This study presents a novel methodology for studying RNA-containing samples using non-covalent nucleic acid-sensitive dyes in MST. This “mix-and-measure” protocol uses non-covalent dyes, such as those from the Syto or Sybr series, which lead to the statistical binding of one fluorophore per RNA oligo showing key advantages over traditional covalent labelling approaches. This new approach has been successfully used to study the binding of ligands to RNA molecules ( Time to shine a light on RNA: this protocol presents an innovative technique to study RNA using nucleic acid-sensitive dyes in MST. Traditional MST methods for RNA studies rely on reporter dyes such as Cy5 or fluorescein, which are synthetically attached to the 3′- resp. 5′-ends, or to modified nucleosides.6,8–11 While this is effective to prevent steric interaction of the fluorophore with the investigated biomolecular interface, it might also reduce the effect of local structural changes (T-jump effects) on the dyes' fluorescence.12,13Understanding the molecular foundations of RNA biomolecular interactions is crucial for the study of their cellular involvement and the development of RNA-targeting therapeutics.14 It is important to note that most of these dyes do not significantly alter overall RNA stability which is a prerequisite for the study of biomolecular interactions.15 To our knowledge, there is only one published study which has used non-covalent SybrGold labelling of DNA oligos using a non-microscale thermophoresis setup (microscope detection) to show that Sybr-DNA duplexes do not dissociate under common thermophoretic conditions.16 In this previous study, however, no relation was made to applications in the recently popular MST experimental set-up nor to method development for nucleic acid analysis.In this study, we present a novel MST-based methodology for the investigation of RNA-containing samples using nucleic acid-sensitive dyes, such as those from the Syto or Sybr series, which statistically bind to nucleic acids through non-covalent interaction.17This led us to hypothesize that such nucleic acid dyes are also suitable for RNA labelling in MST experiments, and hence, we elaborated on the experimental scope, structure–activity relationships and practical applications for RNA–ligand and RNA-modification elucidation. Here, we report a “mix-and-measure” protocol that shows key advantages for relevant RNA-MST applications compared to covalent labelling approaches which are technically more challenging, time-consuming, and expensive . This apS-adenosylmethionine (SAM)-I riboswitch was determined  and SybrGold (1×–10×) concentrations, the stoichiometry and apparent binding constant of the dye to the termined .KD = 85.3 nM). Statistical 1 : 1 binding stoichiometry was concluded as a one-step binding event was observed to display the same apparent binding affinity independent of the RNA concentration. Hence, it was found that the minimum RNA concentration during MST was primarily limited by the binding affinity of SybrGold to the respective RNA sequence. For the SAM-I riboswitch, a significant thermophoretic response was saturated at an RNA concentration of approx. 100 nM, which was strongly distinguished from the unbound dye. Fnorm (2 s): 0.75 vs. 0.96 (It was found that 1× SybrGold produced sufficient fluorescence for MST (Monolith NT.115), and at such SybrGold concentrations, one fluorophore molecule is likely to bind statistically to the RNA of interest . For this purpose, we defined ten RNA categories containing each three RNA oligos which comprise different lengths and structural features (Table S1Fnorm (2 s) was mostly between 0.75 and 0.90 across all combinations  were eventually found for shorter RNA, however, a significant dependency on the dye or the category was not ascertained.As a next step, systematic investigations of structure–activity-relationships between fluorescent dyes and RNA in MST were performed. First, we investigated the effect of RNA length and its secondary structures on the MST characteristics, such as the initial fluorescence level and  Table S1. Across  Table S1. In gene Table S1. The ceninations . Lower FFnorm, suggesting that these are more susceptible to biological and chemical modifications  was mostly <5‰ across all categories. An exception was the use of the dyes Syto 62 and RiboGreen. The data points in the dashed box of We found that shorter RNAs (<20 nt) exhibit more sensitive fluctuations in ications . This caFnorm, more detailed correlation studies with RNA descriptors  were performed. The secondary structures of RNA oligos were predicted by the RNAfold web server.18 By analyzing the relationship between Fnorm and RNA length, we confirmed the trend shown in Fnorm  were generally lower than that of the red Syto dyes. High standard deviations (>5‰) were found only for Syto 62 and RiboGreen. As further correlation parameters, the proportion of hybridized bases  slope of thermophoresis traces (measure of the RNA–dye complex dissociation in the temperature gradient), (3) goodness (R2) of the hyperbolic thermophoresis fit, (4) sum of the least squares error of a fluorescence–time curve triplicate (reproducibility of the whole MST experiment), (5) normalized initial fluorescence (measure for the brightness of the intrinsic dye fluorescence).Another aspect of structure–activity relationships was the analysis of dye-dependent parameters on characteristics of thermophoresis curves described by the following descriptors : (1) sloThese results showed that Syto 60, 62, 63, and 64 exhibited increased susceptibility to photobleaching, with Syto 62 showing a particularly strong effect . In contTo summarize, we found that Syto 59  and SybrGold  are the most suitable (of the investigated) dyes for RNA-MST experiments, as they showed the best fluorescence benchmarks and highest reproducibility rates.1,19,20 Currently, the standard repertoire of bioanalytical techniques includes techniques such as nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC).21,22 However, in recent years, MST has probably become one of the most popular techniques for the characterization of RNA-small molecule interactions using covalently dye-labelled RNA targets.6,8,10 Hence, we aimed to demonstrate four examples where the use of SybrGold and Syto 59  RS, with their natural ligands SAM, S-adenosylhomocysteine (SAH), and preQ1, respectively.23–25 For the SAM-I RS, our MST data yielded a KD = 157 nM for SAM and KD = 137 μM for SAH agreeing with previously reported values obtained by ITC measurements.26 For the SAM-VI RS, our MST results for SAM (KD = 953 nM) and SAH (KD = 10 μM) were also consistent with our covalently Cy5-labelled MST experiments  aptamer domains: the s Fig. S5 and lite6,8 For the existing protocols using a covalently labelled RNA target, however, this mostly represents the same technical limitation.While this value is in the same order of magnitude as our previously reported Cy5-labelled RNA affinity Fig. S5, it shouT. petrophila fluoride anion RS, and a synthetic neomycin aptamer.29–31 The AdoCbl RS naturally binds different variants of the cobalamin co-factor family, which are exceptionally large ligands with a molecular weight of >1000 Da. Our MST experiments using an env8 mutant AdoCbl RS with Syto 59 labelling determined a KD value of 2.1 μM with adenosylcobalamin (AdoCbl) as a ligand, which is in agreement with the literature.29 Thus, the non-covalent RNA-MST method is also suitable for non-typically large ligands and their riboswitches.As a next step, we investigated three additional RS aptamer domains against chemically more diverse chemotypes of ligands: a marine metagenome AdoCbl RS, the T. petrophila fluoride anion RS showed that this protocol is also useful for such applications if ligand binding results in a substantial conformational change of the aptamer. Hence, non-covalent RNA-MST experiments with Syto 59 (KD = 67.1 μM) are in the same order as dissociation constants determined by ITC experiments in the literature.30Next, we studied the opposite case; a riboswitch that is able to bind very small ligands. Aptamers against the smallest known ligands are fluoride anion aptamers. The study of the KD,app of 29.6 nM.31As a final example, we examined a neomycin-binding aptamer of synthetic origin. The apparent binding affinity of neomycin to the 27-mer RNA oligo could be determined by in situ labelled RNA samples with their natural ligands mostly does not lead to a change in the fluorescence intensity by displacement of the dye with the ligand , however, we quantified a slight effect on the initial fluorescence at higher ligand concentrations which supports our model of statistical labelling, since for such a short RNA receptor there are fewer opportunities for dye binding that do not overlap with the ligand binding site  similar to a value determined by fluorescence polarization.33Furthermore, we also investigated a macromolecular interaction of RNA with an endogenous protein–ligand. The tRNA34 Currently, the gold standard repertoire of bioanalytical techniques includes mass spectrometric, radiometric, and sequencing methods.35 Due to the simple and fast preparation of an MST experiment, the development of an MST protocol for the quantification of RNA modifications might offer practical advantages. Thus, the two most promising fluorescent dyes (SybrGold & Syto 59) were harnessed to test for the detection of specific RNA nucleoside modifications.The development of methods for the detection and quantification of RNA modifications such as nucleobase methylation is a recent topic of research due to their physiological relevance and disease involvement.Fnorm parameters in dependence on specific RNA modifications. In summary, these model experiments showed that larger or charge-altering RNA modifications  have a more pronounced effect on Fnorm than single nucleoside methylations , which often resulted in scarcely detectable Fnorm shifts , and threonylcarbamoyladenosylation (t6A) on the short non-structured model oligonucleotide AUUAACCUUAA as a fragment of the hmt-tRNALys was investigated.36In the first step, the influence of different physiologically relevant modifications such as pseudouridylation (ψ), 5-uridine fluorination (5FU), 5-taurinomethyl-2-thiourididylation  methylation of the METTL3/14 substrate oligo AACUUAAUGUUUGCAUUGG(m6A)CUUGAGUUA.To derive a relevant biotechnological application from this model concept, an MST-based enzyme assay was developed for the RNA methyltransferase METTL3/14 allowing the tracking of the enzymatic insertion of a single methyl group to an RNA substrate. METTL3/14, which has recently become popular as a potential drug target, is an RNA methyltransferase that transfers a methyl group from SAM to the Nvia fluorescence reporter exists.38,39 Therefore, we sought to determine the intrinsic difference of the substrate's m6A modification in thermophoresis behaviour. For this purpose, we simulated different m6A methylation levels from 0–100% by mixing the synthetic substrate oligo with its methylated analogue at a total RNA concentration of 100 nM , so discrimination was not sufficient for the evaluation of enzymatic turnover. Therefore, we harnessed an antibody-coupled approach to amplify the Fnorm discrimination signal. The addition of an m6A-specific antibody  resulted in a 10-fold dispersion  of the RNA oligos' Fnorm  and mounted in MST capillaries as a crude reaction mixture without further purification of the RNA substrate  