IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2–RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets. IGF2 mRNA-binding protein 1 (IMP1) is a conserved RNA-binding protein that plays a key role in regulating cell motility, morphology and differentiation in the embryo, reviewed by Yisraeli . In adulAt the molecular level, IMP1 regulates the transport, translation and stability of a diverse ensemble of messenger RNAs (mRNAs). In neurons, the best-studied function of IMP1 is its role in mediating the transport and controlled translation of β-actin mRNA ,6. IMP1 IMP1 contains six putative RNA-binding domains—two RNA-recognition motifs (RRMs) and four K-homology (KH) domains Figure —which arThe RNA recognition properties of the KH3KH4 di-domain and its interaction with the β-actin RNA target have been extensively characterized ,18–20. Tet al.  were obtained by expressing KH1KH2 as a N-terminal 6xHis-GST-KH1KH2 fusion protein in Escherichia coli BL21 (DE3) cells (Invitrogen) grown in M9 minimal media supplemented with 15NH4Cl and 12C or 13C-glucose (as required) as the sole nitrogen and carbon sources, while unlabelled samples were obtained by expressing the protein in LB media. The protein was initially purified by immobilised metal affinity chromatography (IMAC) with a Ni-NTA (nitrilotriacetic acid) agarose matrix (ThermoFisher scientific). The His-GST fusion tag was then removed by cleavage with TEV protease and KH1KH2 further purified using a cation-exchange HiLoad SP-Sepharose 26/10 column and then dialyzed into the final NMR buffer (10 mM sodium phosphate (pH 7.4), 50 mM NaCl, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine)). For crystallization and biolayer interferometry (BLI), unlabelled protein was obtained from cells grown in LB media and purified as above with the addition of a final size exclusion chromatography purification step using a HiLoad Superdex75 16/600 column. Protein concentration was determined from the absorbance of the sample at 280 nm and extinction coefficients calculated from the Tyr content of the sequence. In addition to the wild-type protein, two GDDG mutants  were prepared using a QuickChange II Site-directed Mutagenesis Kit (Agilent), sequence-verified by Sanger sequencing , and expressed and purified as above.The KH1KH2 di-domain V194-N369) from human IMP1 (Uniprot Q9NZI8) was expressed and purified in a similar way to that described by Nicastro 4-N369 fr−1  and the concentration determined by measuring absorbance at 260 nm and extinction coefficients calculated from the base composition of the sequence.RNA oligonucleotides were purchased from Dharmacon , de-protected according to manufacturer’s protocol and lyophilized. The RNA was re-suspended in either nuclease-free water or ITC  buffer containing 40–100 u ml1H frequency. All NMR spectra were transformed using NMRpipe  of IMP1 KH1KH2 was estimated from the T1/T2 ratio of residues L200-E352 as described previously, where residues with overlapping resonances were excluded as they could not be fitted reliably. The order parameter (S2) per residue was estimated using a model-free analysis, performed in the program TENSOR assuming an overall isotropic motion . RDCs values were obtained by subtracting the reference value in isotropic solution from the values in anisotropic conditions. The experimental dipolar couplings for the individual amides were then compared to RDCs back calculated from the crystal structure of KH1KH2 using the program Module  were extracted by In-Phase and Anti-Phase (IPAP) experiments  recordedm Module .http://www.ebi.ac.uk/Tools/msa/tcoffee/). Alignment figures were generated by using Jalview / ClustalX.Sequence alignments were made using the T-Coffee multiple sequence alignment program  . The pH was monitored to ensure that it remained within ± 0.1 pH unit of the initial condition. 15N-SOFAST-HSQC spectra were recorded at each point of the titration, and the chemical shift changes of amide resonances in fast exchange were measured and the reported weighted-average values of 15N and 1H chemical shift changes given by Equation  1–4 mM) to 50–75 μM –4 mM to 15N-SOFAST-HSQC experiments in a semi-automated fashion . S fashion . Briefly fashion . Briefly−1 solution of KH1KH2 in 10 mM sodium phosphate, 50 mM NaCl, 0.5 mM TCEP pH 7.4 was mixed in a 1:1 ratio with a mother liquor containing 51.4% PEG1000, 150 mM MOPS (3-(N-morpholino)propanesulfonic acid) (pH 7.0), 60 mM NaI and 4% acetonitrile. Drops were equilibrated with a reservoir of the crystallization solution in a sealed well of a 96-well plate at 18°C. Crystals appeared within 2–3 days and were further optimized by microseeding under the same conditions. Crystals were harvested by transferring into fresh crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen. The crystals belong to the spacegroup P21 with one copy of KH1KH2 in the asymmetric unit (AU). X-ray diffraction data were collected at 100 K at the I02 beamline at Diamond Light Source  and the data processed using the XDS software suite . The equilibrium dissociation constant was determined from each titration series by fitting the measured heat of reaction using a 1:1 binding model, as only one KH domain is capable of binding RNA in the constructs used. The heat of dilution of each RNA oligonucleotide was determined by titration into ITC buffer without protein and was subtracted from each corresponding titration curve prior to fitting the data.All ITC experiments were recorded at 25°C using a MicroCal ITC200 calorimeter  and data were fitted using the Origin 7 package. ITC titrations were performed by successive injections of 2 μl of RNA solution, 0.5 mM for KH1KH2DD titrations or 1 mM for KH1DDKH2 titrations into a cell containing 50 μM of IMP1 KH1DDKH2 or IMP1 KH1KH2DD in 10 mM sodium phosphate (pH 7.4), 50 mM NaCl, 2 mM TCEP and 40–100 u ml−1 bovine serum albumin and RNAse inhibitor 40–100 u ml−1 . Experiments were recorded using a ForteBio OctetRed 96 instrument. 5′-Biotinylated c-myc mRNA-derived RNA (0.5 ng μl−1) was immobilized on Streptavidin-coated biosensors and incubated with varying concentrations (1–0.03 μM) of wtKH1KH2, or KH1DDKH2 or KH1KH2DD mutants. Equilibrium dissociation constants for RNA–protein interactions were determined from plots of the observed association rate constant (kobs) as a function of protein concentration (Bio-layer interferometry (BLI) experiments were performed at 30°C in 10 mM sodium phosphate (pH 7.4), 50 mM NaCl, 0.5 mM TCEP, with 0.5 mg mlntration  using inntration .kon, koff and Kd are the association rate constant, dissociation rate constant and equilibrium dissociation constant for the binding of KH1KH2. Our BLI data determine these values as 1.01 (± 0.054) × 106 M−1s−1, 0.047 (± 0.008) s−1 and 46 (± 8) × 10−9 M, respectively.kon1, koff1 and Kd1 are the association rate constant, dissociation rate constant and equilibrium dissociation constant for the binding of KH1KH2DD. Our BLI data determine these values as 2.74 (± 0.03) × 105 M−1s−1, 0.483 (± 0.016) s−1 and 1.76 (± 0.206) × 10−6 M, respectively.kon2, koff2 and Kd2 are the association rate constant, dissociation rate constant and equilibrium dissociation constant for the binding of KH1DDKH2. These values are not available from the BLI experiments.kC1 is the forward rate for the ring closure step, which follows after the binding of KH2 and kO1 is rate for the ring opening step, where KH1 dissociates whilst KH2 remains bound. As outlined in Nicastro et al. .o et al. , we havekC2 is the ring closure step, which follows after the binding of KH1 and kO2 is the ring opening step, where KH2 dissociates whilst KH1 remains bound. As outlined in Nicastro et al.  as the domains are structurally very similar. The values we used were 2.74 × 105 M−1s−1 / 3 ( = 9.1 × 104 M−1s−1), 2.74 × 105 M−1s−1 and 2.74 × 105 M−1s−1 × 3 ( = 8.22 × 105 M−1s−1).For Kd2 and kon2  in order to find a parameter-set that reproduced the kinetics of binding of the KH1KH2 wild-type.The calculations below were performed for the nine possible combinations of Each of the two possible binding pathways for formation of the closed complex (KH1KH2–RNA) is a reversible bimolecular interaction followed by a conformational change. Therefore, consider first the pathway for formation of the closed complex in which the KH1 domain attaches first  a value for kC2 can be calculated using:Because we have assumed that kC2 values for each of the nine combinations of Kd2 and koff2 . This value depends on the value of kon2, but not on the value of Kd2. The tested kon2 values were 9.1 × 104 M−1s−1, 2.74 × 105 M−1s−1 and 8.22 × 105 M−1s−1, as discussed above, and the resulting koff values were respectively 0.017, 0.026 and 0.049 s−1. As the value of the observed koff is 0.047 s−1, a kon2 value of 8.22 × 105 with a Kd2 value of 100 × 10−6 M adequately reproduces our data.All combinations of constants should give the 5 M−1s−1 is the best kon2 value to reproduce the data and calculate an interval of confidence. We therefore performed simulations using kon2 values on either side of 8.22 × 105 M−1s−1, i.e. 5, 6, 7, 8, 9, 10, 11 × 105 M−1s−1 and tested how the kobs and koff we have