Surprisingly, the left binding arm contains only three nucleotides and forms two canonical base pairs with the RNA substrate. Mutational analysis reveals that a riboguanosine residue 3-nucleotide downstream of cleavage site must not form canonical base pairing for the optimal catalysis, and this nucleobase likely participates in catalysis with its carbonyl O6 atom. Furthermore, we demonstrate that deoxyribozyme 10–12opt can be utilized to cleave certain microRNA sequences which are not preferentially cleaved by 8–17. Together, these results suggest that this novel RNA-cleaving deoxyribozyme forms a distinct catalytic structure than 8–17 and that sequence space may contain additional examples of DNA molecules that can cleave RNA at site-specific locations.Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have found broad applications in biotechnology, DNA computing and environmental sensing. Among these, deoxyribozyme 8–17 is the most common small DNA motif capable of catalyzing RNA cleavage. However, the extent to which other DNA molecules with similar catalytic motifs exist remains elusive. Here we report a novel RNA-cleaving deoxyribozyme called 10–12opt that functions with an equally small catalytic motif and an unusually short binding arm. This deoxyribozyme contains a 14-nucleotide catalytic core that preferentially catalyzes RNA cleavage at UN dinucleotide junctions ( Many deoxyribozymes catalyzing a wide variety of chemical reactions have been discovered by in vitro selection7. Among them, DNA molecules that catalyze the sequence-specific cleavage of RNA have attracted significant attentions due to their immediate use as molecular tools in practical applications8. RNA-cleaving deoxyribozymes have been incorporated into technologies that impact problems ranging from biotechnology and medicine to DNA computing and metal ion sensing20. Most of these utilize the general-purpose RNA-cleaving deoxyribozymes 8–17 and 10–23 isolated by Santoro and Joyce21. Under the optimal conditions, 10–23 cleaves phosphodiester bonds with a catalytic efficiency of 109 M−1min−1, making it the fastest nucleic acid enzyme known21. Deoxyribozyme 8–17, though slightly less efficient, represents an important model for RNA cleavage by a DNA sequence. This deoxyribozyme has been isolated independently from multiple in vitro selection experiments, suggesting that it may be one of the simplest solutions to the problem of how a DNA molecule can fold into a shape that can cleave RNA at a specific location27.Deoxyribozymes are single-stranded DNA molecules that catalyze specific chemical reactions28. One could imagine that the simplicity of the 8–17 motif, which contains only four conserved residues in a catalytic core of 15 nucleotides, would make it relatively easy to rediscover deoxyribozymes containing such motifs by in vitro selection29. However, it is also possible that sequence space contains a large number of different DNA molecules that can cleave RNA. Such deoxyribozymes could be quite rare relative to the 8–17 motif or previously overlooked because they lack an obvious substrate binding site or catalytic motif28.Whether DNA molecules could fold into shapes with similar catalytic activity as 8–17 but unique structural properties is an interesting question that warrants further considerationin vitro selection experiment designed to sample a random pool of DNA for magnesium-dependent RNA-cleaving deoxyribozymes. We then analyzed twenty randomly picked clones for functional activity. This analysis and the following optimization allowed us to identify a deoxyribozyme called 10–12opt with an unusually short substrate-binding arm and an intriguing structural requirement outside the catalytic domain. This novel deoxyribozyme featured a 14-nucleotide catalytic core and a 3-nucleotide left binding arm. Mutational analysis confirmed that the catalytic core formed an essential 3-bp stem loop structure, and also revealed several additional essential residues near the cleavage site. Unexpectedly, a riboguanosine residue downstream of the cleavage site in RNA substrate proved critical for catalysis, and restraining this guanine nucleobase by formation of an rG:dC base pair with deoxyribozyme was detrimental to efficient catalysis. Substitution of guanine by hypoxanthine and 2-aminopurine suggested that the carbonyl O6 atom, but not the 2-amino group, of this critical G residue was necessary and sufficient to support deoxyribozyme-catalyzed RNA cleavage, likely by maintaining active catalytic structure or participating in catalysis directly. In addition to Mg2+, several additional divalent metal ions such as Pb2+, Mn2+, Zn2+ and Co2+ could also support the catalytic activity of deoxyribozyme 10–12opt. Lastly, we demonstrated that deoxyribozyme 10–12opt could be utilized to cleave certain microRNA sequences which are intrinsically not effectively cleaved by deoxyribozyme 8–17.In the current work, we performed an 4OH for 18 h at 55 °C, butanol precipitated, and purified by denaturing polyacrylamide gel electrophoresis (PAGE). The biotinylated DNA-RNA chimeric primer, PCR primers and deoxyribozymes were purchased from Integrated DNA Technologies  or Genscript . The fluorescently labeled RNA substrates were purchased from Takara . The unmodified DNA-RNA chimeric substrate and all-RNA substrate were purchased from Dharmacon , deprotected in acetate buffer according to manufacturer’s protocol, and purified by denaturing PAGE. Klenow Fragment (exo-) DNA polymerase, T4 RNA Ligase 1, T4 polynucleotide kinase and ATP were purchased from New England BioLabs .The DNA library was synthesized on an automated ABI 3400 DNA synthesizer, deprotected in concentrated NHin vitro selection consisted of five steps: primer extension, first strand separation, functional selection, PCR amplification and second strand separation. First, the biotinylated DNA-RNA chimeric primer was annealed to the DNA library in 1x NEBuffer 2  by heating for 5 min