allowed the determination of an apparent KM value for SAM of 226 nM which is in agreement with a 3H-based enzyme assay , METTL3/14 enzyme (10 nM), and a variable concentration of the cofactor SAM (0–1000 nM) were incubated for 2 h at room temperature. Subsequently, the reaction was added with the anti-mubstrate . The rescofactor , which c content  and indi content . The ini Fig. S7A, confirm Fig. S7B and lite50 of 17.2 nM in agreement with the literature-determined value.44To demonstrate the usefulness of the developed METTL3/14 assay for drug discovery applications, we also performed an inhibition experiment with the known literature inhibitor STM2457 , which rin situ labelled RNA samples with orthogonal ligand binding assays, we found that the usage of non-covalent dyes mostly does not alter ligand binding of the RNA molecules, and hence, this new protocol might result in a wide range of opportunities for research applications on the function of RNA molecules. Meanwhile, small modifications that do not significantly affect Fnorm could be enhanced by the use of a modification-specific antibody, as shown for the METTL3/14 assay, allowing the detection of minimal chemical modifications such as single-site methylation of larger RNA molecules. We hope to have laid the first step for the development of further RNA-immuno-MST assays and for numerous studies in this field.In conclusion, we could show that the use of non-covalent nucleic acid dyes is suitable to determine some RNA modifications by a simple “mix-and-measure” protocol. Comparing in vitro transcription  were purchased in HPLC-purified quality  and 1× Q5 reaction buffer (NEB) in a final volume of 200 μL. The reaction was performed using a 3Prime thermal cycler with the following cycling conditions: initial denaturation , followed by 30 cycles of denaturation , annealing  and extension , and a final extension . The PCR product homogeneity was checked on a 0.4% agarose gel to confirm the size and purity.The DNA templates for the IVT were amplified using PCR. The PCR reaction mixture contained 10 nM of template DNA, 2 μM of each forward and reverse primer, 400 μM dNTP Mix (NEB), 3 mM MgClin vitro transcription using the T7 polymerase system. The IVT reaction mixture contained 400 μL PCR product (without further purification), 5 mM of each NTP, 5 mM DTT, 2.5 μg mL−1 BSA, MgCl2 (concentration depends on RNA type and was determined before using micro reactions with different MgCl2 concentrations), 50 U mL−1 T7 RNA polymerase (ThermoFisher) and 1× reaction buffer (ThermoFisher) in a final volume of 1 mL. The reaction was incubated at 37 °C for 2 hours, and then, another aliquot of T7 RNA polymerase was added and incubated for 4 hours at 37 °C.The crude PCR products were used as templates for −1, ThermoFisher), cooled at −80 °C for 2 hours, then centrifuged for 45 min at 4 °C and 16 000 g. The pellet was washed twice with 75% ice-cold ethanol and centrifuged again for 10 min at 4 °C and 16 000 g. The air-dried pellet was redissolved in a small volume of RNase-free water and an equal volume of 2× gel loading buffer  was added.The formed pyrophosphate was removed by centrifugation at 14 000 g for 5 min directly after transcription and the cleared supernatant was transferred to a micro reaction tube for further sample processing. To remove the PCR- and IVT enzymes, a phenol/chloroform extraction was performed twice using ROTI® phenol/chloroform/isoamyl alcohol (Carl Roth) and the standard protocol from the supplier. For a better gel resolution, the extracted samples were desalted using Zeba spin desalting columns (ThermoFisher) following the standard protocol from the supplier. Furthermore, ethanol precipitation was applied to remove chloroform residues and concentrate the RNA. For this, the samples were mixed with 2.5× volume ethanol, 0.1× volume 3 M NaOAc (pH 4.7), and 1 μL glycogen , extracted by cutting out the bands of interest, and the excised parts were aliquoted into small pieces and frozen at −20 °C for 30 min. The frozen sample bands were extracted overnight at 15 °C in a 0.3 M NaOAc (pH 4.7) solution and the samples were centrifuged for 10 min at room temperature and 10 000 g, the supernatant and the gel residues were added into 0.45 μm Nanosep  tubes and centrifuged again for 10 min and 10 000 g. The flow-through was precipitated with ethanol as described above. The pellet was redissolved in RNase-free water and stored until usage at −20 °C. The RNA product homogeneity was checked on a native 10% polyacrylamide gel to confirm the size and purity.2) and supplemented with ligands or additives if needed. Previous to the measurement, the RNA stock solution was heated for 5 min at 75 °C and then cooled down to room temperature in the heating block.The reaction mixtures used for a typical MST experiment contained 100 nM of the investigated RNA and 1× of the respective dye both dissolved in MST buffer  using Monolith standard capillaries. The instrument was calibrated according to the manufacturer's instructions. The laser type was chosen dye-dependently (NanoBlue laser for SybrGold and RiboGreen and PicoRed laser for Syto dyes). General settings were applied for all MST experiments as follows: manual temperature control: 25 °C, fluorescence measurement before MST: 4 s, MST (IR laser) on: 20 s, fluorescence after MST: 2 s, delay: 25 s. The laser power was adjusted to optimize the signal-to-noise ratio and the fluorescence signal. LED and MST power settings were chosen individually for each sample by adjusting the LED power to yield fluorescence signals of at least 300 units (blue laser) or 3000 units (red laser) and the MST power to achieve an appropriate thermophoretic response (standard setting: medium MST power). MST measurements were analyzed using the NT analysis software (version 1.5.41) and exported for statistical analysis and plotting in GraphPad Prism 7.01.c = 10×KD) in a buffer that has been shown to be appropriate for the biological function of the RNA: (1) +RNA/+dye/+ligand, (2) +RNA/+dye/−ligand, (3) −RNA/+dye/−ligand, (4) −RNA/+dye/+ligand. Samples 3 and 4 should not show a significant thermophoresis curve due to the absence of RNA. Sample 1 should optimally result in an MST shift compared to Sample 2 as a function of reference ligand concentration.We recommend the following procedure for assaying new RNA targets: initially, the new experimental setup should be tested with 100 nM RNA and 1× SybrGold (for NanoBlue devices) or 1× Syto 59 (for PicoRed devices). Initial assays include four controls consisting of 100 nM RNA, 1× dye, and a known reference ligand (etc.).If a ligand-dependent MST shift is not achieved with this initial setup, we recommend the following steps with the same assay setup: (1) variation of RNA dyes as described above, (2) variation of RNA concentration, (3) variation of thermophoresis settings  and supplemented with ligands in appropriate concentrations. Data acquisition was performed identically to the setup of the non-covalently labelled MST experiments (vide supra). MST measurements were analyzed using the NT analysis software (version 1.5.41) and exported for statistical analysis and plotting in GraphPad Prism 7.01.MST experiments were performed on a Monolith NT.115 using standard uncoated capillaries. The 5′-Cy5-labelled RNA was heated to 75 °C for 5 min and cooled to room temperature for 60 min. The Cy5-labelled PreQS-adenosylmethionine  in 50 μL of assay buffer . After an incubation period of 2 h at room temperature, the reaction was quenched by the addition of SAH , added with the anti-m6A antibody , SybrGold , and mounted in MST capillaries in triplicates. The laser power was adjusted to 20% for the LED laser and “high” for MST laser. MST measurements were analyzed using the NT analysis software (version 1.5.41) and exported for statistical analysis and plotting in GraphPad Prism 7.01. The decrease in Fnormvs. [SAM] is a measure of the activity of the enzyme, and thus, the MST traces follow the Michaelis–Menten equation which was used for enzyme kinetic analysis: v0 = (vmax × [S]/KM + [S]), where v0 is the initial reaction rate, at a given substrate concentration [S]. vmax is the maximum reaction rate, while KM indicates [S] at which the conversion rate is half-maximal.MST-based METTL3/14 assays were carried out by mixing the RNA substrate AACUUAAUGUUGCAUUGGACUUGAGUUA , the METTL3/14 enzyme  and a variable concentration of the cofactor 50 value of the reference inhibitor STM2457, the assay was performed as described above with the following adaptations. 500 nM SAM was used as substrate. STM2457 was added as DMSO stock . The enzyme reactions were incubated for 60 min at room temperature and quenched with SAH (10 μM). Fnorm values vs. [STM2457] were evaluated using a 4-parameter Hill equation to determine an IC50 value: y(Fnorm) = bottom + ([STM2457]slope) × (top − bottom)/([STM2457]slope + ICslope50).To determine the IC3H-SAM  in 10 μL of assay buffer .43 The reactions were started by the addition of the cofactor 3H-SAM and were carried out at room temperature for 30 min. Aliquots of 8 μL were taken from the reaction mixture and spotted on Whatman glass microfiber filters . The RNA was precipitated on the filters in 5% ice-cold TCA (Sigma Aldrich) for 35 min. The filters were washed twice with 5% TCA at room temperature for 20 and 10 min and once in EtOH for 10 min. After drying, the filters were transferred into scintillation vials and 3 mL of Gold MV liquid scintillation cocktail (PerkinElmer) was added. Scintillation was measured on a scintillation counter  with a measurement time of 1 min. Statistical analysis and plotting were performed in GraphPad Prism 7.01 as described above.The radiometric METTL3/14 assay was carried out as described previously by mixing the RNA substrate AACUUAAUGUUGCAUUGGACUUGAGUUA , the METTL3/14 enzyme  and a variable concentration of the cofactor The datasets supporting this article have been uploaded as part of the ESI.†Elisabeth Kallert: formal analysis, methodology, validation, writing—original draft. Malte Behrendt: methodology, validation. Ariane Frey: methodology, formal analysis. Christian Kersten: conceptualization, writing—review & editing. Fabian Barthels: conceptualization, visualization, writing—review & editing.There are no conflicts to declare.SC-014-D3SC02993J-s001