calculated compared with those obtained by BLI . This comparison  or on the dissociation rate constant (koff).Next, we wanted to establish whether 8.22 × 10mparison  confirmsKd2  is much >KdRegardless, the latter values and corresponding errors can be calculated as follows:kC1 = Kd2 x koff1 / KdTherefore koff1 / Kd = 10.5 ± 1.85 so thatKd2 = 100 × 10−6 M then kC1 = 1050 +/- 185 s−1Kd1-Kd) / Kd = 37.3 ± 7.9Then, the propagation of errors results in kC2 = koff2 × 37.3 ± 7.9 s−1So that Kd2 = 100 × 10−6 M and kon2 = 8 × 105 M−1s−1 (see above) then koff2 = 80 s−1 and kC2 = 2985 ± 632 s−1.If we use These calculated and BLI-derived values (reported in the ‘Results’ section and figures) have been rounded for both a clearer presentation and to be more representative of the experimental precision of measurements.Simulations were performed by numerical integration of the system of ordinary differential equations associated with the model presented here. We used in-house software employing the fourth-order Runge–Kutta method as described in . The com2. Interestingly, many of the amino acids mediating the contacts between the two α3 helices  are conserved in KH1KH2 across species but are very different in KH3KH4  between the two datasets, indicating that the crystal structure is highly representative of the protein conformation in solution.To validate the key contacts between KH1 and KH2 structural elements, we used NMR spectroscopy and assess whether the inter-domain orientation of KH1 and KH2 in the crystal structure corresponds to the conformation in solution. First, using diagnostic NOE cross-peaks from e linker . Then, we Figure  reveals 15N T1 and T2, and 1H-15N NOE experiments and calculated a KH1KH2 rotational correlation time (τc) of 9.8 ± 0.6 ns. This τc is consistent with a globular or quasi-globular protein of ∼20 kDa, the molecular weight of the KH1KH2 di-domain. Also consistent with the structure, the relaxation experiments reported that the inter-domain linker is not flexible but tumbles coherently with the two domains. By contrast, significant motions are observed in the GxxG and variable loops of KH2, which are often flexible in KH domains , where the conserved RNA-binding GxxG-loop of KH2 was mutated to GDDG. The two negatively charged residues prevent the interaction between the loop and the backbone of the RNA and abolish RNA binding by KH2. This mutant can then be used to study directly the KH1–RNA interactions in the context of the entire KH1KH2 structural unit. Fingerprint 1H-15N HSQC NMR experiments and CD-monitored temperature unfolding of the protein confirmed that this double mutation does not disrupt the structure of the domain or cause significant changes in stability (Kd = 0.5 μM to Kd > 200 μM). At position 1, replacement of C with any other nucleotide results in around a 20-fold loss of affinity. Overall, the ITC analysis of the binding of all sequence variants displays a trend that mirrors the SIA results  experiments Figure . The titKd > 50 μM for all tested pentamers Kd between 50 and 150 μM) no binding at all could be measured for other combinations. Therefore, no absolute specificity could be determined in our assays at any of the four positions. Figure Kd of 100 μM). As for KH1, NMR CSP mapping of the protein–RNA interactions confirmed that the RNA binds in the canonical RNA-interaction groove of the domain, with minor contacts been made with the carboxy-terminal of α3  for the concentration-dependent binding as a function of protein concentration to obtain kinetic constants (kon1 = 2.74 ± 0.03 × 105 M−1s−1 and koff1 = 0.48 ± 0.02 s−1) and therefore the affinity (Kd1 = 1.76 ± 0.2 μM) of the KH1–RNA interaction , while the kinetics of the interaction show that the association and dissociation of the domain are faster than those of KH3 and KH4 . Equivalent experiments were recorded on the KH1DDKH2 mutant to assess KH2 binding, but we could not obtain a clear concentration dependent association, most likely because of the low affinity of this domain for MYCRNA.The BLI interferogram series recorded using immobilized MYCRNA and increasing concentrations of KH1KH2DD displayed a fast, concentration dependent, association phase Figure . We analn Figure . This ankon = 1.01 ± 0.05 × 106 M−1s−1 and a koff = 0.05 ± 0.01 s−1, resulting in a Kd of 46 ± 8 nM , as is typically observed in multi-domain RNA recognition, it is however nearly two orders of magnitude stronger than that observed in similar di-domain units, as discussed below. While no values are reported in the literature for the affinity of KH1, KH2 or KH1KH2 for RNA, a full-length IMP1 protein with mutations that impair RNA binding in KH3 and KH4 has been reported to bind RNA with a Kd ∼ 60 nM (6 M−1s−1) with the RNA and a slower dissociation rate constant (∼0.05 s−1).Having explored the affinity and kinetics of individual KH1 and KH2 domains, we analysed the interaction between the wild-type protein KH1KH2 and the MYCRNA, where both domains can engage in the interaction. As observed for KH1 binding, the interferograms showed a fast, concentration-dependent association whose analysis yielded a M Figure . This re ∼ 60 nM . Our stukC1 and kC2, that describe the conformational rearrangements taking place in the two different pathways  and the observed association rate at 0.25 μM added protein (kobs).Next, we employed a kinetic simulation to describe the two-step KH1KH2–RNA interaction in order to provide a mechanistic insight into RNA recognition and re-modelling. In addition, these calculations allowed us to derive a number of important parameters that are not accessible experimentally. In the model, the KH1KH2–RNA interaction is considered as a reversible bi-molecular interaction followed by a concentration-independent conformational change. This can be visualized as the binding of RNA to either KH1 or KH2 followed by looping of the RNA and then a second RNA-binding event at the unfilled site Figure . This mes Figure . The kinkon2 = 8.0 ± 2 × 105 M−1s−1 provides the best fit to the koff and kobs values discussed above  and kC2 (3000 ± 600 s−1), that are a result of the strong coupling between domains, compensate for the short lifetime of the KH2–RNA interaction (koff2 = kO2 ∼ 80 s−1) and lead to a high-affinity (Kd ∼ 46 nM) KH1KH2–RNA interaction.In practice, we tested a range of values for the estimated kinetic constants . This test established that a ed above . AssuminKd1 and Kd2 an alternative complex where a single RNA molecule binds two protein molecules cannot form in significant amounts. Finally, at this protein to RNA stoichiometry, the amount of bound RNA depends on the protein, not the RNA concentration. Overall, our calculations describe previously inaccessibly kinetic parameters and provide a mechanistic insight in KH1KH2–RNA binding.Our simulations require estimates of the protein and RNA concentrations in living cells. The cellular concentration of IMP1 has been quantified in the sub-micromolar range both in a line of highly proliferating cancer cells (K562)  and in dThis work explores the structure and RNA-binding properties of the KH1 and KH2 domains of IMP1 and builds a mechanistic model for RNA recognition. IMP1 function is mediated by the recognition of a diverse ensemble of functional RNA targets and the KH domains have target-dependent roles in IMP1–RNA recognition . Therefode novo from the transcriptome-wide binding sites of a protein is challenging. However, prior knowledge of the target sequence provides a tool to separate different binding modes and ascribe functional implications, as recently shown for the cancer factor RBM10 , the lifetime of KH3KH4 is ∼14 times longer than that of the KH1KH2–RNA complex . The loKd ∼1 μM. In contrast, in KH1KH2 the affinity of KH2 (Kd∼100 μM) is ∼2 orders of magnitude lower than that of KH1 (0.5 μM) and this could influence the RNA-binding mode and its capacity to re-model the RNA. However, we show that, despite the low-binding affinity of KH2, KH1KH2 binds RNA with an overall Kd in the nanomolar range. Our simulations explain that in the second step of KH1KH2 binding, a very fast ‘closing’ of the RNA loop compensates for the short lifetime of the KH2–RNA interaction. Arguably, one of the determinants of this is the fast association of the KH2 domain with RNA, which is 10 times faster than any of the other three KH domains. Other contributions to overall affinity may derive, for example, from positively charged residues on the protein surface (An important consequence of IMP1–RNA binding is the re-modelling of the local RNA structure. The well-described RNA re-modelling by the KH3KH4 di-domains ,18, toge surface , which cThe IMP1 KH1KH2 coordinates and structure factors have been deposited in the Protein Data Bank under accession number 6QEY. NMR resonance assignments have been deposited in the Biological Magnetic Resonance Data Bank under accession number 27777. The plasmid for the expression of IMP1KH1KH2 is available from the authors upon request. The computer code for the kinetic simulations of IMP1 KH1KH2–RNA interactions is available from the authors upon request.Supplementary DataClick here for additional data file.