at 75 °C and cooling to 37 °C. Primer extension reactions contained 1 μM primer-template complex, 100 μM dNTPs, and 0.25 U/μl Klenow Fragment (exo-) DNA polymerase. Reactions were initiated by adding DNA polymerase to a solution containing all the other reagents and heating for 10 min at 37 °C. Reactions were stopped by adding an equal volume of quench buffer .Each round of 2+ ions, the ssDNA library with embedded ribonucleotide residues was allowed to fold and self-cleave in selection buffer  for 3 h at 37 °C. Sequences that were able to catalyze RNA cleavage reactions were collected. To favor the enrichment of deoxyribozymes with fast catalytic rates, the incubation time was decreased incrementally from 3 h in round one to 30 min in round ten.After primer extension, the dsDNA library was immobilized onto pre-equilibrated streptavidin-coated magnetic beads and the template strand was removed by incubation in the presence of 20 mM NaOH for 1 h at 37 °C. After extensive wash using wash buffer  to remove residual NaOH and Mgin vitro selection and amplification, the DNA library was cloned and sequenced to examine the diversity of the molecules that remained in the pool.The recovered functional sequences were amplified by PCR using Taq DNA polymerase and PCR primers P1 and P2. The dsDNA library was made single-stranded by immobilizing the pool onto streptavidin-coated magnetic beads and eluting the template strand with 20 mM NaOH for 1 h at 37 °C. After ten rounds of 32P] ATP with T4 polynucleotide kinase for 1 h at 37 °C. 32P-labeled substrate (500 nM) was incubated with each deoxyribozyme clone (500 nM) in screen buffer  for 3 h at 37 °C. Reaction products were analyzed by 20% denaturing PAGE. Deoxyribozymes 8–17 and 10–23 were included as positive controls, and partial alkaline hydrolysis was performed by incubating the chimeric substrate in the presence of 200 mM NaOH for 4 min at 37 °C to generate a ladder for comparison.The unmodified DNA-RNA chimeric substrate was 5′-end labeled by incubation in the presence of [γ-To evaluate the effect of single-point mutations and to probe the substrate cleavage site selectivity of the deoxyribozyme, 5′-Cy5.5-labeled RNA substrate (100 nM) and deoxyribozyme variants (100 nM) were incubated in screen buffer for 3 h at 37 °C. Following incubation, 1 μL reaction product was mixed with 20 μL ice-cold stop buffer  and resolved on 20% denaturing PAGE. The gel was visualized and analyzed using Dual-channel Near Infrared Laser Scanning Imaging System.2+ (1 mM) and Pb2+ (0.1 mM), the concentration of the divalent metal ion is 3 mM. The reaction pH was maintained at 7.0 except for Pb2+ (pH 6.0). To probe the pH dependency of deoxyribozyme-catalyzed RNA cleavage, similar reactions were carried out but at varying pH conditions. After incubating at 37 °C for 3 h, 1 μL reaction product was mixed with 20 μL ice-cold stop buffer and resolved on 20% denaturing PAGE The gel was then visualized and analyzed using Dual-channel Near Infrared Laser Scanning Imaging System.Deoxyribozyme 10–12opt (100 nM) and Cy5.5-labeled RNA substrate (100 nM) were incubated under different reaction conditions. To study the effect of metal ion on deoxyribozyme catalysis, reactions were carried out with or without 1 M NaCl, in the presence of various divalent metal ions. Except for Zn2, 1 M NaCl. All reactions were started at the same time. At specific time points, 1 μL reaction aliquot was removed from the water bath and quenched by mixing with 20 μL ice-cold stop buffer. The cleavage products were analyzed on denaturing PAGE.Kinetic assays were carried out at 37 °C under a single turnover condition (enzyme:substrate = 100:1) with 10 nM substrate and 1 μM DNAzyme in 50 mM Tris (pH 7.5) containing 50 mM MgClThe RNA substrate was conjugated to an ATP aptamer to facilitate RNA cleavage-based ATP detection. The aptamer-linked substrate (100 nM), in the absence or presence of various concentrations of ATP, was first denatured in screen buffer by heating at 70 °C for 30 s and then allowed to fold by cooling slowly to 50 °C for 50 s and holding at 4 °C. Reactions were initiated by adding deoxyribozyme 10–12opt (1 μM) and the reaction progress was monitored by removing aliquots at specific time points, mixing with ice-cold stop buffer and analyzing on denaturing PAGE.21. Briefly, an RNA-DNA chimeric primer with 12 embedded ribonucleotides was annealed to the library template strand. The DNA library contained a central degenerate region composed of partially randomized nucleobases with a lower frequency of guanine  in the left binding arm had little influence on catalysis, mutations to T5, C6 and T7 caused more dramatic reduction in deoxyribozyme catalytic activity Fig. . ParticuTo determine whether a putative dT:rG wobble pair at position 4 is necessary for catalysis, we constructed four deoxyribozymes and four substrates with varied nucleotide identities at this putative wobble pair position. When these 16 enzyme-substrate combinations were assayed for RNA cleavage, it was discovered unexpectedly that significant cleavage was only observed when RNA substrate contains a guanine at the +4 position and on the opposite position of deoxyribozyme is not cytosine Fig. , suggestIt has not been observed in any previously reported RNA-cleaving deoxyribozymes that a riboguanosine residue downstream of the cleavage site was critical for catalysis. It was even more fascinating that formation of canonical rG:dC base pair at this position completely abolished deoxyribozyme catalytic activity. Intrigued by this observation, we employed modified nucleobase analogue substitutions to investigate the role of nucleobase functional group. We first introduced base mutations to position 4 of deoxyribozyme, using hypoxanthine, 2-aminopurine and 8-oxoguanine as purine analogues and using uracil and 5-methylcytosine as pyrimidine analogues. Consistent with earlier natural base substitution results, RNA substrates with A, U and