The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods. Commonly used protein-based tools to monitor intracellular RNA and DNA can impact steric accessibility and native nucleic acid biology. Here, the authors show that fluorogenic uridine-rich internal loop tagging bPNA probes can be used to label nucleic acids in fixed and live cells. Despite the existence of many useful RNA imaging strategies, there are limitations on the placement of probe binding sites and concerns regarding the overall structural impact of labeling on the RNA of interest. Modification of internal sites in RNA via a chemical reaction or duplex hybridization can be technically challenging or sequence-limited3. While fluorescence in situ (duplex) hybridization5 (FISH) with DNA or PNA probes is broadly used for labeling RNAs, FISH is limited to fixed cell experiments. Molecular beacons may be used with unmodified cells and transcripts in live cell labeling but require electroporation or microinjection strategies and have not been as widely adopted as other methods6. In addition, duplex hybridization to internal sites destroys existing secondary structures via strand invasion or transforms a single-stranded loop into a new duplex stem where none existed previously4, introducing potentially disruptive steric interactions. Beyond chemical modification and FISH, approaches to intracellular RNA tracking may be roughly divided into small molecule dye binding and protein labels. Dye-binding SELEX-derived aptamers  have been inserted into RNAs of interest for tracking and sensing applications13. A variation on this approach exploits RNA binding to dequench dye-quencher conjugates  and can utilize native aptamers to universal quenchers such as cobalamin16; this method may be applied to a wider range of fluorophores without the need for additional aptamer selection. However, all aptamer methods require the insertion of a distinct folded structure within the ROI that has the potential to alter RNA sterics or lifetime. Further, early fluorescent aptamer designs suffered from insufficient brightness, thought to be due to misfolded RNA loops17. In particular, the G-quadruplex motif common to Mango and Spinach aptamers20 is especially labile to intracellular degradation21 and requires non-native levels of potassium and magnesium8, which may result in deficient intracellular aptamer performance1. Bacteriophage-derived RNA hairpins MS2 and PP7 are native sequences that bind to phage coat proteins MCP (MS2 coat protein) and PCP (PP7 coat protein), respectively. MS2-labeling requires insertion of multiple MS2 hairpins into ROIs and with subsequent RNP imaging via binding of MCP fusion proteins that facilitate fluorescent readout (eg.- fluorescent proteins23 (MCP-FPs), MCP-HaloTag24 and MCP-SNAP tag25 fusions). Though effective, the use of constitutively fluorescent dyes and FPs generates a significant background signal, requiring multiple (often >20) MS2 hairpins to generate a sufficient signal to noise. Further, these protein labeling systems carry significant steric bulk (>40 kD for a single MCP-FP) with the risk of altered transcript decay28 and inhibited native contacts. Despite these drawbacks, MS2/PP7 labeling remains the most widely used method for RNA tracking. Similarly, CRISPR-Cas/guide RNA complexes29 can be used to track of DNA and RNA targets via fluorescent readout of protein fusions to Cas variants, but also via formation of sterically encumbering protein binding to native RNA30 targets or genomic loci31.There remains a need for improved and orthogonal methods for localizing and tracking intracellular RNA and DNA molecules in real time4xU4) U-rich internal loop (URIL) can be selectively labeled with a fluorogenic bPNA probe via triplex hybridization. The FLURIL system minimizes the introduction of new RNA secondary structures by use of duplex-for-triplex stem replacement, is considerably smaller than typical RNA aptamer or protein labeling systems, and exhibits improved signal-to-background and stability over comparable methods 39, which presents synthetic melamine bases41 on an α-peptide backbone44  base triples, generating triplex stems that can functionally replace native RNA stems47, tertiary contacts47, block protein readthrough48, direct chemistry49, and modulate lncRNA lifetime50. Optimized51 cationic 4 M bPNAs  while retaining nanomolar affinity45, thus enabling specific intracellular bPNA targeting of URIL-tagged ROIs. via formation of triplex hybrid  stems that are similar in mass to a 4 bp RNA duplex. Via URIL-tagging, bPNA hybridization can place prosthetic groups at internal RNA sites without aptamer selection; this manuscript describes  fluorogen-modified bPNA binding to the URIL (FLURIL) tag. We demonstrate FLURIL tagging of intracellular RNAs and RNPs in both fixed and live cell contexts.Here, in a method we call fluorogenic U-rich internal loop (FLURIL) tagging, we show that intracellular RNAs bearing an 8-nt  emission with PNA-thiazole orange (TO) conjugates55 that place TO at the hybridization interface. Similarly, Dervan has attached TO to pyrrole-imidazole polyamide (Py-Im) minor groove intercalators that fluoresce upon sequence recognition56. While these probes have utility, they are limited with regard to transport and RNA binding57. To study fluorogenic RNA binding in the context of URIL triplex hybridization, we synthesized bPNAs N-terminated with Cy5, difluorohydroxybenzylidene (DFHBI)7 and TO derivatives . Structure-function studies59 indicated that presentation of two melamine bases on the ε-amine of lysine (K2M) results in a solubilizing cationic sidechain that enables strong URIL binding even with short, low molecular weight di and tri-peptides. Even following N-terminal modification with fluorogenic modules, these efficient bPNA probes are in a low molecular weight regime (~1 kD). Moreover, Fmoc-K2M can be obtained on a multigram scale by double reductive alkylation of Fmoc-lysine with melamine acetaldehyde without column purification60, making these bPNA probes are highly accessible and synthetically scalable. We prepared a small family of dye-modified  4 M bPNAs  scaffold where X is an α-amino acid and dipeptide isomers. Fluorogenic turn-on upon RNA complexation was modest for the cyanine and DFHBI dyes  or directly to the ε-amine of lysine (X = KTO). As dipeptide and tripeptide bPNAs of alternating amino acid configuration had previously shown an advantage in triplex hybridization over the homochiral dipeptides due to syndiotactic base presentation62, we set out to test the impact of stereochemistry on the fluorogenic binding when modified with TO on the N-terminus  and ε-amine of the central amino acid (K2MkTO-K2M). Additionally, we synthesized and evaluated the isodipeptide (TO-(αK2M)2) that features a sidechain linkage, with base presentation on the α-nitrogens and TO modification on the ε-amine.We previously reported that cyanine dye  modified bPNAs exhibited fluorescence enhancement upon triplex hybridization to URIL RNA and subsequent hybrid RNA-protein bindingves Fig. . Prior w−4)63. However, when TO-K2MAla-K2M binds to URIL RNAs  does not improve the brightness of the TO-bPNA derivatives  indicates the critical role of bPNA triplex hybridization in fluorogenic binding. All bPNAs gave a strong fluorogenic response to URIL RNAs, but there appeared to be significant differences among bPNA variants58. Further, despite the biophysical advantage previously observed for triplex hybridization with L,D and D,L dipeptide bPNAs51, these fluorogenic variants were not as bright as the tripeptides . Based on these results, we chose the TO-K2MAlaK2M probe (1) for subsequent intracellular studies.With URIL-binding bPNAs in hand, we tested fluorogenic binding in vitro to hairpin and duplex URIL-RNAs Fig. . The absNAs Fig. , emissioNAs Fig.  and 13. NAs Fig. . This TOves Fig. . While tLys with URIL RNA hairpins  was designed to serve as an intracellular negative control that lacks bPNA binding. The U4-tRNA construct was used to determine intracellular fluorogenic triplex hybridization with TO-bPNA. Indeed, treatment of HEK-293T cells with probe 1 in cell culture media following transfection with either U4 or NEG tRNA resulted in a 5- to 10-fold brighter fluorescence intensity in the U4-tRNA transfected cells  along with the TO-bPNA hybrid with U4-MS2 tRNA. Thus, treatment of cell culture with TO-bPNA in media can effectively FLURIL-tag intracellular RNA and RNP targets and track RNP transport from cytoplasm to nucleus. Importantly, without MCP-RFP, green fluorescence from TO-bPNA remains cytoplasmic; without TO-bPNA, MCP-RFP remains localized to the nucleus as expected.To benchmark bPNA labeling of RNA against known RNA tracking strategies, we juxtaposed the U4 URIL with the MS2 hairpin sequence in the tRNARNA Fig. . The U4-asm Fig. . This re69, where it is mislocalized from the nucleus to the cytoplasm70. As TDP-43 is known to bind to repeat sequences of UG/TG72, we co-transfected HEK-293T cells with plasmids encoding URIL-UG repeat RNA (U4-(GU)8, Fig. 73. Upon Treatment with TO-bPNA in cell media resulted in co-localized red (tdTomato) and green fluorescence in the nucleus 8/TDP-43 systems verified, we transfected with swapped RNA-protein pairings that should not result in complex formation: U4-MS2 was co-transfected with TDP-43-tdTomato, and U4-(GU)8 with MCP-RFP  due to its central role in the progression of neurodegenerative diseases such as ALS30. CRISPRainbow is a live cell imaging strategy that uses MS2 or PP7 modified guide RNA (gRNA) with deactivated Cas9 (dCas9) to precisely track endogenous DNA repeats marking specific genomic loci74. Incorporation of MS2/PP7 bacteriophage RNA hairpin sequences into the gRNA enables the dCas-gRNA complexes to be tracked in live cells by fluorescence imaging upon co-expression of phage coat protein (MCP/PCP) fusions with a “rainbow” of fluorescent proteins; sequence-optimization of the bacteriophage hairpin array yielded more stable and brighter imaging systems known as CRISPR-Sirius gRNAs75. We selected two CRISPRainbow/Sirius gRNAs that target proximal intergenic DNA regions  and modified one to utilize FLURIL tagging only, with the other serving as an internal benchmark of MS2 labeling. On the gRNA targeting IDR3, a single U4XU4 bulge (URIL) was installed in a stabilized hairpin  and Hepatitis Delta Virus (HDV) self-cleaving ribozymes77. We thus modified the gRNA plasmid to contain the Hammerhead (HH) and Hepatitis delta virus (HDV) ribozymes on either side of the gRNA under a CMV promoter and inserted it into a dual gRNA plasmid expressing CRISPR-Sirius gRNA (Sirius-IDR2-(MS)8) for dual-color genomic loci targeting  and IDR3 (IDR3-U4 + TO-bPNA) was readily established in U2OS cells by lipofectamine plasmid transfection and staining with dyes delivered in media . Live cell imaging revealed partially overlapped red  and green  foci  enhancement of emission intensity upon bPNA triplex hybridization with structured RNAs URILs in vitro and a substantive increase in quantum yield from ~0.01% in the unbound state to 43% in the RNA complex. This significant fluorogenic response to URIL-RNAs translated to 5–10X intracellular signal-to-noise and a highly usable, low-background cellular imaging of URILs. While an engineered biomolecule (URIL-RNA) is needed, this is a universal requirement for the most broadly used, genetically encoded live cell tracking strategies such as MS2-labeling.Intracellular FLURIL tagging of RNAs in both fixed and live cells was directly verified using established protein labels from endogenous mammalian RNPs as well as bacteriophage MS2 labeling, which remains the gold standard for RNA tracking. In the present study, we have inserted stabilized hairpin structures to host the URIL site and included a PP7 hairpin as a control element in IDR3-U4; however, a URIL could be inserted into a native fold by replacement of an existing native 4 bp stem buttressed by other structures in the ROI, further streamlining the FLURIL-tag footprint. Importantly, RBP fusions with fluorescent proteins (FP) confirmed that FLURIL tags were labeling the correct RNA species, while cells lacking the correct RNA-protein pairing resulted in the segregation of FLURIL tag and RBP-FP fusion signals. Under live cell tracking conditions, FLURIL tags also performed comparably to established MS2-based methods (CRISPR-Sirius) while occupying a considerably more compact molecular footprint. It is particularly noteworthy that the FLURIL tag only requires the replacement of a 4 bp stem with an 8-nt URIL to enable RNP live cell tracking with a cell-permeable, ~1kD bPNA probe.1; however, inhibition