FecB ++ genotype. The results suggested that several key hypothalamic circRNAs may participate in sheep reproduction by influencing gonadotropin-releasing hormone (GnRH) activities or affecting key gene expression indirectly or directly. This study provides a further reference for understanding the differences of sheep fecundity.The hypothalamus plays crucial roles in sheep reproduction. However, the expression profiles of sheep hypothalamic circular RNA (circRNA), which has been proved to exert important functions in many physiological processes, remain largely unknown. In this study, we used RNA sequencing to explore the expression of circRNAs in the hypothalamus of sheep with the FecB ++ genotype. We totally identified 41,863 circRNAs from sheep hypothalamus, in which 333  were differentially expressed in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF), moreover, 340 circRNAs  were differentially expressed in polytocous sheep in the luteal phase versus monotocous sheep in the luteal sheep (PL vs. ML). We also identified several key circRNAs including oar_circ_0018794, oar_circ_0008291, oar_circ_0015119, oar_circ_0012801, oar_circ_0010234, and oar_circ_0013788 through functional enrichment analysis and oar_circ_0012110 through a competing endogenous RNA network, most of which may participate in reproduction by influencing gonadotropin-releasing hormone (GnRH) activities or affecting key gene expression, indirectly or directly. Our study explored the overall expression profile of circRNAs in sheep hypothalamus, which potentially provides an alternative insight into the mechanism of sheep prolificacy without the effects of FecB mutation.Circular RNA (circRNA), as an emerging class of noncoding RNA, has been found to play key roles in many biological processes. However, its expression profile in the hypothalamus, a powerful organ initiating the reproductive process, has not yet been explored. Therefore, we used RNA sequencing to explore the expression of circRNAs in the hypothalamus of sheep with the  Sheep is an important agricultural species, the reproductive traits of which are strictly controlled by reproduction-related hormones, such as gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) . In thisBMPRIB) gene (A746G) and shown to be significantly associated with sheep reproduction. FecB has been found in many sheep breeds [FecB mutation can significantly increase little size by 1, and two copies can increase by 1.5 [FecB BB genotype (two copies of FecB mutation), the FecB B+ genotype (one copy of FecB mutation), or the FecB ++ genotype (without FecB mutation). Sheep with FecB ++ normally have one offspring, but cases of such sheep having two or even three offspring have also been observed [FecB was the first mutation identified in the bone morphogenetic protein receptor IB  of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (IAS-CAAS) , and ethical approval was given by the Animal Ethics Committee of the IAS-CAAS (No. IASCAAS-AE-03).FecB ++ genotype were then divided into a monotocous group  and a polytocous group  based on litter size records . All ofAll of the selected STH sheep were treated with CIDR  for 12 days. Six sheep including three monotocous and three polytocous ones were slaughtered within 45–48 h of CIDR removal (follicular phase); the remaining six sheep were slaughtered in the same way on day 9 after CIDR removal . Therefore, all of the STH sheep were categorized as polytocous sheep in the follicular phase (PF), polytocous sheep in the luteal phase (PL), monotocous sheep in the follicular phase (MF), or monotocous sheep in the luteal phase (ML).The tissue of the whole hypothalamus was obtained immediately after sheep slaughter and stored in liquid nitrogen (−80 °C) for RNA extraction. Total RNA was isolated from the 12 hypothalamic samples using the TRIzol Reagent  in accordance with the manufacturer’s instructions. To obtain high-quality RNA, 1% agarose electrophoresis and an Agilent 2100 Bioanalyzer  were used to examine the integrity and concentration of the extracted RNA. The purity of isolated RNA was also ensured using an Agilent RNA 6000 Nano Kit (Agilent Technologies).All the sequencing data were obtained from Annoroad Gene Technology Co., Ltd. . Samples of 3 μg of total RNA from each hypothalamus were pooled to construct the RNA library. First, ribosomal RNA was removed using the Ribo-Zero™ Gold Kit . Then, a fragmentation buffer was added to break the RNA into fragments, which were used as templates to synthesize the first strand of complementary DNA. The second strand of cDNA was also synthesized in the presence of dNTPs, ribonuclease H, and DNA polymerase I. The obtained double-stranded cDNA was processed with end-repair, the addition of base A and sequencing adaptors, and Uracil-N-Glycosylase (UNG) enzyme digestion. Subsequently, the polymerase chain reaction was performed to construct an RNA library. Finally, the desired RNA fragments were selected for sequencing (Illumina HiSeq 2500).The clean reads were obtained from the raw reads after removing the reads with adaptor contamination , low-quality reads , and reads with a rate of N  greater than 5%.http://www.ensembl.org/index.html). Then, the BWA-MEM algorithm, which can align reads to the genome rapidly and efficiently and also supports the mapping segmental alignment of sequences to genomes, was applied to map clean reads to the Ovis aries reference genome (Oar v.3.1). Subsequently, a “CircRNA Identifier (CIRI)” strategy [First, the reference genome and annotation files were downloaded from ENSEMBL   was usedp < 0.05, which was determined by the hypergeometric test method, was considered to reflect significant enrichment.We performed Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses using host genes of circRNA due to a lack of circRNA annotation. A particular GO term or KEGG pathway with http://www.mirbase.org/index.shtml) was searched to identify sites on DE circRNAs to which miRNAs bind to build a circRNA-miRNA interactome using Cytoscape software [http://www.targetscan.org/vert_72/).Many studies have reported that circRNAs could function as sponges of miRNAs to modulate gene expression ,21. To esoftware . We also®480II system . Finally, the data obtained from RT-qPCR were calculated with the normalization of β-actin  were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.  . Then, reviously ).We obtained a total of 1,460,254,556 raw reads after sequencing; the mapped reads numbered 1,459,435,298, so the mapping rate in each sample reached nearly 100% . We idenp < 0.05 to identify DE circRNAs. In total, we identified 333 DE circRNAs in polytocous sheep in the follicular phase versus monotocous sheep in the follicular phase (PF vs. MF), where 162 were upregulated, while 171 were downregulated , a key member of the SMAD family, was shown to be a host gene of oar_circ_0018794 and particularly associated with the TGF-β signaling pathway. Moreover, SMAD2 was found to be highly enriched in rat hypothalamus [FGF2), which is a source gene of oar_circ_0008291 and enriched in the MAPK signaling pathway, is a critical neurotrophic factor and mitogen for hypothalamic cells in vitro, and its expression decreases at the beginning of puberty in female rats [FGF2 significantly increased nearly twofold after fasting in rats [FGF2 may cooperate with leptin to mediate GnRH activities, although the detailed mechanisms involved require further validation.CircRNAs have been revealed to act at the promoter region of host genes to enhance their transcription by interacting with U1 small nuclear ribonucleoproteins (snRNPs) and RNA polymerase II . Therefothalamus  and to pthalamus  and mousthalamus . In addithalamus , indicatthalamus . Fibroblale rats , moreove in rats . Interes in rats . Conside in rats , we specARAP2), which was enriched in pathway of endocytosis and was a host gene of oar_circ_0034134, was found to reduce glucose uptake and affect sphingolipid metabolism after ARAP2 knockdown [ARAP2 knockdown also mediated the cytoplasmic signaling of estrogens [EIF5) and ATR serine/threonine kinase (ATR), both of which generate the circRNAs oar_circ_0010234 and oar_circ_0013788. EIF5 was reported to promote GTP hydrolysis and translation initiation complex assembly in eukaryotic translation initiation [ATR could be a sensor to respond to DNA damage [ATR results in early embryonic lethality in mice [EIF5 and ATR, may be enhanced by circRNAs to promote the expression of some other genes related to hormone activities such as GnRH release.Regarding PL vs. ML, the most enriched pathway was endocytosis, which mainly mediates the responses to receptor activity . ArfGAP nockdown . In addinockdown . It shoustrogens , which mitiation ,46, and itiation . ATR couA damage  and be rA damage . Additio in mice . TherefoDIAPH1) was also reported to mediate the biosynthesis of cortisol by modulating mitochondrial trafficking [DIAPH1. Further experiments may confirm its functions in mediating GnRH pulsatile secretion.The competitive endogenous RNA network, as an emerging strategy, has been widely used to understand complex processes such as cancer and reproduction ,52. In officking . In addifficking , suggestIn this study, we established the first circRNA expression profile in sheep hypothalamus. We also identified several key circRNAs, such as oar_circ_0018794, oar_circ_0008291, oar_circ_0015119, oar_circ_0012801, circRNAs-oar_circ_0010234, and oar_circ_0013788, through functional enrichment analysis and oar_circ_0012110 through a ceRNA network. Most of these circRNAs may function by influencing GnRH activities or altering key gene expression indirectly or directly. This study presents an integral circRNA analysis in sheep hypothalamus and provides a reference for understanding sheep prolificacy.