C residues at the +4 position were not efficiently cleaved, no matter what nucleobase analogue was used at position 4 of the deoxyribozyme Fig. . Only RNHaving observed that the first three residues of deoxyribozyme left binding could be mutated without affecting its catalytic activity, and that the fourth position had a strong and unusual preference against cytosine, we wondered what the impacts of different substrate-binding arm lengths on deoxyribozyme catalytic activity would be. Therefore we synthesized deoxyribozyme constructs of various binding arm lengths. As expected, the catalytic activity of deoxyribozyme gradually decreased with incrementally shortened right binding arm, presumably due to weakened binding affinity to the substrate. However, when the length of left binding arm was varied, the deoxyribozyme exhibited maximal catalytic activity when the arm length was between four and seven base pairs Fig. . FurtherWe studied the RNA cleavage site selectivity of deoxyribozyme 10–12opt. When assayed against RNA substrates with 16 different cleavage site dinucleotide junctions, 10–12opt exhibited preferential cleavage activities, with 5′-UN-3′ as the most favorable cleavage junction in the order of UU > UG > UA > UC. As shown in Fig. Considering the deoxyribozyme showed strong substrate sequence requirement and preference at positions −1, +1 and +4 Figs , 4B, we 2+, and as expected the catalytic activity of 10–12opt increases in a magnesium-dependent manner ) where Ymax was the yield of a 24-hour reaction. As illustrated in Fig. kobs) of 1.5 × 10−2 min−1 and 2.7 × 10−3 min−1 towards RNA substrates with UU and UA cleavage site junctions, respectively.To quantify the catalytic activity of 10–12opt, we measured the reaction rates of deoxyribozyme-catalyzed RNA cleavage under single turnover conditions. Briefly, 10 nM RNA substrate was incubated with 1 μM deoxyribozyme (substrate/enzyme ratio = 1:100) in 50 mM Tris (pH 7.5) buffer containing 50 mM MgClGiven that deoxyribozyme 10–12opt features an unusually short left binding arm and selectively cleaves RNA substrates containing UNGRG consensus sequence, we reasoned that such deoxyribozyme could be used to cleave microRNA (miR), which is intrinsically short in length and plays diverse regulatory roles in biology. We searched the human microRNA database for microRNA sequences that meet the above requirements, and chose miR-676-3p, miR-3658 and miR-3675-5p as substrates because they each contain UU, UG and UA cleavage site junctions, respectively. For comparison, deoxyribozyme 8–17 was included because it exhibited low catalytic activity at such cleavage sites. As expected, deoxyribozyme 10–12opt could effectively cleave all three microRNA substrates  rendered deoxyribozyme catalytically inactive , or when this guanine was restrained in a perfect duplex by formation of a canonical rG:dC base pair, the catalytic activity of the deoxyribozyme was greatly diminished. This has not been observed in 8–17 or any reported RNA-cleaving deoxyribozymes. Furthermore, deoxyribozyme 8–17 has four conserved residues  with tertiary interactions within its catalytic coreive Fig. . Lastly,ive Fig. . All the37. A uracil residue near the ligation site was found to be critical for catalysis, and initially suspected to form a base pair with the deoxyribozyme nucleotide G or A. However, substitution and compensatory mutational analysis indicated that this residue was not interacting with deoxyribozyme via a simple base pairing.Almost all RNA-cleaving deoxyribozymes discovered thus far feature two substrate-binding arms and a central catalytic core. Initially, deoxyribozyme 10–12opt appeared to adopt a similar structure. However, careful mutagenesis analysis suggested that it might adopt a different catalytic conformation. It was found that substitution to or deletion of the first three nucleotides  of the 7-bp left binding arm had little influence on deoxyribozyme catalytic activity Figs , suggest38. Altogether, these results suggested that the role of the unusually short left binding arm was not limited to substrate binding. Instead, the requirement that a canonical base pair must not form at position 4 indicated that that at least the riboguanosine nucleotide was directly involved in catalysis. Since our deoxyribozyme 10–12opt features an unusually short substrate-binding arm, it could be potentially useful in assays where RNA substrate is short and near-terminus site-specific cleavage is desired. Moreover, 10–12opt cleaves all-RNA substrates preferentially at UN dinucleotide junctions, and thus could be practically complementary to the previously reported 8–17 and 10–23 deoxyribozymes.Since the first three nucleotides could be deleted without compromising catalytic activity and T7 was not involved in base pairing with the RNA residue at +1 position, we reasoned that deoxyribozyme 10–12opt contained a 3-nucleotide left binding arm and 14-nucleotide catalytic core. Interestingly, only two nucleotides (T5 and C6) formed canonical base pairing with the RNA substrate, and the residue at position 4 must not form WC base pairing with G +4 on the RNA substrate for optimal catalysis. We speculated that the riboguanosine nucleotide on RNA substrate played a critical role in maintaining the deoxyribozyme catalytic structure or participated directly in the catalytic mechanism using its carbonyl O6 atom as a potential hydrogen bond acceptor. In fact, RNA substrates with non-guanosine nucleotides at +4 position were not efficiently cleaved by any 10–12opt variants. Moreover, deoxyribozyme mutant with cytosine at position 4, which formed a canonical dC:rG base pair, exhibited little or reduced catalytic activity, implying that at least some flexibility of G +4 was necessary for optimal catalysis and restraining this essential guanine in a perfect duplex conformation lead to significantly reduced catalytic activity. To the best of our knowledge, this RNA-cleaving deoxyribozyme