of transcript degradation by insertion of bacteriophage (MS2/PP7) hairpins is less of an issue in mammalian cells28. In contrast, FLURIL tags add negligible additional mass to the RNA target and a structural perturbation that may be as minor as the replacement of a duplex stem with a triplex stem. Prior work has demonstrated that this replacement does not disrupt proximal RNA domains, retaining tertiary interactions as well as catalytic function. In addition to minimizing the addition of obtrusive non-native structures, FLURIL tagging also avoids the use of labile21 G-quadruplex domains found in SELEX-derived dye-binding aptamers such as Spinach and Mango. Furthermore, as fluorogenic dye binding is driven by bPNA triplex hybridization to the URIL rather than direct RNA recognition of the dye, prosthetic groups50 may be used to decorate RNPs without the technical demand of a SELEX campaign. In particular, other fluorogens in the cyanine family could potentially be used in FLURIL tagging, thus expanding the color range available without laborious aptamer selection procedures. Analogously, the aptamer Pepper11 binds a family of fluorogenic dyes with significant structural variation, suggesting that minimal specific contacts are needed to access fluorogenic binding.Though there remain many appealing aspects to MS2 labeling, including multiplex and multicolor approaches, the use of protein fusions and multiple RNA hairpin sites encumbers the RNA of interest with substantial steric bulk, adding 41–47 kD of mass per MCP-FP and MCP-HaloTag fusion, respectively. The steric bulk and RNA secondary structures required for protein labeling have raised concerns that this could affect native transcript processing and tertiary contacts41 are underway. Despite these caveats, the present work demonstrates the utility of FLURIL tagging, as well as its appealing compatibility with popular methods such as MS2/Halotag labeling. We anticipate that FLURIL tagging will be a useful tool that may also be used in conjunction with other methods  to facilitate RNA tracking while minimizing the steric burdens of RNA labeling.The unique advantages of FLURIL tagging are counterbalanced by some limitations. As mentioned above, a URIL motif must be installed in the RNA of interest, requiring non-native RNA expression by transfection. The efficiency of tracking, as described herein, thus depends on the efficiency of transfection and vector design, which can lead to variability in labeling outcomes; additionally, transfection yields elevated, non-native transcript levels. These issues may be alleviated with genome editing, though this comes with a significant cost and effort per experiment. Currently, FLURIL tagging has only been demonstrated with a single color, though efforts to expand both color selection and sequence scopeAll chemicals were used without further purification from commercial sources as indicated unless otherwise noted. DNAs and RNAs were purchased from Integrated DNA Technologies (IDT). Nucleic acid strands shorter than 20 nt were used without further purification. Otherwise, the DNAs/RNAs were purified by TBE-urea denaturing gel. DNA stock solutions were serially diluted in MilliQ water, and concentrations were determined by measuring solution absorbance at 260 nm on a Thermo Fisher Nanodrop 2000. Sample fluorescence was measured on a Thermo Fisher Nanodrop 3300. Cell lines were acquired from ATCC  and cultured according to ATCC protocols.RNAs for in vitro fluorescence studies shown below were purchased. All RNAs were annealed prior to use. Duplexes were annealed together from A and B strands indicated.12-U4-12 A: 5’-CGCAUAGCUCAGUUUUGACUCGAUACGC-3’12-U4-12 B: 5’-GCGUAUCGAGUCUUUUCUGAGCUAUGCG-3’12-U6-12 A: 5’-CGCAUAGCUCAGUUUUUUGACUCGAUACGC-3’12-U6-12 B: 5’-GCGUAUCGAGUCUUUUUUCUGAGCUAUGCG-3’RNAI U6: 5’-GGCAGCUUUUUUUUGGUAGUUUUUUGCUGCC-3’RNAII WT: 5’-GCACCGCUACCAACGGUGC-3’The following RNA sequences were delivered into cells by plasmid transfection for intracellular labeling.U4 tRNA: 5’-GCCCGGAUAGCUCAGUCGGUAGAGCAGCGGCCGUUUUCGCUCCGGCGUUUUCGGCCGCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA-3’U4-MS2 (MBSV5) tRNA: 5’-GCCCGGAUAGCUCAGUCGGUAGAGCAGCGGCCGUUUUCGCACAUGAGGAUCACCCAUGUGCGUUUUCGGCCGCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA-3’NEG tRNA: 5’-GCCCGGAUAGCUCAGUCGGUAGAGCAGCGGCCGCGCGCGCUCCGGCGCGCGCGGCCGCGGGUCCAGGGUUCAAGUCCCUGUUCGGGCGCCA(GU)8-U4 RNA: 5’-CGGCCGUUUUCGCUCCGGCGUUUUCGGCCGGUGUGUGUGUGUGUGU-3’RNAs were annealed with bPNA at a 1:1 ratio and incubated for 30 min following slow cooling . Sample fluorescence was measured using a Thermo Fisher Nanodrop 3300 (relative fluorescence units (RFU), excitation = 470 nm, emission = 522 nm, 2 μl sample volume). All fluorescence values are the average of triplicate measurements, starting from fresh sample preparation. Error bars indicate standard deviation. Data were processed using OriginPro and Graphpad Prism.78. All dye samples  were prepared at 1 μM with hybrid samples prepared at 1:1 TO:RNA ratio . Sample concentrations were determined by UV absorbance (Cary UV-Vis-NIR Spectrophotometer) and adjusted to obtain matched sample absorbances, not to exceed 0.04 at 470 nm (±0.0002). The fluorescence was measured by Quantamaster 8000 spectrofluorometer and corrected for anisotropy with a vertical polarizer in the excitation path (470 nm) and a polarizer in the emission path (507 nm) set at the magic angle (54.7°), with excitation and emission slit widths = 5 nm)79. The integrated emission of the bPNA hybrid was 49.7% that of fluorescein, indicating a relative quantum yield of 43%.Relative quantum yield was calculated against fluorescein (quantum yield = 92%)80. The ligation product was transformed into DH5α for amplification, and the resulting mixture was inoculated on an agar plate with ampicillin selection. After 16 h, several colonies were picked and amplified in LB media containing ampicillin. The harvested plasmid was isolated by miniprep (Qiagen) and verified by sequencing. Tomato-TDP43 and MCP-TagRFPt plasmids were obtained from Addgene (#28205 and #64541)81 and amplified in the corresponding E.coli cell lines.The DNA inserts corresponding to modified tRNA scaffolds  were annealed into duplexes from which 2 µg were digested with SalI and XbaI , following Thermo Fisher protocol. Vector pAV U6 + 27 (1 µg) was separately digested with SalI and XbaI (12 h), and all digested products were gel purified (band isolation from with Qiagen QIAquick Gel Extraction Kit). Purified products were ligated (DNA insert:linearized vector=5:1) with T4 DNA ligase (Thermo Fisher), following published protocol5/ml. After one day of incubation, the cells were transfected with corresponding plasmids (1000 ng each per dish) with Lipofectamine 3000 (Invitrogen) following the online protocol, and the cells were incubated for 24 h. Culture medium was removed and the fresh medium containing 1 µM bPNA-dye was added to the attached cells. The cells were incubated at 37 °C for 2–8 h, medium discarded, and Hoechst 33258 (Invitrogen) in fresh culture medium was added at the final concentration of 200 ng/ml to the cells and incubated for 15 min at 37 °C. Cells were washed with PBS, fixed with 4% formaldehyde in PBS for 15 min at room temperature and rinsed with PBS. The coverslip was transferred to glass slides for fluorescence microscopy and imaged under Olympus FV3000 systems  405 nm, (emission) 422 nm, Voltage: 355 V, detection range: 420–470 nm; Green: (excitation) 488 nm, (emission) 520 nm, Voltage: 580 V, detection range: 499–599 nm). Three or more replicate data sets were obtained that showed consistent findings, starting from cell seeding.HEK-293T cells were cultured based on ATCC protocol. Cells were seeded to a 35-mm culture dish (Thermo Fisher) with a clean coverslip (Thermo Fisher) attached to the bottom at a concentration of 5 × 102 and humidity supplement. A laser quad-band filter set for TIRF  was used to collect fluorescence signals simultaneously. Data acquisition was carried out with CellSens 4.1.1 software. Localization precision was ~5 nm in 4 s, ~6 nm in 16 s, and ~10 nm in 80 s82. The video was recorded at 136 ms per frame with a total of 96 frames and 100 ms exposure time. Image size was adjusted to show individual nuclei, and intensity thresholds were set on the basis of the ratios between nuclear foci signals to background nucleoplasmic fluorescence.Cell imaging (U2OS) was carried out on an Olympus IX83 microscope equipped with three EMCCD cameras (Andor iXon 897) mounted on a 4-camera splitter, four lasers , mounted with a ×1.6 magnification adapter and ×60 apochromatic oil objective lens (NA 1.5), resulting in a total of ×96 magnification. The microscope stage incubation chamber was maintained at 37 °C with COFiji83 and Mathematica (Wolfram) software. To achieve subpixel registration accuracy, parameters for shifting, scaling, and rotating camera images were determined by the least-squares fitting of fluorescent bead images . The experimental data from each channel were processed through an affine transformation and overlapped in false-color channels for visualization. The locus trajectory was obtained by tracking the locus position over time, and graphs were generated by OriginPro (OriginLab version 2019b).The images were registered and analyzed by Further information on research design is available in the Supplementary InformationReporting SummarySupplementary Movie S1


These overlapping promoters were predicted in silico with rather high scores, effectively bound RNA polymerase in vitro and in vivo and were capable of initiating transcription in sense and antisense directions. The genome-wide correlation between in silico promoter scores and RNA polymerase binding in vitro and in vivo was higher for promoters located on the antisense strands of the genes, however, sense promoters within the uxuR gene were more active. Both regulatory RNAs synthesised from the divergent promoters inhibited expression of genes associated with the E. coli motility and chemotaxis independent of a carbon source on which bacteria had been grown. Direct effects of these RNAs were confirmed for the fliA gene encoding σ28 subunit of RNA polymerase. In addition to intracellular sRNAs, promoters located within the uxuR gene could initiate synthesis of transcripts found in the fraction of RNAs secreted in the extracellular medium. Their profile was also carbon-independent suggesting that intragenic uxuR transcripts have a specific regulatory role not directly related to the function of the protein in which gene they are encoded.Small non-coding and antisense RNAs are widespread in all kingdoms of life, however, the diversity of their functions in bacteria is largely unknown. Here, we study RNAs synthesised from divergent promoters located in the 3′-end of the  Although non-coding transcripts have already been studied for quite a long time, their new biotypes  and waysThe first discovered and the best characterised regulatory RNAs in bacteria are small regulatory RNAs (sRNAs), ranging from dozens to hundreds of nucleotides in length. They can form complementary or partly complementary duplexes with mRNAs or their regulatory regions, e.g., ribosome binding sites, thus affecting translation or stability of mRNAs .More recently, regulatory functions were described for the fragments originating from tRNAs (tRFs) and the 3′-ends of mRNAs . Very invia the extracellular membrane vesicles (EV), which protect them from degradation by nucleases  seemed