gland) . The tray gland) . Howevery gland) . In the y gland) .P-gp, originally related to multiple drug resistance 1 (MDR1), was further defined as a membrane transporter expressed in many tissues and organs, playing a mainly protective role. In the male reproductive system, P-gp is one of the key components of the blood-testis barrier, where it operates in endothelial cells, and thus limits xeno-and endobiotics penetration. Sertoli and Leydig cells are also rich in P-gp . It has SLC2A9 gene coding for glucose transporter 9 (GLUT9), was the highest (of the studied) expressed SLC transporters in the seminal vesicles. However, due to unavailability of the reliable antibodies, its immunolocalization and protein abundance were not studied. However, its high expression suggests that, as in other tissues and organs, it is mainly expressed in the kidneys, liver, and intestines, mediated urate transport [ransport . A studyransport . GLUT9 iransport . Apart fransport . Fructosransport . Determiransport .SLC7A5) is another ubiquitously expressed transporter, which carries a wide range of neutral amino acids, especially with large branched or aromatic side chains . Experimental studies defined LAT1 expression in the male reproductive system, where the carrier was localized in the epididymis [LAT1 (ididymis . In the ididymis .SLC16A1) were evidenced in the seminal vesicles. The transporter is ubiquitously expressed, and it takes lactate, pyruvate, butyrate, acetoacetate and β-hydroxybutyrate as predominant substrates [In the present study, the expression and immunolocalization of MCT1  and influx carriers (LAT1 and MCT-1). Based on the current study observations, as well as the literature data, a hypothetical distribution of membrane transporters and carriers in the seminal vesicles can be outlined  of ithelium . The aboithelium .The present study for the first time provides a broad expression characteristics of membrane transporters and carriers in human seminal vesicles. The information presented may enable better understanding of pathophysiological processes in the organ. Active and passive transport across the membranes can regulate seminal fluid composition , which in turn can affect sperm quality and male fertility potential. From other organ studies it is known that various factors modulate transporter expression and function, and likewise in seminal vesicles, an inflammation process, drugs, toxins can affect transporters and carriers. Interactions between membrane transporters and microbiome is one of the emerging areas of investigation. Observations from the gastrointestinal tract suggest that microbiome products, e.g., short-chain fatty acids, can interact with enterocyte transport functions, and after absorption in remote organs (the blood–brain barrier or kidneys). Experimental findings provide evidence that diet or antibiotics can modify seminal fluid microbiome, and thus the microbiome can be another mediator of interactions between environmental factors and membrane transporter functions ,37.The results of the present study have some limitations, e.g., the analysis of samples from prostate cancer subjects, which might affect the picture of the examined transporters in seminal vesicles. Likewise, relatively elderly samples were analyzed, and the panel of membrane transporters and carriers was assessed only in subjects over 50 years of age, which may vary from younger individuals. Therefore, the results from the present study should be analyzed with some restrictions, and more studies are needed to define the role of membrane transport in physiology and pathological states of human seminal vesicles.The study provides, for the first time, information about the expression of membrane transporters and carriers in the seminal vesicles. The localization of transporters can in part explain the contribution of the seminal vesicles to the composition of seminal plasma, and confirm the secretory ability of the epithelium. Expression of the ATP-binding cassette transporters, which are expressed not only in secretory epithelia, but also in many barrier organs , suggests that the seminal vesicle epithelium may also provide protective role, limiting penetration of toxic molecules to seminal plasma.