holds the shortest binding arm thus far, and the RNA substrate is the first one with an essential guanine residue away from the cleavage site, which must not form WC base pairing with deoxyribozyme. While not entirely identical to our shortened deoxyribozyme 10–12opt, one of Breaker’s oxidative DNA-cleaving deoxyribozymes also has an unusual and rather short triplex binding arm structure40. As these experiments progress, it will be interesting to learn how deoxyribozymes are distributed in sequence space and how well sequence copy number correlates with functional activity.Deoxyribozyme 10–12opt provides an interesting example of a DNA molecule that catalyzes RNA cleavage at a site-specific location, which represents a step forward in the discovery of diverse DNA architectures that cleave RNA. To our knowledge, 10–12opt is the first example of an RNA-cleaving deoxyribozyme that contains only three nucleotides as its left binding arm. Within this region, a guanine on RNA substrate proves critical for catalytic activity and it must not form a canonical rG:dC base pair with deoxyribozyme for optimal catalysis. All these unusual sequence requirements and preferences suggest that 10–12opt forms a discrete catalytic architecture, different from all known RNA-cleaving DNA enzymes. This discovery provides the motivation to search combinatorial libraries for DNA molecules with even more diverse structural features. Obtaining an unbiased view of the fitness landscape should be possible with recent advances in next-generation deep sequencing technologies, which allow for a large sampling of DNA sequencesWe have identified a novel small RNA-cleaving deoxyribozyme with an unusually short (3-nucleotide) left substrate-binding arm, which preferentially catalyzes RNA cleavage at UN junctions. A riboguanosine nucleotide within the left binding arm proves critical for catalysis, and its O6 atom is likely involved in catalysis. Taking advantage of its short left binding arm, it was further demonstrated that this deoxyribozyme could be used to catalyze site-specific cleavage of short microRNA sequences containing the UNGRG consensus sequence near the 3′ end. This deoxyribozyme represents a unique catalytic DNA structure capable of promoting site-specific RNA cleavage.supplementary info


We demonstrated that Com recognizes a sequence motif within a hairpin-loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so-called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19-mer self-complementary double helix composed of the canonical and non-canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin form.The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification (momification) of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com–RNA interactions by  RNA-binding proteins play important roles at every stage of RNA life cycle: transcription, splicing, editing, export, degradation and regulation of translation . Many ofThe bacteriophage Mu Com-RNA complex can be considered one of the model systems for studying the interaction of a ZnF domain with RNA. The Com protein consists of 62 amino acids, including an N-terminal CCCC zinc finger module where four cysteine residues are involved in zinc ion coordination, and a C-terminal intrinsically disordered segment ,14. Com In this work, we aimed at establishing the preferred RNA sequence recognized by Com and determining the structural basis of Com-RNA interactions. We attempted to co-crystallize Com with its natural RNA target, as well as with its variants designed computationally to form either a monomeric hairpin-loop structure or a homo-duplex form. Thus far, we obtained crystals and solved the structure of an RNA homo-duplex form. While the high-resolution structure of the Com-RNA complex remains to be determined, we propose a low-resolution structural model of Com-RNA interactions based on the available experimental data.Haemophilus sputorum Mu-like prophage Com gene sequence (GI:400376712) was optimized for Escherichia coli expression and synthesized by GeneArt Gene Synthesis (Thermo Fisher Scientific). The gene was subcloned into prokaryotic expression pGEX4T vector . E. coli BL21(DE3) (New England Biolabs) was used to overexpressed the glutathione S-transferase (GST) fusion-tagged Com that included a two-residue linker (S-H) between the GST tag and Com. Expression was carried out in LB medium, induced with 1 mM isopropyl-D-1-thiogalactopyranoside solution (IPTG) at OD600 of 0.6 and conducted at 37°C with shaking at 200 rpm for 4 hours. The LB medium was supplemented with 100 μM ZnSO4 (Sigma-Aldrich) upon induction. The fusion protein was purified by GST affinity with Glutathione-Agarose beads (Sigma-Aldrich) according to the manufacturer protocol and stored in +4°C.The et al.  labeled synthetic RNAs. RNAs were labeled with γ 33P ATP  using T4 PNK Kinase (Thermo Fisher Scientific), phenol/chloroform extracted, precipitated, desalted and separated from the unincorporated label on MicroSpin G-25 Columns . RNA samples were annealed in buffer containing 50 mM Tris, pH 7, 2.5 mM MgCl2 by heating at 80°C for 15 min and cooling 1°C per min until it reached room temperature .The RNA-protein interactions were determined by the Electrophoretic Mobility Shift Assay (EMSA). We used the affinity purified GST-fused Com and . ,45. 44,4S1 FigUnit cell boundary is shown in black with axes marked. Some nucleotides are labeled for indication of the orientation. Figure prepared in PyMol .(TIFF)Click here for additional data file.S2 FigNon-canonical base pairs are represented in red spheres with the exception of the G•G pair represented in green. (A) Intra-strand hydrogen bonding and base step distances. (B) Inter-strand C1α-C1α distances, and N-C1α-C1α and C1α-C1α-N angles (see text for details). Two numbers are shown when double conformation is present in the structure.(TIFF)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(PDF)Click here for additional data file.