Salmo trutta L.) liver. The storage time and temperature had an effect on RNA integrity, but RNA suitable for quantitative reverse transcription PCR (RT-qPCR) (RIN > 7) was still obtained from samples preserved at − 20 °C for 6 months. Freeze-thaw cycles of tissue or RNA did not compromise the integrity of RNA. RNA degradation had an effect on RT-qPCR results, and the effect depended on gene. The RT-qPCR analysis of historical samples from a bleached kraft pulp mill effluent exposure in 1984 revealed no significant cyp1a induction. Recommendations are given for the preservation and handling procedures of samples designated for transcriptomic analyses.As transcriptomic studies are becoming more and more common, it is important to ensure that the RNA used in the analyses is of good quality. The RNA integrity may be compromised by storage temperature or freeze-thaw cycles, but these have not been well studied in poikilothermic fishes. This work studied the effects of tissue storage time and temperature, and freeze-thaw cycles of tissue and extracted RNA on RNA integrity in brown trout ( The studies using transcriptomics are becoming more and more common in ecotoxicology. To make sure that the results are trustworthy, it is absolutely crucial to use good-quality RNA in the analyses. The RNA is considered fragile, and the recommendation is to flash-freeze the samples in liquid nitrogen, to preserve them at − 70 °C or lower, and to avoid freeze-thaw cycles both of the tissue sample and of the extracted RNA  tissues and found that the effect depended on tissue, liver being the tissue with very rapid RNA degradation  was preserved at room temperature, fridge, normal freezer, deep freezer, or liquid nitrogen for various time periods up to 6 months. In addition, the effect of freeze-thaw cycles on RNA integrity was studied by analyzing RIN after repetitive thawing and freezing liver tissue and purified RNA. The effect of RNA integrity on RT-qPCR results was investigated by room temperature incubation of liver samples, resulting in RNA with varying RINs. RNA was also extracted from historical samples that had been stored for 28 years in liquid nitrogen, and RT-qPCR run for them.The aim of this study was to investigate how tissue handling, storage time, storage temperature, and freeze-thaw cycles affect the quality of RNA of fish liver. Liver tissue of brown trout  with the mean body weight of 1006 g in Laukaa fish farm of the Natural Resources Institute Finland  in November 2012. The brown trout were taken from water of < 5 °C and the sampling was performed in a low-temperature room. The fish were stunned with a blow to the head, weighed, and their length was measured, after which they were sacrificed by nuchal break. Livers were removed and cut to 50–100 mg pieces randomly, avoiding connective tissue areas. They were placed in microcentrifuge tubes, and either immediately frozen in liquid nitrogen or kept on ice. In addition, larger liver samples of up to 1.5 g were taken, and immediately frozen and preserved in liquid nitrogen. The samples were transferred to the University of Jyväskylä (4 h) either in an ice bath or frozen in liquid nitrogen, and then stored at room temperature (+ 20 °C), fridge (+ 4 °C), normal freezer (− 20 °C), or deep freezer (− 80 °C). The RNA extraction was carried out after 1, 5, 31, and 180 (6 months) days of storage. The transfer and storage conditions are summarized in Table The new liver tissue samples were taken from six four-year-old male brown trout . The uppermost supernatant was transferred to a new tube, and the RNA was precipitated by adding 500 μl isopropanol. After centrifugation , the precipitate was washed twice with 75% ethanol. The precipitates were stored under 75% ethanol at − 20 °C until processed further. After the ethanol was removed, the precipitates were let dry for 5 to 10 min at room temperature and dissolved in 200 μl of nuclease-free water (Thermo Fisher Scientific Inc.). The dissolved RNAs were stored at − 80 °C.The RNA was extracted with Tri reagent (Molecular Research Center) according to the manufacturer’s protocol. Pieces of liver (50–100 mg) were homogenized with plastic homogenization sticks in 1 ml Tri reagent in microcentrifuge tubes. After 5 min incubation at room temperature, 200 μl chloroform was added and the tube was shaken vigorously. The tubes were let stand for 5 to 15 min at room temperature, after which they were centrifuged  and purity  of the RNAs were measured with NanoDrop 1000 (Thermo Fisher Scientific). When necessary, the RNAs were diluted to reach the concentration suitable for the RNA integrity analysis (25–500 ng).The concentration  and BioAnalyzer (Agilent) according to the manufacturer’s instructions. The software did not need modification for assessing RINs in brown trout samples.Brown trout liver tissue (ca. 1.5 g pieces) stored in liquid nitrogen was cut to 50–100 mg pieces when still frozen , was taken to room temperature and let to melt. Pieces of 50 to 100 mg were taken at 0 min (when the tissue was still frozen), and at 10, 20, 30, 40, 50, 60, 80, and 100 min, and the RNA was extracted with Tri reagent as described above.cytochrome P450 1a was chosen as the target gene. The primers were designed with AmplifX  and checked for specificity with Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The features of the primers are shown in Table 60S ribosomal protein l17 (rl17) and nadh dehydrogenase 1 alpha subcomplex subunit 8 (ndufa8) were chosen as reference genes because their expression in effluent-exposed and control brown trout was the most stable of a set of reference genes tested .Quantitative reverse transcription PCR (RT-qPCR) runs were conducted for all room temperature incubated samples, for three new trout samples, and for historical samples that had a RIN ≥ 6. As the historical pulp and paper mill effluents were known to contain substances that induce its transcription, Ts) were always over 38. Melt curves showed a single peak, confirming formation of only one PCR product. The expression values were calculated with the 2C−ΔΔT method, as the efficiencies of all genes were almost identical and close to 100%  according to the manufacturer’s instructions. The RNA was reverse transcribed to cDNA , after which the cDNAs were diluted 1 + 9 with sterile water. Each RT-qPCR reaction consisted of 5 μl of the diluted cDNA, 0.75 μl of forward and reverse primers , 6 μl sterile water, and 12.5 μl iQ SYBR Green Supermix (Bio-Rad). The reactions were run in duplicates in clear 96-well PCR plates (Bio-Rad), on a CFX96 Real-Time PCR cycler (Bio-Rad). The protocol was 3 min at 95 °C; 40 cycles ; 10 s at 95 °C and melt curve from 65 to 95 °C. No template controls were run for all genes on all plates, and their cycles of threshold , using the data from the room temperature incubated historical liver samples. Analysis of covariance (ANCOVA) was used to compare the linear regressions for CT as a function of sample melting time between different genes  to determine whether they were statistically similar. One-way ANOVA with repeated measures was used to test the effect of melting time on RIN. Because the data violated the assumption of sphericity, a Greenhouse-Geisser correction was used.Linear regression curves for C10 transformation was performed for the expression values to meet the requirements of ANOVA . One-way ANOVA followed by Tukey’s test was used to test whether the effluent exposure had an effect on the levels of cyp1a mRNA. IBM SPSS Statistics 20 was used for the statistical analyses.Logp < 0.001 for both). The RINs were different between all storage temperatures . Transfer temperature (ice/liquid nitrogen for 4 h) had an effect on RIN in samples stored at − 20 °C  but not in those stored at − 80 °C .High storage temperatures of liver samples resulted in poor-quality RNA, whereas all storage temperatures below zero yielded RNA with RIN > 7 Table . Even thFreeze-thaw cycles had no effect on RNA integrity. The RNA extracted from liver samples that had been thawed five times was still intact with RIN > 9.5 Table . Similarp = 0.001)  and cyp1a and rl17  . The more degraded the RNA (the lower the RIN) was, the higher the CT value of every gene, which means that less target was present in the samples with a lower RIN  Fig. . When qP18) Fig. , Table 4cyp1a expression did not differ between treatments in the historical liver samples of brown trout . Preservation of samples at + 4 °C caused significant RNA degradation in 24 h (mean RIN < 7) and nearly complete loss of integrity in 5 days (mean RIN < 4). Previous studies have shown that it may be possible to extract good-quality RNA from human and rat tissues kept at room temperature for several hours after surgical removal . A normal freezer may thus be suitable for short-time preservation if a deep freezer is not available. Long-time preservation of samples at − 80 °C is recommended, as other studies have found that RNA integrity is not compromised by several years of storage at − 80 °C  (Vuorinen and Vuorinen −1 (in fat), respectively  masked the effect of the effluent. Another possibility is that the BKME effluent contained estrogen-like compounds that repressed cyp1a expression , and with similar RIN values.These recommendations apply to tissues with relatively low RNase activity, such as the liver, kidney, and brain. Tissues with high RNase content  may need to be treated in a different way to obtain good-quality RNA.


X-ray Birefringence Imaging is shown to be a sensitive method for spatially resolved mapping of molecular orientations in liquid-crystalline materials. The X-ray Birefringence Imaging (XBI) technique, first reported in 2014, is a sensitive method for spatially resolved mapping of the local orientational properties of anisotropic materials. We report the first application of the XBI technique to characterize molecular orientational ordering in a liquid crystalline material, demonstrating significant potential for exploiting XBI measurements to advance structural understanding of liquid crystal phases. The X-rays transmitted through the sample are interrogated using a diffraction-based polarization analyzer (set at a Bragg angle as close as possible to 2θ = 90°), and the resultant X-ray intensity is recorded in a spatially resolved manner using an area detector. The XBI technique was first demonstratedX-ray optic axis corresponds to the C–Br bond orientation.time-averaged resultant of the orientational distribution of the C–Br bonds.The experimental set-up for XBI  to align the liquid crystal phases and a variable temperature capability, controlled by passing an electric current through the graphite outer sample holder . Because the experimental set-up  carried out in the present work  gives phχ (defined in xy-plane) perpendicular to the propagation direction (z-axis) of the incident X-ray beam. The angle χ denotes rotation of the magnetic field around the z-axis, and thus specifies the direction of molecular alignment of the liquid crystal phases relative to the direction of linear polarization of the incident X-ray beam. For χ = 0°, the magnetic field is horizontal .For the isotropic liquid phase, the XBI images  are unifχ = 45° and cooled in small increments in the temperature region near the phase transition to the nematic phase, until the first change in X-ray intensity was observed in the XBI data.χ = 45° . As the effective optic axis for XBI studies at the Br K-edge is dictated by the resultant direction of alignment of the C–Br bonds, the X-ray birefringence behaviour for the nematic phase is clearly interpreted in terms of a high degree of orientational ordering of the molecules, with a resultant C–Br bond orientation effectively parallel to the magnetic field.For the nematic phase, the variation in X-ray intensity measured from the XBI images as a function of χ with minimum brightness at χ = 0° and maximum brightness at χ = 45° and χ = –45°. As shown in χ. Significantly, the maximum intensity is higher than the maximum intensity for the nematic phase, indicating that, as expected for a more ordered phase with partial translational ordering, the smectic A phase has a higher degree of orientational ordering of the C–Br bonds  in the direction of the applied magnetic field.The XBI behaviour for the smectic A phase 184 °C;  is very In this experiment, XBI data were also recorded at selected lower temperatures, but did not include a measurement that could be unambiguously identified as the smectic B phase.χ-scans at selected fixed temperatures was found to be problematic in the case of liquid-crystalline phases, as it was often observed in such experiments that the domain structure could occasionally change suddenly and unpredictably on changing the value of χ, reflecting the fluid nature of these phases under gravity. Under such circumstances, it is difficult to extract reliable information on the characteristic differences in X-ray intensity between the different liquid crystal phases. Instead, we have found that a much more reliable protocol for exploring the changes in the degree of ordering in the liquid crystal phases as a function of temperature is to record the XBI images with the orientation of the applied magnetic field fixed at χ = 45° while scanning through the temperature range of interest. The results from an experiment of this type are shown in i.e. starting in the isotropic liquid phase) to 108 °C at a rate of 1 °C min–1, with the XBI images recorded continuously during the cooling process . At the highest temperature, which is above the clearing temperature, the intensity is very low as a result of the isotropic orientational molecular distribution in the isotropic liquid phase. On decreasing the temperature, the intensity increases substantially between 216 °C and 205 °C, representing the transition from the isotropic liquid into orientationally ordered phases (from Iso → N → SmA). The upper part of ca. 0.2 to 0.4). The fact that the sharp rise in intensity between 216 °C and 205 °C covers a significantly wider temperature range than the Iso → N → SmA events observed by DSC and optical microscopy may reflect a combination of the temperature gradient across the sample plus the kinetics of alignment in the presence of the magnetic field. On decreasing the temperature within the SmA phase, the intensity shows a gradual increase until a visible transition into the SmB phase is observed from a further sharp  increase in intensity, followed by a significant decrease in intensity upon crystallization.The type of experiment described above, in which XBI data were measured by changing the sample orientation by S = = 〈½(3 cos½(3 cos2 θ – 1) − 1)〉, where , where θ is the angle between the director and the individual long molecular axes. The value of S is often determined by measurement of the optical birefringence . As an example, Fig. S3S, and while our interpretations of the changes in X-ray intensity as a function of temperature have invoked this relation at a qualitative level, our future research will include the derivation of a more quantitative framework for determining values of order parameters from XBI data.The order in liquid crystal phases can be quantified by a series of order parameters, of which the most commonly used is the orientational order parameter, The results reported in this paper represent the first experiments to study the orientational ordering of liquid crystal phases using the XBI technique, and demonstrate clearly the capability of the method to identify the direction of preferential molecular alignment in these phases and to establish, at least qualitatively, the relative degree of molecular orientational ordering in the different liquid crystal phases observed. The type of experiment reported in –1 or 10 °C min–1.Synthesis of 4′-octyloxy--4-yl-4-bromobenzoate  was carrχ-scans at fixed temperature  is limited by the resolution of the CCD-based detector. The spatial resolution in the horizontal direction is limited by the dynamical diffraction extinction depth of the polarization analyzer. The background intensity distribution in the XBI images is discussed elsewhere.31All XBI experiments were carried out on beamline B16 at Diamond Light Source with the sample mounted on a five-circle, vertical-scattering, Huber Eulerian diffractometer.perature , the areperature , the areThere are no conflicts to declare.Supplementary informationClick here for additional data file.