to have the most predictable mechanism of action, forming complementary duplexes with the mRNAs of adjacent or overlapping genes . Their gDespite this seeming simplicity, till now the mechanisms of aRNAs action are not very well understood. A series of genome-wide studies led to a suggestion that in prokaryotic genomes antisense transcripts are just by-products of noise without any significant functions . The comuxuR gene coding for a transcriptional regulator of hexuronate metabolism. UxuR belongs to the GntR family of regulators with the helix-turn-helix N-terminal DNA binding domain and the C-terminal ligand binding/dimerization domain , transporters , and regulators  of hexuronate metabolism  was used as the model organism. For RNA extraction, cells were grown to OD650 of 0.3–0.4  under constant shaking at 37°С in Minimal Salts (MS) media supplemented with 5% LB, 0.2% D-glucuronic acid or 0.2% D-glucose. The uxuR gene disruption was made as described in  registered at ± 250 bp from the most prominent transcription start site was taken. We analysed only those antisense (n = 663) and co-directed (n = 241) promoters that cannot initiate transcription of adjacent genes and do not overlap with any of the known or predicted gene promoters.To calculate correlations between the PlatProm scores and efficiency of RNAP binding, we analysed two ChIP-chip datasets made with σ70- RNAP . Maximal2, 50 mM NaCl, BSA (5 mg/mL), 1 pmol of PCR-generated DNA-fragments and 2–8 pmol of σ70-RNA polymerase . Interaction was allowed for 30 min, then 20 μg/mL heparin was added to prevent any non-specific binding. Samples were loaded on a prewarmed gel under very low voltage. Gels were then stained with ethidium bromide, visualised under the UV-light and photographed.Ability of RNAP to interact with the predicted intragenic promoters was estimated by electrophoretic mobility shift assays (EMSA). DNA fragments were PCR amplified with primers indicated in uxuR gene to form open complexes, potassium permanganate footprinting was used  as described above. Transcript starts for the RNAs synthesised from within the uxuR gene were localized by single round transcription in vitro as previously described .To test the ability of promoters within the was used . 0.2 pmoescribed . To asseTotal RNA from the exponentially growing cells (4 h of growth) was isolated using the RNAqueous RNA isolation kit  according to the manufacturer’s protocol. Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA library prep kit for Illumina . Sequencing was performed as 50 nt SE on the Illumina HiSeq 4,000 machine.650 = 0.4 was centrifuged at 4,500 rpm + 4°С. Supernatant was filtered through two 0.22 μm PES filters and aliquoted 500 μL/1.5 mL Eppendorf tube. Then, equal volume of the TriZol reagent  was added to each tube, and RNAs were extracted using the miRNeasy SerumPlasma kit .Fraction of small exoRNAs was isolated as described in . In brieThe RNA quality was checked using 4% polyacrylamide gel with 8 M urea, and quantity measured on a Qubit fluorometer . Sequencing libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Kit for Illumina . Sequencing was performed as 150 + 150 PE on the Illumina NextSeq 500 machine. Sequencing was performed at the Skoltech Core Genomics Facility.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then were trimmed using the Trim_Galore! tool. . The rRNA sequences were removed using SortMeRNA (https://academic.oup.com/bioinformatics/article/28/24/3211/246053). The reads were then aligned to the E. coli genome using STAR-aligner (https://pubmed.ncbi.nlm.nih.gov/23104886/). The aligned reads were translated into counts using the featureCounts tool (https://pubmed.ncbi.nlm.nih.gov/24227677/). The differential expression analysis was carried out with the NOISeq tool (https://pubmed.ncbi.nlm.nih.gov/26184878/). Raw reads are available in NCBI GenBank (SRA) under accession PRJNA882437.The RNA sequencing data was processed using the following pipeline. Fastq files first underwent quality analysis using the FastQC tool . Next, the reads were aligned with Bowtie two and sorted with SAMtools, after which the number of reads per gene was calculated using featureCounts, which is a part of the Subread package (http://subread.sourceforge.net/). The featureCounts output count data normalisation  was performed via the edgeR package (https://bioconductor.org/packages/release/bioc/html/edgeR.html) and then used as the input data for constructing a linear regression model and scatter plots. The regression models and scatter plots were implemented via the R programming language, and the ggplot2 packages (https://cran.rproject.org/web/packages/ggplot2/index.html) and ggrepel (https://cran.rproject.org/web/packages/ggrepel/index.html). Raw reads are available in NCBI GenBank (SRA) under accession PRJNA883224.The quality of the sequencing data of secreted small RNAs was validated uxuR-aRNA (5′-UUUAUCCAGCGGCCAUGAAUC-3′), and uxuR-exoRNA  were added to 10 mL of cultures. Total RNA was isolated using the TriZol reagent  and treated with DNAse I  according to the manufacturers’ protocols. Reverse transcription was made using respective gene specific primers and RevertAid MMul-V reverse transcriptase . A DT-Lite thermocycler  and qPCRmix-HS SYBR  were used for quantitative PCR (qRT-PCR). Primers used for reverse transcription and amplification were as follows: flgK_RT 5′-TTACTCACCAGCGTTTGCAG-3’; flgK_PCR 5′-CTGGTGTGCAGCGTGAGTAT-3’; fliA_RT 5′-GCGTTGCGGCCAAGTTCCTG-3’; fliA_PCR 5′-CTATGCTGGATGAACTTCGCA-3’. No PCR products were detected in negative controls in the absence of reverse transcriptase. Data obtained from at least three biological samples and analysed in three statistical replicates were calculated by the ΔCt method. The error bars indicate the standard deviations of the respective mean values.Cultures were grown till mid-exponential phase (4 h of growth) in the same conditions as had been used for RNA-seq. To test the influence of small RNAs on the gene expression, 1 μmol of artificially synthesised RNAs, UxuT (5′-CAAGGGUAAACGUUCCUUGCGCUUUCUUAAAUUAAGAAGUCGCAAUGAGUAUUACUUUGUAAAUUGCAGGGUAUUGUUU-3′), Targets for sRNA binding were found using IntaRNA . UxuT wain vivo  > 1, in silico and in vivo data. However, only 54% of putative antisense promoters were able to bind RNAP at the same cut off level    . However0.00001) .in vitro for 35 DNA fragments, containing intragenic promoters, of which 21 were antisense. Examples of all types of observed modes of interaction with RNAP are shown in lacZ gene, in vivo, in these experiments transcription complexes were formed in the absence of transcription factors or ligands which can affect RNA polymerase binding. As a result, statistically significant correlation with PlatProm scores was observed for all sets of promoters . Transcription from the antisense promoter was also detected giving a product of around 105–110 nt if the template was restricted by primer 1, around 195–200 nt for the templates with primer 2, and around 275–280 for the templates with primer 3. The respective product is initiated at the P1 promoter. Thus, both antisense P1 and co-directed P2 promoters are capable of productive transcription, with P2 being preferred by RNA polymerase in the studied conditions. Antisense promoters with lower scores located closer to primer 2 . In the latter case, the UxuR protein could not be produced, but all the possible promoters for small RNAs located closer to its end were still present. Keeping in mind that UxuR is a critical regulator of hexuronate metabolism, RNA sequencing was made for cultures growing on D-glucose or D-glucuronate, a key intermediate of the Ashwell pathway. On D-glucuronate, all genes coding for transporters, enzymes and regulators of hexuronate metabolism  were predictably activated, and the same was observed here and earlier on both carbon sources upon deletion of uxuR reflecting repressor function of its protein product  in the expression efficiency were detected for the genes of four operons linked with cell motility and chemotaxis, flgBCDEFGHIJ, fliFGHIJK, fliDST and tar-tap-cheRBYZ. They were all highly activated, suggesting that RNAs might act as their repressors, while genes responsive for cell resistance to extreme acid conditions were vice versa inhibited . Similar, albeit less pronounced changes were observed upon bacterial growth with D-glucuronate, indicating that the regulatory effects of the uxuR RNAs products could be not very much dependent on a carbon source.More interestingly, for at least 59 genes expression critically changed upon deletion of whole xuR gene  have a pfliA gene coding for σ28 and thus controlling flagellar genes, and of flgK encoding structural flagellar hook-associated protein was checked. qRT-PCR was made with the RNAs isolated from the wild type K-12 MG1655, K-12 MG1655 ΔuxuR and K-12 MG1655 ΔuxuR with addition of one of three candidate RNAs: uxuR-aRNA synthesised from P1; co directed RNA UxuT (uxuR Terminator) synthesised from P2; and the most abundant secreted RNA (exoRNA) detected in . Among other affected genes were genes responsible for regulation of transcription (sfsB and ygiV) and translation (raiA), small RNAs FnrS and GadF, as well as rRNAs and tRNAs.A change of a carbon source significantly affected profiles of exoRNAs . PredictuxuR gene was detected  and codirected (P2) promoters in the uxuR 3′-UTR , and those involved in chemotaxis   with energy of −12.33 kcal/mol, thus apparently affecting its stability. Also, this interaction might occur close to a putative transcription start at 2,001,544  . The inf qRT-PCR . Expresswas used . Accordiwas used , we may fliA, and CsgD, serving as a repressor . As such, we can speculate that UxuT might interfere with FlhDC preventing this activator from binding and thus also inhibiting fliA expression. Further RIL-seq will be the best option to test if these interactions can indeed happen in the living cell.UxuT, in turn, may bind the promoter region strictly between the sites for FlhDC, an activator of epressor . In contor sRNAs . Due to uxuR gene represents a nice example where three different types of transcripts with a regulatory potential were found: the 3′-UTR RNA UxuT transcribed from the promoter located in the rho-independent terminator of uxuR, aRNA transcribed divergently from the overlapping promoter, and exoRNA fragment produced from the antisense promoter located 148 bp upstream . These data suggest that small RNAs synthesised from within the uxuR gene, both intracellular and extracellular, might have specific functions unrelated to carbon metabolism. Many exoRNAs are synthesised from within genes coding for regulatory proteins that are not highly expressed themselves. We also noticed that they are often mapped to the genes overlapping with promoter regions of functional genes and small RNAs, suggesting some interplay with other regulatory elements of the bacterial genome.Interestingly, almost no genes coding for enzymes of hexuronate metabolism were affected by deletion of promoters for small RNAs, except n source . In lineuronates . At the uronates  it was cIn summary, although our findings are in line with a concept of widespread antisense transcription, they do not support the idea that bacterial aRNAs are simply the products of transcriptional noise. Both intracellular and secreted extracellular small RNAs may interfere with other regulatory events in bacterial cells, and their precise functions are yet to be understood.