This study used piecewise growth modeling to describe the developmental trajectories of self-rated health (SRH) in the elderly and longitudinal associations with activities of daily living (ADL), educational level, economic status, age, and gender. Data were drawn from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), collected over 12 years (from 2002 to 2014) at five waves. A total of 16,064 Chinese elders  were analyzed. Results showed two phases of development for SRH; specifically, the decreasing trend of SRH was from slow  to fast . Descriptives showed that the turning point age was at the age of 83.69 . ADL were positively associated with SRH within each time point (wave of data). Female elders had a higher initial state  of SRH than did male elders, and poorer economic status was associated with worse initial status of SRH. Self-rated health (SRH) in elderly people has been documented and understood as an important indicator of the elder’s overall health over time . In 2016The elderly population is increasing globally . SRH is Given that SRH has an important effect on public health, it is important to identify the determinants of SRH. These may include gender , educatiFew studies have discussed SRH from a longitudinal perspective, rather, most SRH studies have been conducted using cross-sectional data . Cross-sIn sum, this study intended to examine (1) the trajectory patterns and the rate of change of SRH over time in Chinese elders, and (2) the longitudinal associations between SRH and ADL, educational level, economic status, age, and gender. It is hoped that the present study could provide scientific research evidence for the importance of improving the quality of life in elderly people considering their own perceptions of SRH.M = 86.33, SD = 11.70) at baseline. Men averaged 83.76 (SD = 10.80) and women 88.23 (SD = 11.98) years of age. There were 1,680 elders contributing complete data at all five waves . 834 had data at four waves , 1,677 had data at three waves , 3,984 had data at two waves , and 7,889 had data at one wave . Because our models involved within time point (wave) associations, we included these elders with 1 time point data in our analysis.Participants included a large, random sample of Chinese elders involved in the CLHLS . The CLHChinese Longitudinal Healthy Longevity Survey is accessible to the public for research purposes . PermissVery good through 5 = Very bad). Higher scores indicated lower in elders’ SRH.Self-rated health was measured using the sing-item question, “What do you think of your own health?” on 5 point response scales . We created a composite score for each elder by taking the sum of all these 14 items (scores were reversed whenever necessary), with higher scores indicating more help needed. Internal consistency for the ADL items was good at each wave .Activities of daily living was measured using the Katz index . The ChiMale; 1 = Female), age, self-reported education level (years of schooling) and economic status (from 1 = very rich to 5 = very poor), assessed at the 2002 wave .Covariate variables included gender  using maximum likelihood estimation with robust standard errors . We usedDescriptive statistics and bivariate correlations among primary variables are displayed in b = 2.603 (0.007), p < 0.001, and the average growth in Chinese elders’ SRH was 0.01 in the first phase , b = 0.013 (0.002), p < 0.001, and the average growth in Chinese elders’ SRH was 0.02 in the second phase , b = 0.020 (0.004), p < 0.001. As expected, Chinese elders’ SRH decreased by 0.01 units each studied wave period in the first phase and decreased by 0.02 units in the second phase each studied wave period. The rate of change (declining) of SRH was from slow to fast. Results also indicated that there was non-trivial variation in the amount of Chinese elders’ SRH at the initial time point, b = 0.256 (0.022), p < 0.001, and Chinese elders’ SRH may be varied over time both in the first phase, b = 0.003 (0.001), p = 0.022, and in the second phase, b = 0.009 (0.002), p < 0.001. The amount of Chinese elders’ SRH at the initial time of measurement was associated with changes over time in the first phase (but not with changes in the second phase). In this study, the turning point of development for SRH was at time point 3 (the 2008 wave). The average age of the study elders was 83.69 .We examined the model fit indicators for the unconditional growth models . Results showed that the piecewise growth model fit the data best . Two phaWe tested the effects of ADL (time varying) within each wave (within person) and also the time-invariant covariates gender, age, education level, and economic status  in our two-stage piecewise growth model. n = 1,680). Similarly, two phases of development for SRH was identified. Specifically, the average starting amount of SRH (the intercept) was 2.39 units, b = 2.388 (0.020), p < 0.001, and the average growth in elders’ SRH was 0.02 in the first phase , b = 0.017 (0.004), p = 0.002, and the average growth in elders’ SRH was 0.03 in the second phase , b = 0.033 (0.005), p < 0.001. Results also indicated that there was variation in the amount of Chinese elders’ SRH at the initial time point, b = 0.178 (0.037), p < 0.001, and elders’ SRH varied over time in the second phase, b = 0.008 (0.002), p < 0.001, but not in the first phase (p = 0.498). Results provide the first evidence for two phases of development for SRH in Chinese elderly and help to understand their perceptions of their own health functioning.To ensure that the pattern of results was robust, we tested the piecewise growth model using the sample who had five wave data , octogenarians , nonagenarians , and centenarians . Results showed that there were positive associations between SRH and ADL for each of these four groups of Chinese elderly sample. However, the estimated relationship weakened with age .We did not provide a hypothesis for the age-specific relationship between SRH and ADL of the Chinese elderly sample. However, we conducted a series of linear growth models using the data widetolong command to examine the possible age-specific relationship . To do so, we categorized the sample into four age groups – young elders . This finding is consistent with what the literature has documented in other cultures e.g., . The decThis study provides evidence that several variables contribute to Chinese elders’ SRH. Consistent with other studies e.g., , we founThis study showed that Chinese elderly people with a poorer economic status had a worse initial SRH . SeveralThis study also showed that female elders had a worse initial state of SRH than did male elders in the sample. First, a relatively low proportion of the sample (10.5%) contributed complete data at all five waves. We considered several covariate variables . Other variables could also be included, for example, family and community information norms that might be related to elders’ SRH. Second, data were elders self-reported and self-evaluated. Future