Perturbations within the secretory pathway were verified by an increased secretion of ER-resident PDI and decreased cell viability under ER stress conditions, suggesting KDELR1-KO cells to be severely impaired in maintaining cellular homeostasis.KDEL receptors (KDELRs) represent transmembrane proteins of the secretory pathway which regulate the retention of soluble ER-residents as well as retrograde and anterograde vesicle trafficking. In addition, KDELRs are involved in the regulation of cellular stress response and ECM degradation. For a deeper insight into KDELR1 specific functions, we characterised a KDELR1-KO cell line (HAP1) through whole transcriptome analysis by comparing KDELR1-KO cells with its respective HAP1 wild-type. Our data indicate more than 300 significantly and differentially expressed genes whose gene products are mainly involved in developmental processes such as cell adhesion and ECM composition, pointing out to severe cellular disorders due to a loss of KDELR1. Impaired adhesion capacity of KDELR1-KO cells was further demonstrated through  This process is largely driven by pH differences between two compartments: the slightly acid pH environment of the Golgi allows binding of KDEL-bearing chaperones, subsequently initiating KDELR interaction with Arf-GAP and coatomer components to trigger COPI-mediated retrograde transport5. In contrast, the neutral pH in the ER lumen causes the ligand/receptor-complex to dissociate and KDELRs are recycled back to the Golgi via anterograde transport6. Initially described in yeast, three KDELR homologues  with different ligand specificities and expression profiles were subsequently identified in mammalian cells8. Further studies revealed an important signalling function of mammalian KDEL receptors in order to balance membrane fluxes and to maintain Golgi homeostasis. Thereby, KDELR activation by ligand binding causes an interaction with Golgi-associated G proteins: Gq mediates the activation of Src family kinases (SFKs) and, thereby, stimulates anterograde transport processes to the plasma membrane9. This Golgi-to-plasma membrane trafficking was recently shown to be likewise facilitated by an interaction of KDELR with the monomeric Gα0 subunit, subsequently activating Rab1/Rab3 GTPases10. An interaction with Gs results in PKA activation and promotion of retrograde vesicle trafficking. For longer-term regulation of both transport directions, PKA further activates transcription factors to enhance the expression of genes encoding COPI and COPII components11.The secretory pathway represents a highly organised multi-organelle compartment which enables the transport of newly synthesised secretory proteins as well as membrane proteins to their final cellular destination and place of activity. These transport processes include massive membrane fluxes from one compartment to another and need to be counterbalanced by tightly connected cellular control mechanisms. A central key player in the regulation of vesicle transport from and to the Golgi is the eukaryotic KDEL receptor (KDELR), a seven transmembrane protein which was initially described to be responsible for the retrieval of soluble ER residents from the Golgi back to the ER14. In this context, a specific mutation in KDELR1 affecting its interaction with protein phosphatase 1 (PP1) leads to an increased stress response and enhanced apoptosis of T-cells15. In contrast, KDELR overexpression has been demonstrated to result in MEK1/ERK-dependent autophagy activation, enabling the cell to minimise the accumulation of proteins such as SOD1, α-synuclein, and huntingtin which are associated with neurodegenerative diseases13. In addition, KDELRs have also been shown to regulate invapodia formation and extracellular matrix degradation in a Src family kinase-mediated signal transduction process by activating focal adhesion kinases (FAK)17.Due to their relevance in controlling intracellular transport processes, KDELRs are associated with cell stress conditions and the regulation of a cellular stress response, while its particular outcome highly depends on the KDELR expression level and the respective cell type20. To facilitate the analysis of such novel KDELR functions, we focused on a genome-wide characterisation of a KDELR1-knockout (KO) HAP1 cell line. Transcriptome analysis revealed strong alterations in the expression of genes whose products are involved in cellular development, adhesion and extracellular matrix (ECM) composition. In this regard, differences concerning the adhesion and migration behaviour of KDELR1-KO cells could be confirmed by in vivo scratch and adhesion assays. Furthermore, enhanced secretion of protein disulphide isomerase (PDI) and increased sensitivity to ER stress were also observed as characteristic KDELR1-KO phenotype.Furthermore, recent studies also suggest a regulated transport pathway of KDELRs to the plasma membrane which might include novel signalling functions at the cell surface after extracellular ligand binding21. KDELR1-KO was generated via the CRISPR/Cas technology using ATGAATCTCTTCCGATTCCT as guide RNA sequence. KDELR1-KO cells maintain a 1 bp deletion in exon 1 which causes a frameshift and thereby a non-functional KDELR1 gene. Both, HAP1 wild-type and HAP1 KDELR1-KO cells were commercially obtained from the company Horizon which provides additional information about the cell line and the experimental approach to generate a particular KO strain on the following homepage: https://www.horizondiscovery.com.HAP1 represents a near-haploid human cell line derived from the male chronic myelogenous leukemia (CML) cell line KBM-7 containing a single copy of each chromosome, except for a heterozygous 30-megabase fragment of chromosome 152.HAP1 cells were cultivated in IMDM (Iscove’s Modifed Dulbecco’s Medium) medium (Gibco) supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  at 37 °C and 5% CO10 µl cell suspension was transferred into a reaction tube, washed once with 1 × PBS and resuspended in sterile water before freezing at −80 °C overnight. 1 µl of the cell lysate was used as PCR template to amplify the genomic KDELR1 sequence within its first exon. The reaction was carried with the „Phusion Hot Start II HighFidelity DNA Polymerase“-Kit (Thermo Scientific) and the following primers: forward: AGCTCCAGCCTTTGCTCCCCCTCCCAAA, reverse: CCCAAACCCTTCCTGAGTCC TGCGACGT. The PCR product was cleaned up using the „Wizard® SV Gel and PCR CleanUp System“ (Promega) and sequenced by “GATC Biotech AG”.® Poly(A) mRNA Magnetic Isolation Modules“ and created strand specific cDNA libraries using the „NEBNext® UltraTM II Directional RNA Library Prep Kits for Illumina®“ (New England BioLabs) using 1 µg total input RNA and 8 PCR cycles. Quality of the cDNA libraries was determined via Bioanalyzer . Sequencing of the samples was carried out in a „HiSeq. 2500 Platform” (Illumina) using high-output mode (100nt SE reads). Reads were demultiplexed with bcl2fastq (v1.8.4) and trimmed for adaptor contamination and low-quality bases with the cutadapt (v1.4.1) wrapper trim_galore (v0.3.3)22.RNA isolation was performed with the Direct-zol RNA MiniPrep Kit (Zymo Research) which includes DNAse digestion. RNA integrity was checked by denaturing gel electrophoresis. We enriched for poly-A RNA using „NEBNext23 was used for the quantification of gene expression levels in HAP1 wild-type and KDELR1-KO cells. We used gene annotation from the Ensembl database24 version 95 for the analysis with Salmon. Using estimated gene expression counts from Salmon, DESeq. 2 software was used to estimate differentially expressed genes25. p-values have been corrected for multiple testing using the Benjamini-Hochberg procedure.Salmon software26 using the GO annotation database version 1.227.For the analysis of Gene Ontology (GO) enrichment we used the Ontologizer software (version 2.1)6 HAP1 wild-type and KDELR1-KO cells were seeded in 6-well plates containing IMDM medium and incubated at 37 °C and 5% CO2. After reaching approximately 80% confluency, the cell network was slightly disintegrated by a scratch in the middle of the well using a 1,000 µl pipette tip. The medium was changed to FCS-free IMDM, and the scratch area was measured immediately (=time point 0 h). After 24 h and 48 h incubation in FCS-free medium, scratch areas were measured and compared to the respective area at time point 0 which was set to 100%.1 × 10Collagen type I (Sigma) was diluted in sterile water to a working concentration of 100 µg/ml. For the coating of 96-well plates, 100 µl of the working solution was added to each well and incubated at 37 °C for 5 h. After removal of the remaining solution, the coated surface was allowed to dry. Wells were washed with sterile water and dried for storage at room temperature until use. Laminin (Sigma) was diluted in 1 × PBS to a final concentration of 100 µg/ml, and 100 µl were added to each well of a 96-well plate. Plates were sealed to prevent evaporation and incubated at 37 °C for 5 h. The residing solution was removed, the wells were washed with 1 × PBS and covered with 1 × PBS for storage at 4 °C until use.4 cells of HAP1 wild-type and its KDELR1-KO variant were seeded in collagen-coated, laminin-coated or uncoated wells of a 96-well plate with IMDM medium, respectively. After adhesion for 4 h, unbound cells were removed by washing with 1 × PBS. Adherent cells were fixed with 2% paraformaldehyde  for 15 min, washed twice with 1 × PBS and stained with 0.1% crystal violet solution (Merck) in 10% ethanol for 10 min. After 5 washing steps with sterile water, absorption was measured in a plate reader „SpectraMax® Paradigm® Multi-Mode Microplate Platform“ (Molecular Devices) with the „Multi-Mode Analysis Software 2010“ (Molecular Devices).2 × 104 cells each) were seeded in 96-well plates with phenol red-free DMEM medium (Gibco) supplemented with 10% FCS and 1% Pen/Strep and cultivated for 24 h. Subsequently, cells were treated with 1 or 1.5 µg/ml thapsigargin  or with the corresponding amount of DMSO in the negative control sample. After 24 h incubation, cell viability was determined via MTT assay. In brief, a solution of MTT -2,5-diphenyl-2H-tetrazolium bromide, Sigma) was added to a final concentration of 0.4 g/l (in 1 × PBS) and the 96-well plates were incubated for 3 h at 37 °C and 5% CO2. Arosen crystals were dissolved by adding 100 µl solubilisation solution  and slow shaking at 20 °C. Absorption was measured at 570 nm using „SpectraMax® Paradigm® Multi-Mode Microplate Platform“ (Molecular Devices) with the „Multi-Mode Analysis Software 2010“ (Molecular Devices). Calculation of mean values and standard deviations were performed with Microsoft Excel.HAP1 wild-type and KDELR1-KO cells  and concentrated by centrifugation using VivaSpin columns . The total protein amount of the concentrated supernatant was determined by NanoDrop measurement  and equally adjusted for both samples. SDS-PAGE was performed in 10% Tris-Tricine gels under non-reducing conditions, and subsequent semi-dry blotting onto PVDF membranes was carried out in transfer buffer . After transfer, membranes were blocked in 3% BSA in TBS-T  overnight at 4 °C, and subsequently incubated with antibodies against PDI , 1:1,000 in 3% BSA/TBS-T) for 1 h at 20 °C. Incubation with HRP-coupled secondary antibody  was performed for 1 h at 20 °C. Chemiluminescence reaction was performed by a short incubation with “SuperSignalTM West Femto Maximum Sensitivity Substrate“ (Thermo Scientific) and signals were detected and quantified with an „Amersham Imager 600“ .HAP1 cells were incubated in IMDM medium (Gibco) without FCS for 24 h at 37 °C and 5% CO11. In addition, KDELRs were shown to contribute to the regulation of cellular stress response and ECM degradation16. Since our recent studies strongly indicated an additional co-localisation of KDELRs at the plasma membrane, a broader spectrum of novel KDELR functions can be predicted. To investigate such KDELR1-specific functions, we characterised a KDELR1-KO HAP1 cell line, which holds a deletion of thymine at position 16 within the genomic KDELR1 sequence that leads to a premature stop in translation, resulting in a KDELR1-KO . We cultivated KDELR1-KO and wild-type cells in FCS-free medium for 24 h and collected the cell-free culture supernatant for subsequent dialysis and concentration. The amount of PDI detected by western analysis was nearly doubled in the supernatant of KDELR1-KO cells compared to wild-type Fig. . Since o14, and that KDELR1 regulates eIF2α de-phosphorylation via PP1 interaction15. Regulation of cellular stress response in KDELR1-KO cells is, therefore, negatively affected at different levels, leading to substantial difficulties of a cell to compensate ER-stress conditions.Since KDELRs are known to be involved in the regulation of cellular stress response and transport defects are also associated with cell stress, we assumed KDELR1-KO cells to have difficulties in counterbalancing stress conditions. To induce ER-stress, KDELR1-KO and HAP1 wild-type cells were incubated with the SERCA inhibitor thapsigargin for 24 h and subsequently analysed for cell viability using MTT assays. Indeed, a strong decrease in the viability of KDELR1-KO cells compared to wild-type was observed, indicating severe defects in the regulation of a cellular stress response Fig. . A typicOur study provides the first complete transcriptomic characterisation of a KDELR1-KO cell line to get a global insight into the defects of these cells. Within the analysed HAP1 cell line, a KDELR1-KO causes significant transcriptomic changes in four rather general developmental processes supporting the wide-ranging functions of KDELR1 in maintaining cellular homeostasis. Significant enrichment in five more specific GO terms concerning cell adhesion and ECM formation points to a vital necessity to counterbalance KDELR1 loss in these processes. We validated these findings by subsequent molecular analysis of protein secretion and cell adhesion. An impaired adhesion capacity of KDELR1-KO cells was confirmed by adhesion assays and could be rescued by collagen- or laminin-coating of the surface. The strongly increased adhesion ability of KDELR1-KO cells on coated surfaces compared to wild-type supports the transcriptomic changes to improve adhesion processes and compensate for corresponding defects. Since KDELRs are key players in regulating protein/vesicle trafficking processes within the secretory pathway, this transport might also be disturbed in KDELR1-KO cells. Here, mislocalisation of proteins of the secretory system was demonstrated by increased PDI secretion which might contribute to the observed increased sensitivity of KDELR1-KO cells to ER stress conditions. Thus, KDELR1-KO cells seem to be severely affected in cellular homeostasis and require compensation in cell adhesion processes at the transcriptional level. Considering the diverse expression level of KDELR1 and its various distinct functions in different cell types, other characteristics of a KDELR1-KO are likewise possible, especially for varying tissue types. Such analysis could represent an interesting and fruitful approach for future studies.Supplementary Information


By contrast, much less research has been conducted on 5‐methylcytosine (m5C), which was detected in tRNAs and rRNAs and more recently in poly(A)RNAs. In this review, we discuss recent developments in the discovery of m5C RNA methylomes, the functions of m5C as well as the proteins installing, translating and manipulating this modification. Although our knowledge about m5C in RNA transcripts is just beginning to consolidate, it has become clear that cytosine methylation represents a powerful mechanistic strategy to regulate cellular processes on an epitranscriptomic level.It is a well‐known fact that RNA is the target of a plethora of modifications which currently amount to over a hundred. The vast majority of these modifications was observed in the two most abundant classes of RNA, rRNA and tRNA. With the recent advance in mapping technologies, modifications have been discovered also in mRNA and in less abundant non‐coding RNA species. These developments have sparked renewed interest in elucidating the nature and functions of those “epitransciptomic” modifications in RNA. N6‐methyladenosine (mRNA Processing > RNA Editing and ModificationRNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional ImplicationsRNA Processing > tRNA ProcessingRNA Turnover and Surveillance > Regulation of RNA StabilityThis article is categorized under: Pseudou6A) as the most abundant and to date best understood mRNA modification  tail that assists the progress of nuclear export, stability as well as translation . We will discuss methods for its detection, its distribution in different types of RNA, its effects on RNA function and the enzymes responsible for its deposition. Most of our current knowledge about m5C in RNA comes from research on the abundant rRNAs and tRNAs. Although we will briefly touch on these findings, in depth discussion of rRNA and tRNA methylation can be found in several excellent recent reviews  in acidic pH resulting in deamination and formation of uracil‐sulfonate which converts to uracil (U) under basic pH conditions thus causing a C‐to‐U conversion that can be detected by sequencing , RNA‐bisulfite‐sequencing (RNA‐BS‐seq) allows for the determination of the extent of methylation of a specific C position in RNA. However, one big caveat of RNA‐BS‐seq is its failure to react with Cs in a base‐paired conformation. Considering the low overall m5C occurrence in RNA and specifically in mRNA  was originally used to identify the mg Figure  was used2.35C occurrence without previous knowledge of the nature of the responsible methylating enzyme, aza‐IP and miCLIP were designed to identify the target sites of RNA cytosine methyltransferases (RCMTs). Both techniques take advantage of the catalytic mechanism of RNA methyltransferases which includes a transient covalent linkage of the enzyme to the methylation target cytosine. In aza‐IP, 5‐azacytidine (5‐azaC) is incorporated into RNA by feeding cells with the modified nucleoside. If this occurs at an RCMT target site, it traps the RCMT because the covalent adduct with the target C cannot be resolved. Thus, antibodies against the RCMT (or a tagged version) can be used to immunoprecipitate the protein along with the covalently bound RNA, which is sequenced. Enrichment analysis is then performed to identify methylated RNAs, and sites with increased C‐to‐G transversion signatures (resulting from a ring opening of 5‐azaC during the protocol) correspond to the sites of methylation  is based on the use of a modified RCMT in which the cysteine that is responsible for releasing the RCMT from the RNA substrate is mutated resulting in a covalently linked RNA‐protein complex. Similar to aza‐IP, immunoprecipitation with an antibody against the RCMT is used to enrich bound RNA, which is then subjected to deep sequencing