Furthermore, we identified Rap guanine nucleotide exchange factor 4 (RAPGEF4) as a potential downstream target of NOP2 in HGSOC. Finally, our findings suggest that the regulation of NOP2 and RAPGEF4 may depend on m5C methylation levels.RNA methyltransferase nucleolar protein p120 (NOP2), commonly referred to as NOP2/Sun RNA methyltransferase family member 1 (NSUN1), is involved in cell proliferation and is highly expressed in various cancers. However, its role in high-grade serous ovarian cancer (HGSOC) remains unclear. Our study investigated the expression of NOP2 in HGSOC tissues and normal fimbria tissues, and found that NOP2 was significantly upregulated in HGSOC tissues. Our experiments showed that NOP2 overexpression promoted cell proliferation  Standard treatments used to tackle ovarian cancer include cytoreductive surgery and chemotherapy based on carboplatin-paclitaxel combinations.4 Despite the advancements in surgery, chemotherapy, biochemical markers, genetic testing, and targeted therapies, almost all women suffer from ovarian cancer recurrences,5 and complications from other diseases are frequently a concern for advanced stage patients.6 As a result, researchers are exploring new mechanisms for early detection and improved diagnosis and therapy of HGSOC.Ovarian cancer is among the deadliest tumors in women, having the second highest incidence and highest mortality rate among female reproductive system tumors. It is also among the top five most lethal tumors worldwide.6A, while research on m5C methylation is still in the early stages. RNA methylation plays a key role in various biological processes, such as nuclear RNA export capacity, RNA stability enhancement, RNA shearing and RNA-protein interaction regulation.7 Abnormal RNA methylation levels are associated with tumors,8 cardiovascular system diseases9 and neurological diseases.10 There are different types of RNA methylation, comprising N1-methyl-adenosine (m1A), m5C, N6-methyl-adenosine (m6A), 7-methyl-guanosine (m7G) and Um.11 m5C is methylation on the fifth C in the RNA cytosine. It is predominantly present in untranslated regions (3‘UTR and 5‘UTR), GC-enriched regions, adjacent to conserved AU(m5C)GANGUAGO sequences12 and near protein binding sites of AGO.13 RNA methylation is a reversible process whose regulation depends on writers (methyltransferases), erasers (demethylases), and readers (binding proteins). In m5C methylation, NOP2 is a vital methyltransferase that still lacks extensive research. NOP2 was initially explored as an important protein for ribosome synthesis and processing and 60s ribosomal subunit synthesis in budding yeast.14 Recently, NOP2 has been associated with ribosome biogenesis in humans15 and increased expression in most cancers resulting in a poor prognosis.16 NOP2 was found to have elevated expression in ovarian cancer compared to normal controls and was associated with poor prognosis.17 Moreover, NOP2 enhances the proliferation, migration and invasion in colon cancer.18 However, research on the mechanism of NOP2 in HGSOC has not yet been reported.RNA methylation is a significant post-transcriptional modification of RNA. Current research focuses mainly on m19 Recent studies have identified the cAMP signaling pathway as being closely related to tumorigenesis and progression and as a potential therapeutic strategy.20 Epac proteins are guanine nucleotide exchange factors (GEFs) for Rap1 and Rap2 and are associated with tumor development.21–23 Epac inhibitors are already available as a treatment to fight tumors.24The cyclic adenosine monophosphate (cAMP) signaling pathway belongs to the cyclic nucleotide system, a signaling pathway that regulates the concentration of the second messenger through receptor binding to extracellular signals. The subsequent downstream responses include the induction of specific intracellular responses. cAMP is one of the signaling molecules that translates the extracellular signals into specific intracellular responses. Three primary targets of cAMP are protein kinase A (PKA), cAMP response element-binding protein (CREB), and exchange protein directly activated by cAMP (Epac). These processes involve gene transcription, cell migration ability, cell proliferation, cell death and play a crucial function in normal physiological activities and disease development.in vitro, as well as the proliferation ability of cells in vivo and in vitro. Our experiments reveal that NOP2 regulates Rap guanine nucleotide exchange factor 4 (RAPGEF4) in HGSOC as a potential downstream gene. We further discovered that NOP2-induced proliferation of HGSOC cells was contingent upon the expression of RAPGEF4, which in turn was regulated by m5C methylation. Our findings indicate that a regulatory mechanism of NOP2 on RAPGEF4 could account for HGSOC carcinogenesis and progression via a novel mechanism.As a result of our study, we found that NOP2 was overexpressed in HGSOC compared to FT tissues. Furthermore, NOP2 could promote the migration and invasion of HGSOC cells 25 was downloaded from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Ovarian cancer cell line data was downloaded from Expression Atlas (https://www.ebi.ac.uk/gxa/home)26 and the accession number is PXD030304.27 The m5C regulator-associated somatic mutation and Copy number variation (CNV) analyses of pan-cancer and ovarian cancer (OV) were performed through the cBioPortal website .28 This data analysis included 398 TCGA ovarian cancer samples. Kaplan – Meier overall survival (OS) analysis of NOP2 expression in OV patients was performed through Kaplan-Meier Plotter .29Matrix files in SOFT format for Gene Expression Omnibus (GEO) dataset (GSE10971)30 The package is also implemented to calculate the up-regulated differentially expressed genes (DEGs) in different clusters. DEGs were determined for genes with p < .05 & |log2FC|≥1.The differential analysis of genes was implemented by the limma R package (version 3.52.4).Between 2017 and 2022, 63 HGSOC tissues and 13 FT tissues were collected through Shanghai First People’s Hospital. All operations were performed after informing the patients and obtaining their consent. Ethical approval was obtained from the Medical Ethics Committee of Shanghai First People’s Hospital (ethics number: 2017KY068).We fixed the collected tissue specimens in tissue fixative for more than 24 h. We removed the tissues from the fixative in a fume hood and trimmed the tissues with a scalpel, placing the prepared tissues and corresponding labels in an embedding box. The tissues were dehydrated by sequential immersion in graded concentrations of ethanol and xylene. All dehydrated tissues were submerged in pre-heated liquefied wax and immersed overnight. Tissue wax blocks were subsequently made on a HistoCore Arcadia . Finally, we submitted the prepared tissue wax blocks to Servicebio  for tissue microarrays.We cooled the prepared wax blocks on ice, and then placed the cooled blocks in a paraffin slicer  for slicing (4 μm thickness). Finally, we float the sections on warm water at 40°C to flatten the tissue, lift the tissue with a slide and bake the sections at 60°C. Sections can be stored at 4°C after cooling down naturally.The previously prepared sections or tissue microarrays were placed in a Thermostatic Incubator  at 60°C for 3 hours or overnight to melt off the surface sealing wax. Sections or tissue microarrays were sequentially deparaffinized and rehydrated by immersion in xylene and a concentration gradient of ethanol in a fume hood. Citrate Antigen Retrieval Solution  was used for antigen retrieval in boiling water for 7 min. 3% hydrogen peroxide solution was used to eliminate endogenous peroxidase activity in tissues. Tissues were blocked with 10% goat serum  for an hour. Then, the anti-NOP2 antibody  diluted to 1:300 was added for overnight incubation at 4°C after PBS rinsing. After the tissue sections were returned to room temperature and rinsed with PBS, we used the immunohistochemistry kit  for secondary antibody incubation and staining. Then, tissue sections were stained again in hematoxylin  for 3 min and terminated staining in distilled water. Tissue sections were dehydrated by sequential immersion in a gradient of ethanol and xylene. Finally, the slices were blocked with neutral resin . Figures were captured with a fluorescent microscope . The results were analyzed and calculated the average optical density (AOD) values by Image-Pro Plus . We open the picture in the software, click measure → intensity, click new → std. optical density → options → image in the intensity box, and then select the blank place in the figure, click the ok button. We change the incidental level to the value of blank place and then click measure→count/size→select colors, click the icon of the pen on the left side, select the immunohistochemistry picture of the positive protein expression region, and click close after the selection. Finally get the mean of area and mean of integrated optical density (IOD) by clicking Measure→Select Measurements, selecting iod→ok→count inside and clicking view→statistic in the table. AOD=mean of IOD/mean of area.Hey (RRID: CVCL_0297), Caov3 (RRID: CVCL_0201), 293T (RRID: CVCL_0063) and Tubal Epithelial Cells (RRID: CVCL_F597) were provided by National Collection of Authenticated Cell Cultures . Hey, Caov3, 293T and Tubal Epithelial Cells were cultured using high sugar DMEM medium  containing 10% fetal bovine serum . The growth environment was 37°C and 5% CO2 in a CO2 - Incubator .The plasmid of NOP2 knockdown was synthesized by Tsingke Biotechnology  and the plasmid has been validated by DNA sequencing. The plasmid of NOP2 and RAPGEF4 overexpression was synthesized by Genomeditech  and the plasmid has been validated by DNA sequencing. DNA sequencing report in Supplementary Material. Briefly, the shRNA primer pair for NOP2 was derived from shRNAlibrary (TRC) and inserted into pLKO.1 vector to generate the shNOP2 plasmid. The open reading frame of NOP2 was copied and inserted into PGMLV vector to generate the NOP2 overexpression plasmid. The open reading frame of RAPGEF4 was copied and inserted into the pcDNA3.1 vector to generate the NOP2 overexpression plasmid. Cells transfected with the NOP2 knockdown plasmid are referred to as shNOP2–1 and shNOP2–2, and those transfected with the control plasmid are referred to as shNC. Cells transfected with the NOP2 overexpression plasmid are referred to as NOP2, and those transfected with the control plasmid are referred to as NC. Cells transfected with both the NOP2 knockdown plasmid and RAPGEF4 overexpression plasmid are referred to as shNOP2- RAPGEF4, and those transfected with the control plasmid are called shNOP2-NC.293T was cultured in 6-cm dishes in advance to a cell density of 50%. Transfection was performed using lipofectamine 3000 reagent  according to the manufacturer’s protocol. 