study should include objective measures to assess SRH. Third, in this study, we used piecewise growth modeling. When we entered several covariates, individual differences existed. Future study could use piecewise growth mixture modeling to classify participants into different categories (membership) and then explore the study relationships. The current data did not allow us to do so due to the limited portion of complete data over the five waves. Finally, it seemed sufficient to use linear growth modeling to examine the relationship between SRH and ADL in the current data, but piecewise growth modeling indicated better model fit . HoweverIn this study, we examined the developmental trajectories of SRH in Chinese elderly people and the predictive role of ADL, gender, age, education level (years of schooling), and economic status. Two phases of development for SRH were identified. The decreasing trend of SRH was slow in the first phase (waves 1 to 3) and was fast in the second phase (waves 3 to 5). Our results suggest that the trend of SRH (declining) should not be understood as linear. ADL was a protective factor for SRH within each time point. Women’s physical health should be an important focus.All authors have contributed significantly to the work and agreed to the current version of the manuscript.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.


Previous studies have shown that affording people choice increases their satisfaction with subsequent experiences: the choice effect. However, it remains unclear whether the choice effect occurs in the hedonic response to foods and beverages. Thus, the present study aimed to demonstrate the choice effect on the palatability perception. Ready-to-serve curries and tea were presented as options in Experiment 1 and Experiment 2, respectively. Experiment 1 failed to demonstrate significant differences among palatability ratings for a curry chosen by participants and for a curry chosen by the experimenter. However, Experiment 2 demonstrated that participants perceived a tea chosen by themselves as more palatable than another tea chosen by the experimenter, regardless of the fact that the two cups of tea were identical. Intriguingly, the effect was obtained only when the number of options was neither too small nor too big. These results indicate that the exercise of choice from an optimal number of options, even when the choice is ostensible and illusory, makes people perceive their chosen foods and beverages as being more palatable. Some implications for the domain of food business are also discussed. It is generally believed that our hedonic responses to foods and beverages are simply determined by their physical and chemical properties. However, a growing body of studies has demonstrated that hedonic responses to foods and beverages are significantly affected by contextual factors such as the order of tasting ,2, plateWhen the hedonic responses to foods and beverages are investigated by researchers, several different aspects, such as palatability, pleasantness, liking, or preference are discussed. Generally, palatability and pleasantness are thought to be hedonic experience of the sensory aspects of foods and beverages, while liking and preference are thought to be cognitive judgement or resultant selection behavior ,11,12. Tthe choice effect in the present study, is applicable to the food domain, appropriately affording people choice can lead them to perceiving a food or beverage as being more palatable. If the choice effect occurs in food perception, people who are afforded the chance to choose would perceive their chosen food as being more palatable than others who are merely given the food. Moreover, people would perceive a food chosen by themselves as more palatable than another food chosen by others, even when the foods are the same.In our everyday life, we, as consumers, often struggle to choose the best item from a number of items in supermarkets or online marketplaces. Choice is customarily believed to be an opportunity to match a preference with the available options. Contrary to this belief, recent psychological studies have demonstrated that choice affects the response to its outcome; people who were afforded a chance to choose were more satisfied with their chosen option or performed better in their chosen activity or task compared to their counterparts who were not afforded the chance to choose but were simply given the same option or activity ,14,15,16This study aims to demonstrate the choice effect on the palatability perception of foods and beverages. The present study consists of two experiments. A variety of ready-to-serve curries and tea were presented as options in Experiment 1 and Experiment 2, respectively. Participants were asked to choose one item to taste and evaluated its palatability. In Experiment 1, the choice effect was examined in terms of expectation before tasting. In Experiment 2, the choice effect was examined in terms of the number of options presented.This experiment aimed to demonstrate the choice effect on palatability perception of the chosen curry and to examine its relationship with expectation. In our daily lives, people normally choose one item from various options because they expect that the item will provide them with the greatest experience or utility among the available options; choice often concurs with greater expectation. Since expectations about foods or beverages are known to affect the actual experience of foods or beverages ,18,19, tIn this experiment, nine kinds of ready-to-serve curries were presented as options. Participants were asked to rank the curries based on their expected palatability , and choose one curry they wanted to taste from three curries ranked medium . Participants were randomly allocated into one of the three experimental conditions: the mid-choice condition, the high-given condition, or the low-given condition. In the mid-choice condition, participants were told that they were going to be served the curry they had chosen. In the high- and low-given conditions, on the other hand, participants were told that they were going to be served a higher- or lower-ranked curry that the experimenter had chosen. Regardless of what the participants chose or believed to taste, the curry that was actually served and tasted was identical across the conditions, which enabled us to control the choice outcome and examine the choice effect more directly ,21. If tageM = 20.7 years) participated in this experiment. Participants were randomly allocated into the mid-choice (n = 10), high-given (n = 10), or the low-given condition (n = 11).Thirty-one university students , Ginza Curry , Indian Curry Beef Spicy , Bon Curry , and Curry Youbi . In the choice session, these visual stimuli were presented as options and participants were asked to choose one curry to taste  or Curry B , was presented. This aimed to examine whether the possible effect would be restricted to a certain type of curry. The identity of the