Figure . Because5C data sets.Taken together, all available methods suffer from different drawbacks caused by a variety of reasons ranging from the biological features of the RNA itself to the experimental procedures and the data analysis approaches , rRNA , mRNA (messenger RNA), snRNA , miRNA (microRNA), lncRNA (long noncoding RNA) or eRNAs (enhancer RNA) from many species and in all three domains of life. There are differences, however, in the occurrence of m5C in specific RNA types in different species. For example, m5C appears not to be present in tRNA and mRNA from bacteria, while it has been found in eukaryal and archeal tRNA and mRNA  tRNAMet in mice , which is important for the decoding of AUA methionine codons during mitochondrial translation  between large and small subunit. This contact was proposed to be important during translation, when eL41 acts as a rotation center for 40S subunit movement  and of the brain was reported  mapping to 1,650 mRNAs were detected in ESCs and 2,075 m5C sites mapping to 486 mRNAs in the brain. Another recent publication on HeLa cell and mouse cytosine methylomes identified ~3,600 sites within ~2,000 mRNAs in HeLa cells and 2,500–4,400 sites  in different mouse tissues  may all influence the outcome of m5C methylome analyses. Hence, further improvement of the methodology is clearly needed to obtain highly reliable data sets. This will also address the second question of whether mRNA actually contains m5C. However, although it is possible that the number of true m5C sites in poly(A)RNA in the end will be considerably lower than suggested now, multiple lines of evidence suggest that mRNA is indeed subject to cytosine methylation . Conversely, methylation of specific cytosines in the 3′UTRs of the cell cycle regulators CDK1 and p21, respectively, was shown to promote translation of these mRNAs in vitro and in a reporter gene system in vivo  has differential effects on mRNA function.Together the findings from these studies focusing on m3.3.65C has also been detected in other RNA types, specifically in long non‐coding RNAs (lncRNA) and smaller non‐coding RNAs, such as enhancer associated RNAs (eRNAs) or vault RNAs (vtRNAs). For lncRNAs, m5C was found near a protein binding domain in human HOTAIR in various cancer cell lines as well as in the functionally important A‐region of the lncRNA XIST. Importantly, m5C can interfere with binding of XIST to the chromatin regulatory PRC2 (polycomb repressive complex 2) complex in vitro  as a methyl group donor. Members of this group of proteins can be found in all domains of life targeting proteins, DNA, RNA, lipids and small molecules for methylation. To date all confirmed m5C‐specific RCMTs belong to either the DNMT2 or the NOL1/NOP2/sun (Nsun) subgroups of methyltransferases , enables the enzyme to covalently bind the target cytosine by attacking the carbon 6 atom of the pyrimidine ring, thus activating the non‐nucleophilic carbon 5  has been investigated already two decades ago, showing a critical involvement in 60S biogenesis in yeast . Rcm1p, which is localized in nucleoli, was identified as the enzyme modifying C2278 in 25S rRNA  can be found. These genes are transcribed but give rise to shorter open reading frames, and it is currently not clear if the corresponding transcripts are translated . Overall, all individuals showed significant delay in development and speech. Height and weight were below the 5% percentile but muscle tone was increased in all limbs. Although patients had smaller heads, computer tomography of the brain did not show any abnormalities. Expression of mutant NSUN2(G679R) protein in mouse brain prevented its localization to the nucleolus in Purkinje cells of the cerebellum , suggesting target recognition by the enzyme in a sequence‐specific manner. Dnmt2‐deficient mice, flies and plants were viable and fertile and were morphologically indistinguishable from wild‐type organisms  Dnmt2‐deficient flies exhibited shortened life‐span, while dDnmt2 overexpression resulted in increased stress resistance  substrates and inhibit the action of Dcr‐2 on long double stranded RNAs thus regulating siRNA pathways  in the genome , thus affecting translation of chromatin regulatory proteins involved in the silencing of mobile elements. However, it is also interesting to note that the Dnmt2‐deletion phenotype could be rescued by expression of a catalytically inactive Dnmt2 transgene, which raises the possibility that Dnmt2 might also engage in an RCMT‐independent function in this process ‐tRNAMet ‐dependent dioxygenase ALKBH1/ABH1 to generate 5‐formylcytidine (f5C) at this position  and aza‐IP Nsun6 was identified as a tRNA‐specific RCMT with specificity for tRNAThr and tRNACys in human cells. It introduces an m5C at position 72 in the 3′ acceptor stem. Nsun6 binding to its tRNA substrates requires the presence of a 3′‐CCA sequence in order to carry out methylation, because tRNACys and tRNAThr mutants lacking the 3′‐CCA sequence, were no longer modified at C72 by Nsun6 , 5‐formylcytosine (f5C) and eventually to 5‐carbonylcytosine (ca5C). While f5C and ca5C are mostly considered intermediates on the way to DNA demethylation, hm5C has been demonstrated to have regulatory potential on its own. For RNA, a recent report has shown that hm5C can be found in mRNAs from Drosophila, especially from the fly brain. The Drosophila Tet enzyme is responsible for generation of this mark and Tet‐mutant flies exhibit impaired brain development and reduced hm5C levels (Delatte et al., 5C has also been shown for cells of mouse and human origin (Fu et al., 5C epitranscriptomic map are still obscure awaiting discovery in the years to come.Research on RNA modifications, particularly in mRNA and other lower abundance RNA species, is an emerging and highly dynamic field with new discoveries reported almost on a monthly basis. Yet despite all the exciting new additions to our knowledge, the field is still in its infancy. With respect to mThe authors have declared no conflicts of interest for this article.mRNA methylation by NSUN2 in cell proliferationMapping and significance of the mRNA methylomeRNA methylation in nuclear pre‐mRNA processing


Small‐molecule drugs are used extensively in clinics for cancer treatment; however, many antitumor chemical drugs dissolve poorly in aqueous solution. Their poor solubility and nonselective delivery in vivo often cause severe side effects. Here, the application of RNA nanotechnology to enhance the solubility of hydrophobic drugs, using camptothecin (CPT) for proof‐of‐concept in targeted delivery for cancer treatment is reported. Multiple CPT prodrug molecules are conjugated to RNA oligos via a click reaction, and the resulting CPT‐RNA conjugates efficiently self‐assemble into thermodynamically stable RNA three‐way junction (3WJ) nanoparticles. The RNA 3WJ is covalently linked with seven hydrophobic CPT prodrug molecules through cleavable ester bonds and a folic acid ligand for specific tumor targeting while remaining soluble in aqueous solutions without detectable aggregation at therapeutic dose. This CPT‐RNA nanoparticle exhibits efficient and specific cell binding and internalization, leading to cell apoptosis. Tumor growth is effectively inhibited by CPT‐RNA nanoparticles; the targeted delivery, strengthened by tumor ligand, further enhances tumor suppression. Compared with the traditional formulation, solubilization of CPT in aqueous buffer using RNA nanoparticles as a carrier is found to be safe and efficacious, demonstrating that RNA nanoparticles are a promising platform for the solubilization and the delivery of hydrophobic antitumor drugs. Robust RNA nanoparticles with a defined three‐way junction structure are used to solubilize a hydrophobic antitumor drug in aqueous solution and improve the safety profile. Targeted delivery of the therapeutic RNA‐drug nanoparticles further increases the tumor suppression efficacy in human tumor xenograft models. This work demonstrates the feasibility of RNA nanotechnology for the solubilization and the delivery of hydrophobic chemotherapeutic drugs. Cancer is caused by the uncontrolled growth and division of cells and is one of the most fatal diseases worldwide.Water‐soluble RNA nanoparticles have recently been developed for the efficient delivery of therapeutics.We herein report the use of water‐soluble RNA 3WJ nanoparticles to solubilize hydrophobic CPT for targeted delivery in human tumor xenograft models. CPT is a potent small‐molecule antitumor drug,Figurea‐3alkyne and 3WJb‐4alkyne). CPT conjugation resulted in increased hydrophobicity of CPT‐RNA conjugate compared with RNA as evidenced by the extended retention time on reverse phase high‐performance liquid chromatography .To conjugate CPT to RNA oligos, we acylated the 20(S)‐hydroxyl group with 6‐azidohexonic acid to form a CPT prodrug. The ester linkage is used for enzymatic release of parent CPT and the azido group is for subsequent conjugation to RNA oligos with alkyne groups via copper(I)‐catalyzed alkyne‐azide cycloaddition. To maximize the therapeutic effect, multiple CPT prodrug molecules were conjugated to one RNA oligo Figure1a. Thea‐3CPT) in water, indicating that CPT‐RNA conjugate was completely dissolved  still showed no visible precipitate  of free CPT in water.Poor solubility of CPT in aqueous solutions has hindered its clinical use in cancer treatment. Although two CPT derivatives are commercially available in the market, a lot of research effort is focused on improving its aqueous solubility by conjugating CPT to hydrophilic moieties including peptides,d Figure d. In cond Figure d. To contion 8 × 0−3m CPTFigureTm) of 63.5 °C in physiological phosphate‐buffered saline (PBS) buffer , and the resulting products were analyzed on a denaturing PAGE. The CPT release yield increased over incubation time and 50% of CPT release was observed at about 7.7 h .Ester bond has been proven to be enzymatically labile in the presence of endogenous esterase and was widely used as a cleavable linker in prodrug design.