293T was incubated at 37°C, 5% CO2 for 48 h. Subsequently, the supernatant was collected and filtered after centrifugation at 1,000×g for 3 min by centrifuge  to obtain the lentiviral solution. The lentiviral solution was diluted in different concentration gradients  and added into six-well plates of Hey and Caov3 cells that were cultured to 40% cell density in advance, respectively. The cells were cultured at 37°C, 5% CO2 for 48 h and subsequently replaced with 4 ml of DMEM containing puromycin (2 mg/ml) for screening. After 24-48 h of incubation at 37°C with 5% CO2, the most surviving cells in the six-well plate were selected for expansion. Subsequent validation was performed by Western Blot and qPCR assays. The sequence of short hairpin ribonucleic acid (shRNA) is as follows:shNOP2–1: 5′- GACGATGCTGATACGGTAGAT −3′shNOP2–2: 5′- CACTGTACCTTCTGTCACAAA −3′Cells were incubated in RIPA lysis buffer  containing 1% phenylmethanesulfonyl fluoride (PMSF) and 0.1% protease inhibitor cocktail  at 4°C for 30 min, centrifuged at 4°C for 15 min at 13,000×g. Protein concentration was determined using BCA Protein Assay Kit  according to the manufacturer’s protocol. Finally, SDS-PAGE Protein Loading Buffer (5X)  was added at a proportion of 4:1 and incubated at 95°C for 10 min.PAGE Gel Fast Preparation Kit  was used to prepare a 10% gel. 25 ug of total protein per well was sampled and transferred to methanol-activated polyvinylidene fluoride membranes  after electrophoresis. After the end of the transfer, the membrane was put into 5% nonfat milk , blocked at room temperature for 1 h, and then rinsed three times with 0.1% Tris-HCl plus Tween-20 (TBST) for 5 min each. The membranes were incubated with primary antibody and secondary antibody, and rinsed three times after each step. The membranes were tested using the ECL luminol kit  by Chemiluminescent imaging system . The antibodies and dilutions used in the experiment are as follows:31Anti-NOP2 antibody (10448–1-AP) diluted to 1:1000, anti-GAPDH antibody (60004–1-lg) diluted to 1:50000, Anti-rabbit (SA00001–2) diluted to 1:2000 and anti-mouse (SA00001–1) diluted to 1:2000 secondary antibodies were provided by Proteintech (China); Anti-RAPGEF4 antibody (HPA028968-25UL) diluted to 1:1000 was provided by Sigma-Aldrich (USA). Relative protein levels were compared quantitatively with GAPDH using ImageJ software .Pre-denaturation: 95°C, 30s, cycle number: 1Denaturation: 95°C, 10s; annealing & extension: 60°C, 30s, cycle number: 4032. Primers’ sequences used were as follows:Melting curve stage: step 1: 95°C, 15s; step 2: 60°C, 60s; step 3: 95°C, 30s mRNA expression was normalized and calculated using the 2-ΔΔCt methodTotal RNA was extracted from Hey and Caov3 cell lines using TRIeasy™ LS Total RNA Extraction Reagent  based on the manufacturer’s protocol. RNA was reverse transcribed into cDNA using reverse transcription reagent . NOP2, RAPGEF4 and GAPDH were detected by qRT-PCR amplification using Universal SYBR qPCR Mix  under QuantStudio 6 and 7 Flex and ViiA 7 Real-Time PCR Systems . The qPCR cycling conditions were as follows:NOP2, Forward primer: 5’-TGTCTGAGCTGGTGGAGTTCTTAG-3’Reverse primer:5’-ACCCCACGATTGATTAGAGCC-3’GAPDH, Forward primer:5’-CAGGGCTGCTTTTAACTCTGGTAA-3’Reverse primer:5’- GGGTGGAATCATATTGGAACATGT-3’MeRIP-RAPGEF4, Forward primer:5’-TTTTGTTCGTATTGTTTTTTATTGC-3’Reverseprimer:5’-CATAAATCTTCCTCAACAACTATCGA-3’5C primary antibody  diluted to 1:2500 and the anti-mouse secondary antibody diluted to 1:2000. Finally, the membranes were assayed using the ECL luminol kit  by chemiluminescent imaging system .RNA was extracted from NOP2 overexpression and knockdown cells and the corresponding control cells and its mass was calculated. Then the RNA secondary structure was disrupted by incubation at 95°C for 5 min. Setting different concentration titers, RNA was added to the nylon membrane  by 2ul drops per well. After cross-linking at 254 nm UV for 30 minutes and thermal cross-linking in an oven at 60°C for 1 h, the membrane was blocked with 5% nonfat milk. This was followed by incubation with anti-mAccording to the manufacturer’s protocol, cell proliferation assays were assessed using Cell Counting Kit-8 . The cells were added to 96-well plates at 2000 cells per well and then processed at 0,24,48, and 72 hours. CCK-8 reagent was added to the cells and incubated in the incubator for 1 h. Measuring the optical density (OD) at 450 nm using the Varioskan LUX multimode microplate reader . DMEM containing 10% FBS was used as a blank control.A total of 800 transfected Hey and 1500 Caov3 cells were seeded in six well-plates during their logarithmic growth phase. They were incubated in DMEM medium containing 10% FBS at 37°C with 5% CO2 until 14 days or the vast majority of colonies with cell numbers > 40. The cells were then fixed with 4% paraformaldehyde  for 30 min and stained with crystal violet staining solution  for 20 min. Figures were captured with a camera 5 Hey cells and 200 μl of serum-free medium containing 1 × 105 Caov3 cells were added to the upper chambers, and 700 μl of DMEM medium containing 10% FBS was added to the lower layer of the chambers. Hey cells cultured for 24 hours (Caov3 cells at 48 hours) were subjected to migration assay and cultured for 48 hours (Caov3 cells at 72 hours) were subjected to invasion assay. Finally, the cells were then fixed with 4% paraformaldehyde for 30 min and stained with crystal violet staining solution for 20 min. Figures were captured with a fluorescent microscope 60 μl of Matrigel matrix  diluted to 1:6 with serum-free medium was added to the upper transwell chambers  and placed in 37°C for 1 hour to allow it to solidify before use. 200 μl of serum-free medium containing 1 × 107 cells, 100 μl PBS) were injected into the axillary region of BALB/c female nude mice. After tumor formation, tumor volume (1/2 × length × width2) was measured every 2 days. Tumor tissue was collected after 3 weeks and tumor volume and weight were measured. Tumor tissues were embedded, sectioned and stained as described above. All procedures were approved by the Experimental Animal Center of Shanghai First People’s Hospital (ethics number: 2022AW028).BALB/c female nude mice, 4–6 weeks old, were purchased from the Experimental Animal Center of Shanghai First People’s Hospital and randomly divided into two groups (seven mice in each group) for the experiments. Stable NOP2 knockdown Hey cells and the control cells  at high temperature, 94°C for 5–7 min. The fragmented RNA was incubated with reverse transcriptase  to synthesize the cDNA. These complex double strands of DNA and RNA were then converted into DNA duplexes using E. coli DNA polymerase I  with RNase H  for two-strand synthesis. At the same time, dUTP Solution  was spiked into the second strand to complement the ends of the double-stranded DNA to flat ends. A base is then added to each end to enable it to be ligated to a junction with a T base at the end, and the fragment size is screened and purified using magnetic beads. The second strand was digested with UDG enzyme  and then denatured by PCR-pre-denaturation at 95°C for 3 min, 98°C for a total of 8 cycles of 15 s each, annealing to 60°C for 15 s, extending at 72°C for 30 s, and a final extension retained at 72°C for 5 min to form a fragment size of 300 bp ±50bp (strand-specific library). Finally, it was bipartite sequenced using illumina NovaseqTM 6000  according to the standard operation, and the sequencing mode was PE150. The final data obtained were filtered to obtain high-quality sequencing data (Clean Data) by Cutadapt .33 The parameters were as follows:1. removing reads containing adapters; 2. removing reads containing polyA and polyG; 3. removing reads containing more than 5% of unknown nucleotides (N); 4. removing low quality reads containing more than 20% of low quality  bases. Then sequence quality was verified using FastQC  including the Q20, Q30 and GC-content of the clean data. We compared the Clean Data to the human reference genome using the HISAT2 (version 2.2.1) R package.34 Three biological replicates were included for each sample in this experiment. Functional analysis of DEGs was performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) using the ClusterProfiler (version 4.4.4) R package,35 and gene set enrichment analysis (GSEA) was performed using the GSEABase  R package.36 The threshold for screening DEGs is as follows: |log2(Foldchange)| > 1, q-value <0.05.RNA was extracted from stable NOP2 knockdown Hey cells and the control cells using TRIzol  according to the manufacturer’s protocol. RNA-seq performed by LC Sciences . The detailed experimental procedure is as follows: The amount and purity of total RNA was controlled with a NanoDrop ND-1000  and the integrity of RNA was examined by a Bioanalyzer 2100 . Concentrations > 50ng/μl, the value of RIN > 7.0, and total RNA >1 μg were sufficient for downstream assays. The polyadenylated (PolyA) mRNA was specifically captured by two rounds of purification using oligo (dT) magnetic beads . Captured mRNA was fragmented using the Magnesium Ion Interruption Kit  to assess the distribution of NOP2 in the cells. In brief, the apposed cells were treated with EDTA solution and collected by pipetting. Cytoplasmic proteins were obtained by adding cytoplasmic protein