presented curry was counter-balanced across the participants. The curry was prepared and delivered to participants just before tasting (20 g of curry sauce with 50 g cooked rice).In the tasting session, either of the two types of curries, Curry A  on a 55-mm visual analog scale (VAS). The scale was anchored “seems not palatable at all” and “seems extremely palatable” at the left and right ends of the scales.Participants were then presented with the curry, and asked to taste and evaluate how palatable the curry was  on the 55-mm VAS. The scale was anchored “absolutely not palatable” and “extremely palatable” at the left and right ends of the scales. Regardless of what participants believed to taste, in actuality, the curry presented in this tasting session was determined beforehand: Curry A or Curry B .To obtain the expected palatability rating and the perceived palatability rating, the length (mm) from the left edge of the VAS to a mark participants had made was measured for each evaluation and converted into rating ranging from 0 to 100.a priori multiple comparison of the expected palatability rating and the perceived palatability rating (the function pSDCFlig in the package NSM3 in R). To examine the relationship between the expected palatability rating and the perceived palatability rating, Spearman’s rank correlation coefficient ρ was calculated based on the global data as well as the subsets of the experimental conditions (the function cor.test in R). Probability values of less than 0.05 (p < 0.05) were considered statistically significant.Statistical analyses were performed by the software R . Since the sample size in this experiment was small, non-parametric statistical analyses were conducted. To assess the differences among the experimental conditions, Steel-Dwass test, known as a non-parametric equivalent of Tukey-Kramer test, was used as W = −3.34, p = 0.0475). However, the mid-choice condition did not significantly differ from the high-given  or the low-given conditions .The results for the expected palatability rating are shown in ps > 0.10).The results for the perceived palatability rating of the sampled curries are shown in Spearman’s rank correlation coefficients between the indices were calculated based on the global data, as well as the subsets of the experimental conditions . HoweverThis experiment aimed to demonstrate the choice effect on the palatability perception of curry and examine its potential relationship with expectation. However, this experiment failed to demonstrate both the significant choice effect and its relationship with expectation.The expected palatability rating for the high-given condition was significantly higher than that for the low-given condition. The rating was intermediate for the mid-choice condition. This result indicates that the experimental manipulation of expectation was successful.We predicted that, if the choice effect is simply driven by greater expectation of the item, participants in the high-given condition would perceive the curry as being more palatable than those in the mid-choice and low-given conditions. However, the perceived palatability ratings were not significantly different among the conditions. Furthermore, there were no significant correlations between the expected palatability rating and the perceived palatability rating. These results indicate that expectation had little or no effect on palatability perception in this experiment.There was a tendency that the perceived palatability rating for the mid-choice condition was higher than those for the high- and low-given conditions when the sampled curry was Curry B. This tendency was consistent with another prediction that, if the choice itself plays a key role in the choice effect, participants in the mid-choice condition whose choice was accepted would perceive the curry as being the most palatable.medium-ranked curries). This instruction may have made participants perceive the number of options as three rather than nine, which is thought of as too small a number of options to increase consumers’ satisfaction [Since the sensory characteristics of the sampled curries were not well investigated in the present study, it is difficult to fully interpret why the tendency was obtained only when a certain curry was sampled. One possible explanation for the ambiguous result is that the choice effect was attenuated by certain factors. For instance, in this experiment, participants were presented with nine curries. The number of options had been determined based on previous studies, suggesting that eight to ten options are ideal to increase consumers’ satisfaction with their choice ,22,23. Hsfaction ,22,23. TThis experiment aimed to further examine the hypothesis that choice plays a key role in the choice effect, by manipulating and controlling the number of options. In addition, whereas Experiment 1 examined the choice effect in the between-participant design, this experiment examined it in the within-participant design, which aimed to exclude participants’ individual differences of tea preference.In this experiment, several kinds of tea were presented as options. The number of options varied among experimental conditions: three , nine (medium), and twelve (large) options. Participants were asked to choose a preferable tea bag from the options. Participants were told that they were going to taste and evaluate the tea they had chosen  as well as another tea that the experimenter had chosen at random . However, regardless of what the participants chose or believed to taste, the two cups of tea that were served were identical throughout the experiment; all participants tasted and evaluated the same tea twice. This manipulation, as in Experiment 1, aimed to control the choice outcome and examine the choice effect more directly ,21. It wIt has been suggested that consumers’ satisfaction with their choice increases when the sense of self-determination of their choice is stronger, and also that the sense of self-determination increases as a function of the perceived variety in the option assortment . TherefoageM = 19.5 years) participated in this experiment. Participants were randomly allocated into the 3-option condition (n = 18), the 9-option condition (n = 17), or the 12-option condition (n = 15).Fifty university students  were used as visual stimuli. In the 3-option condition, three kinds of tea bags were used: to taste .Koucha-no-Jikan , was presented twice. The tea was stored in a refrigerator and prepared just before tasting. The tea (100 mL) was delivered to the participants in a clear cup.In the tasting session, an iced straight tea with low sugar, The experimental procedure is summarized in Participants were then asked to evaluate how much of a sense of variety they perceived in the assortment . This evaluation was conducted, based on previous studies ,24, by aas well as another tea that was randomly chosen by the experimenter . After that, participants were asked which tea they wanted to taste. Once