−9, 100 × 10−9, and 400 × 10−9m) were incubated with KB cells for flow cytometry analysis. In the presence of FA ligand, the fluorescent FA‐7CPT‐3WJ nanoparticles showed strong binding to KB cells in a concentration‐dependent manner, compared to the one without FA  is frequently overexpressed on the tumor cell surface, and the dissociation constant between FR and its ligand, FA, is found to be in nanomolar range.Figure−6m (CPT concentration). However, IC50 for HepG2 cells was much larger . This discrepancy on IC50 further supports the targeted delivery caused by FA–FR interaction. In addition, CPT‐RNA nanoparticles presented a delayed inhibition on tumor cell growth compared with CPT (IC50 was around 0.05 × 10−6m). More specifically, when comparing FA‐7CPT‐3WJ at 72 h with CPT at 48 h, samples of higher concentrations have comparable performance. This was also supported by the comparison of FA‐7CPT‐3WJ at 48 h with CPT at 24 h. This delayed action may be caused by the slow release of CPT from RNA nanoparticles. Different mechanisms of cell internalization for FA‐7CPT‐3WJ and CPT could also lead to this delayed inhibition. Furthermore, inverted microscope images showed that both FA‐7CPT‐3WJ‐ and 7CPT‐3WJ‐treated cells exhibited poor cell growth after 48 h incubation, as characterized by enlarged nuclei and elliptical membranes, as well as the reduced cell density comparable to those of CPT and CPT prodrug treatment groups . In contrast, FA‐3WJ‐ and 3WJ‐treated cells exhibited healthy cell growth, indicating the safety of using RNA nanoparticles as drug carriers. These results demonstrated that parent CPT drug was released from the RNA nanoparticles and retained its pharmacological activity to inhibit tumor cell growth.To determine the half maximal inhibitory concentration (IC50) of CPT‐RNA nanoparticles, dose‐dependent MTT ‐2,5‐diphenyltetrazolium bromide) assay was performed at different time points, and inverted microscope was used to compare cell morphology after 48 h. The cell viability results demonstrated CPT‐RNA nanoparticles inhibited KB cells growth in a concentration‐dependent manner Figure4a, whiCaspase‐3 as an early cell apoptosis marker was also monitored during the treatments. Elevation of caspase‐3 is reflected by the increased fluorescence signal from the caspase‐3 fluorogenic substrate. Consistent with the MTT assay, caspase‐3 measurement confirmed tumor cell apoptosis induced by CPT‐RNA nanoparticles Figure b. This c3, mice bearing KB xenograft were randomly divided into four groups of PBS, CPT, 7CPT‐3WJ, and FA‐7CPT‐3WJ (n = 5) and administered via IV injection at the dose of 4.3 mg kg−1  on day 1, 3, 5, and 7 for a total of four injections. CPT dissolved very poorly in aqueous solution and many previous studies used intraperitoneal injection or intramuscular injection with special formulations. To ensure comparable administrations, free CPT drug was formulated in a reported method (10% DMSO and 5% Tween 80)FigureGiven the demonstration of specific tumor cell binding and apoptosis effect, we set out to study in vivo tumor suppression in human tumor xenograft model. After the tumors grew to approximately 50 mmIn summary, RNA nanoparticles were used for solubilizing hydrophobic antitumor drug and for targeted delivery in a human tumor xenograft model. High loading of the poorly soluble CPT to the hydrophilic RNA nanoparticles dramatically improved drug solubility in physiological conditions while maintaining therapeutic effect. The resulting therapeutic RNA nanoparticles had the advantages of well‐defined structure, precise drug loading, high thermal and chemical stability, and targeted delivery. The potent CPT was enzymatically released from the RNA vehicles by esterase in vivo, triggering tumor cell apoptosis and inhibiting tumor growth effectively. The in vitro and in vivo data collectively demonstrated the feasibility of RNA nanoparticles for the safe and efficacious targeted delivery of hydrophobic antitumor drugs.Detailed experimental procedures are summarized in the Supporting Information. All protocols involving animals were approved and performed under the supervision of The Ohio State University Institutional Animal Care and Use Committee (IACUC).P.G. is the consultant of Oxford Nanopore Technologies and Nanobio Delivery Pharmaceutical Co. Ltd; he is the cofounder of Shenzhen P&Z Bio‐medical Co. Ltd and its subsidiary US P&Z Biological Technology LLC, as well as ExonanoRNA, LLC and its subsidiary Weina Biomedical LLC in Foshan.SupplementaryClick here for additional data file.


Nearly all classes of coding and non-coding RNA undergo post-transcriptional modification, as more than 150 distinct modification types have been reported. Since RNA modifications were first described over 50 years ago, our understanding of their functional relevance in cellular control mechanisms and phenotypes has truly progressed only in the last 15 years due to advancements in detection and experimental techniques. Specifically, the phenomenon of RNA methylation in the context of ncRNA has emerged as a novel process in the arena of epitranscriptomics. Methylated ncRNA molecules may indeed contribute to a potentially vast functional panorama, from regulation of post-transcriptional gene expression to adaptive cellular responses. Recent discoveries have uncovered novel dynamic mechanisms and new layers of complexity, paving the way to a greater understanding of the role of such phenomena within the broader molecular cellular context of human disease. Up until recently, the central dogma  had supppseudouridine  and ribosomal (rRNA) RNA, novel detection technologies and deep sequencing analysis , 2016, hFigure 1).RNA methylation is a reversible, post-transcriptional RNA modification, affecting several biological processes, such as RNA stability and mRNA translation , through6-methyladenosine (m6A) is the most abundant internal modification detected to date in mRNA (writers), decode (readers), and remove (erasers) methylation  sites in human and mouse. Finally, The methyl group in m-pairing , is high-pairing . It is a-pairing . While nnd ncRNA , particund ncRNA . Recentl1-methyladenosine (m1A) in total RNA date back more than 50 years   as well O-methylation occurs in 3′ termini and is found to be important in plant biogenesis of small RNA, inter alia miRNA and siRNAs  is an epitranscriptomic modification that involves the 5th carbon atom of cytosine as a target for methylation in poly(A) RNA, rRNA, tRNA, snRNA, and lncRNA   for m6A detection  (5C-methyltransferases (m5C -MTases) NSUN2 and NSUN4   coupled lidation . In alteRIP data . Indeed,6A sites  and anti6A sites , most an6A sites . For insetection . Such insolution . In the nd rRNA) , degradand rRNA) . An altend NSUN4 . By mutavia differential RT profiles, with or without chemical treatment. The RiboMeth-seq methodology  as a result of the intervention of METTL3 during pri-miRNA processing. The same authors also showed that m6A marks in pri-miRNAs allow for the RNA-binding protein DGCR8 to identify its specific substrates, promoting the beginning of miRNA biogenesis. 6A mark, binding to m6A marks in pri-miRNAs, thus promoting pri-miRNA processing. Additionally, the effects of RNA demethylation on miRNA expression have also been investigated. 6A demethylase FTO, providing indirect evidence of co-transcriptional processing in the methylation of mRNAs and miRNAs. Finally, Chen T. et al. (2015) discovered that miRNAs positively regulate m6A installment on mRNAs via a sequence pairing mechanism. Methylation events in miRNAs add a new layer of complexity in the regulation of post-transcriptional gene expression and warrant future studies in order to fully elucidate the roles and functions of modified miRNAs.2′OMe has been detected at the 3′-end of miRNAs (only in plants) and found to confer stability and protection from 3′-uridylation and degradation . m6A witing site . In anot6A writer METTL16 at its 3′-triple-helical RNA stability element (6A (6A has been further shown to destabilize the hairpin stems in the transcript, making them more flexible and solvent-accessible (5C sites have also been detected in MALAT1 (5C site which has been verified with a 100% modification rate (6A events have been associated to XIST-mediated transcriptional repression (5C sites can prevent XIST-protein interactions, although it may not be a conserved mechanism (Although the majority of focus has been recently devoted to modifications in mRNA, 1000s of lncRNA transcripts have been detected containing a substantial number of modifications . Evidenc element  specificment (6A . The precessible  as well cessible . Severaln MALAT1 , but no n MALAT1  possesseion rate . Finallypression  while m5echanism . More deGR wrote and set up the manuscript. GN and DV wrote and reviewed the content. SN-S supervised and reviewed the manuscript writing and development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.