extraction reagent, incubating on ice for 15 min, and centrifuging at 16,000×g for 5 min. Then the nuclear extraction reagent was added and after 30 min of intermittent vigorous vortex, the nuclear proteins were obtained by centrifuging at 16,000×g for 10 min. The final obtained nuclear and cytoplasmic proteins were verified by Western Blot assay. Anti-LaminB1 antibody  diluted to 1:5000 was used for the nuclear proteins.5C MeRIP kit  was used to perform specific enrichment of m5C-modified regions in the transcriptome according to the manufacturer’s protocol. Briefly, RNA was first extracted from cells using the method proposed above. RNA was fragmented (RNA fragment size ~ 200 nucleotide), and the fragmented RNA was incubated with pre-prepared immunoprecipitated magnetic beads for 1 h. The precipitated RNA was eluted from the magnetic beads and purified. Finally, the measurement of m5C methylation enriched fragment RNA and input levels by qRT-PCR. MeRIP-qPCR-related primers for RAPGEF4 were obtained by screening through the UCSC genome browser website (http://genome.ucsc.edu/cgi-bin/hgGateway)37 and Meth Primer website (http://www.urogene.org/methprimer/).38The GenSeq@mThe CO-IP assay was completed using the Protein A+G Magnetic Bead Immunoprecipitation Kit  according to the manufacturer’s protocol. Briefly, the cells were lysed with a lysate containing inhibitors. The pre-prepared beads were incubated with the antibody for 1 h at room temperature. The samples were then incubated overnight at 4°C with the antibody-conjugated magnetic beads. Finally, the precipitated protein samples were eluted from the magnetic beads. The antibody used in CO-IP assay is Anti-NOP2 antibody  diluted to 1:30. The final result was detected by Western Blot assay.https://www.r-project.org/). P < .05 was regarded as statistically significant . All the graphs were produced using GraphPad Prism software .All experiments in this study were repeated three times. Any special cases will be described separately. All of our data were first analyzed for normal distribution by performing the Shapiro-Wilk test. The unpaired and two-tailed student’s t-tests was used for normally distributed data. The Mann-Whitne test was used for non-normally distributed data. All statistical analyses were conducted with SPSS software . Analysis of RNA-seq data and GEO data was performed by the R  or interacting with small ubiquitin-like modifier (SUMO)-2/3,40 and has been implicated in breast, cervical, gallbladder, esophageal squamous cell, and uveal melanoma cancers,41–45 as well as being related to worse prognosis in pancreatic cancer and squamous carcinoma of the head and neck.47 However, NOP2 has not been extensively studied in tumors. NOP2 has been described to enhance the cell proliferation, migration and invasive ability of colon cancer,18 and is associated with poor prognosis in renal clear cell carcinoma, gastric adenocarcinoma and ovarian cancer.49 To date, mechanistic studies of NOP2 in ovarian cancer have not yet been reported. In this study, we found that the protein level of NOP2 was higher in HGSOC than in FT. Subsequent experiments revealed that NOP2 significantly enhanced HGSOC cell proliferation, migration, and invasion in vitro, and tumor growth in vivo.RNA m50 and is relevant to the pathogenesis of glioma.51 Although RAPGEF4 is not the only target of NOP2, our experiments demonstrated that RAPGEF4 is one of the downstream genes regulated by NOP2. In our study, we found that the expression levels of NOP2 and RAPGEF4 were positively correlated, and the expression of RAPGEF4 was decreased in the NOP2 knockdown cell lines. And there is a direct or indirect relationship between NOP2 and RAPGEF4. Our results provided a possible regulatory mechanism of NOP2 and RAPGEF4 in HGSOC.We subsequently identified the cAMP signaling pathway as a major pathway regulated by NOP2 in HGSOC based on RNA-seq and KEGG analysis. The cAMP signaling pathway has a broad role in cells. EPAC proteins, including two isoforms Epac1 and Epac2, are one of its major downstream targets. Epac2 is also known as RAPGEF4. Epac proteins are involved in several biological processes, including gene transcription, cell proliferation and apoptosis, and tumorigenesis and development. RAPGEF4 can affect the sensitivity of lung cancer cells to cisplatin by altering cAMP levels through the Epac2-Rap1A-Akt pathway52 NSUN2 is currently the most extensively studied RNA methyltransferase and is primarily associated with RNA stability. The functional mechanism of NOP2 has been poorly studied to date, with current research mainly focused on nucleolus generation and cell proliferation.53 In our experiments, we found that NOP2 was involved in the proliferation, migration, and invasion of HGSOC. NOP2 is predominantly found in the nucleus, but is also present in the cytoplasm. Current studies of NOP2 have focused on its role in ribosome synthesis and processing, however, our findings suggest that there may be another way in which NOP2 acts in HGSOC that can influence the progression of HGSOC. In NOP2 knockdown cells, both total RNA level and mRNA m5C methylation level of RAPGEF4 is reduced. The elevated expression of RAPGEF4 rescued the decrease in cell proliferation, migration, and invasion caused by reduced NOP2 expression. Further experiments revealed a direct or indirect relationship between NOP2 and RAPGEF4. Based on these results, we propose a regulatory mechanism of NOP2 on RAPGEF4 that is dependent on the m5C methylation level.According to previous studies, RNA methyltransferases have been demonstrated to have a major function in mRNA transcription, translation and nuclear export, particularly in tumors.5C methylation levels.In this study, we explored the effect of NOP2 on the cAMP signaling pathway in HGSOC cells and found an effect of NOP2 expression on the prognosis and proliferation of HGSOC. Eventually, we demonstrated that there might be a regulatory mechanism between NOP2 and RAPGEF4 dependent on mClick here for additional data file.


An effective loss-of-function study is necessary to investigate the biological function of long non-coding RNA (lncRNA). Various approaches are available, including RNA silencing, antisense oligos, and CRISPR-based genome editing. CRISPR-based genome editing is the most widely used for inactivating lncRNA function at the genomic level. Knocking out the lncRNA function can be achieved by removing the promoter and the first exon (PE1), introducing pre-termination poly(A) signals, or deleting the entire locus, unlike frameshift strategies used for messenger RNA (mRNA). However, the intricate genomic interplay between lncRNA and neighbor genes makes it challenging to interpret lncRNA function accurately. This article discusses the advantages and disadvantages of each lncRNA knockout method and envisions the potential future directions to facilitate lncRNA functional study. While deleting the promoter and first exon of lncRNA has been extensively utilized and demonstrated efficacy in deactivating lncRNA function, the intricate nature of lncRNA gene loci and their interactions with nearby or overlapping genes pose challenges in achieving precise lncRNA knockouts without affecting the expression of nearby or overlapping genes (Long non-coding RNA (lncRNA) is a type of RNA transcript that is over 200 nucleotides long and lacks protein-coding potential. Like messenger RNA (mRNA), lncRNA is transcribed mainly by RNA polymerase II and undergoes post-transcriptional modifications . The useng genes . In thisMost lncRNAs are transcribed by RNA polymerase II, which undergoes post-transcriptional modifications such as 5′capping, 3′poly(A) tailing, and intron splicing . LncRNAsLINC00948 lncRNA encodes a peptide called MLN, which interacts with SERCA to regulate the phenotype in skeletal muscle  and Cas proteins , with del muscle .XIST, HOTAIR, ANRIL, etc., interact with PRC2 components in the nucleus, regulating H3K27 methylation and gene silencing. These lncRNAs play an essential role in dosage compensation, imprinting, and histone accessibility  and modulates the surface localization of cadherin 5 (CDH5) (in-cis interacting lncRNA. The lncRNA Upperhand (Uph) and the protein-coding gene Hand2 localize on chromosome 8 in the mouse genome in a divergent direction  . This in5 (CDH5) . Anotherirection . There igulation .XIST, was discovered in the cell nucleus that dominates the X-inactivation by silencing one of the two female X chromosomes and preventing the dosage effect  inhibition, and CRISPR-based genome editing . CRISPR-in vitro transcribed (IVT) RNA, and purified protein  signal downstream of the TSS, which causes the detachment of RNA polymerase II from the DNA strand and disassembly  Liu et . Only ontrans- and cis-action. The BESST knockout technique could distinguish between sequence-dependence and transcription-dependence in the mechanism of lncRNAs across their genomic loci , including head-to-head, tail-to-tail, and entirely overlapped . Therefomic loci . Howevermic loci .in vivo, including RNA silencing and antisense oligo  or 6-thioguanosine (6SG)  . The infMoshe is detected in the cardiac mesoderm stage (E8.5 to E9.5) along with the expression of Gata6, and continues to be expressed and increases in the atrioventricular septum (E12.5)  lineage genes, which may play a critical role in ASD. Another level of spatial expression of lncRNAs is subcellular localization. LncRNAs exhibit distinguished biological functional and underlying mechanisms in different cell compartments. Localized lncRNAs in the cell nucleus are primarily involved in epigenetic and transcriptional regulation, whereas cytoplasmic lncRNAs are typically involved in post-transcriptional regulation, protein-protein interactions, etc.  . Knock-dns, etc. . Accordins, etc. .Last, but not least, the expanding range of RNA-binding proteins (RBPs) also broadens our view of lncRNA functioning. RBPs are proteins containing one or more RNA binding domains (RBDs) that can directly interact with RNA strands and alter their structure and function . Several