participants indicated their choice, they were asked to evaluate how much they felt that the choice was based on their own will . This evaluation was conducted, based on previous studies ,25, by aThe experimenter prepared the tea in another room, and then participants were sequentially and separately presented with the chosen tea and the given tea. The order of presentation was counter-balanced across the participants. Each tasting session was separated by about three minutes. Regardless of what participants believed they tasted, in actuality, the two cups of tea were identical. To make participants believe that they were tasting two different teas, tea bags of the chosen tea and the given tea were presented together with the cups of tea. Participants were asked to taste and evaluate the palatability of the tea  on the 100-mm VAS anchored “absolutely not palatable” and “extremely palatable” at the left and right ends of the scales.After the tasting session, participants were asked to verbally report anything special they had felt or noticed during the experiment. Participants were then informed the true purpose of the experiment.n = 5) reported that they had noticed the chosen and the given tea being identical. They might have also noticed the true purpose of this experiment, which could have altered their response and rating. Therefore, one participant in the 3-option condition, three participants in the 9-option condition, and one participant in the 12-option condition were excluded from the following analyses.Some participants  from the left edge of the VAS to a mark participants had made was measured for each evaluation. In addition, to directly examine the choice effect on palatability perception, the palatability rating for the given tea was subtracted from that for the chosen tea for each participant, and it was used as a choice effect score .The perceived variety in the assortment was measured by the two different items. Since internal reliability of the two items was high (Cronbach’s α = 0.808), scores for the first item (“How different are the tea options from each other?”) and reverse scores for the second item (“How similar are the tea options to each other?”) were averaged to create an index of perceived variety.The sense of self-determination was also measured by the two different items, but internal reliability was not high (Cronbach’s α = 0.492). Therefore, scores for the two items (“I selected this particular tea because I wanted to” and “I selected this particular tea because I had no choice”) were individually analyzed as indices of the sense of self-determination.a priori multiple comparison of the choice effect score, the perceived variety in the assortment, and the sense of self-determination (the function pSDCFlig in the package NSM3 in R). To examine whether the choice effect scores for each condition significantly differed from zero, one-sample Wilcoxon signed rank test against zero was also conducted (the function wilcox.test in the package ggpubr in R). Probability values of less than 0.05 (p < 0.05) were considered statistically significant.Since the sample size in this experiment was small, non-parametric statistical analyses were conducted. To assess the differences among the experimental conditions, as in Experiment 1, Steel-Dwass test was used as W = 3.40, p = 0.0428), though the 9-option did not significantly differ from the 12-option condition . There was no significant difference between the 12-option and the 3-option conditions . Moreover, one-sample Wilcoxon signed rank tests revealed that the median for the 9-option condition was significantly higher than zero (p = 0.01915), but that for the 3-option (p = 0.48) and 12-option conditions (p = 0.78) did not differ from zero.The results for the choice effect score among the experimental conditions are shown in ps > 0.10).The results for the perceived variety in the assortment among the experimental conditions are shown in ps > 0.10).The results for the sense of self-determination among the experimental conditions are shown in This experiment aimed to examine the hypothesis that participants would perceive the chosen tea as more palatable than the given tea, and also that the choice effect would be obtained only when the number of options was nine, but not three or twelve. The result supported the hypothesis: the choice effect score for the 9-option condition was significantly positive and the highest, but the scores for the 3-option or the 12-option conditions were not significant. This result was consistent with previous findings ,22,23 thIt has been suggested that consumers’ satisfaction with their choices increases when the sense of self-determination of their choice is strong, and also that the sense of self-determination increases as a function of the perceived variety of the assortment . Thus, wA growing body of research has shown that our hedonic responses to foods and beverages are not simply determined by their physical and chemical properties ,7,8,9,10Experiment 1 aimed to demonstrate the choice effect on the palatability perception of a chosen curry and examine its relationship with expectation. The result showed that the expected palatability rating was high for the high-given condition where the participants believed that they were tasting a curry highly ranked by themselves. However, the perceived palatability ratings were not different among the experimental conditions. Moreover, contrary to previous studies suggesting a strong link between expectation and the actual experience of foods and beverages ,18,19, tThe result of Experiment 1 also showed a tendency that the perceived palatability rating was high for the mid-choice condition where participants believed that they were tasting a curry they had chosen from the medium-ranked curries, but it was not significant or consistent. We speculated that the key of the choice effect was the exercise of choice itself, but the choice effect was attenuated since participants might have perceived the number of options as three rather than nine, which is thought of as being too small to increase participants’ satisfaction with their choice ,22,23.Experiment 2 examined this hypothesis by manipulating and controlling the number of options. The results showed that when the number of options was nine, but not three or twelve, participants perceived the chosen tea as more palatable than the given tea. This result is surprising since the two cups of tea (ostensibly served as the chosen tea and the given tea) were identical. Therefore, it is indicated that when choice is exercised from an optimal number of options, the choice effect on palatability perception does occur. To our knowledge, this is the first demonstration of the choice effect on the palatability perception of beverages.Consistent with the suggestion from previous studies ,22,23, tThe present study focused on the palatability perception of foods and beverages. Palatability (or pleasantness) perception is thought to be hedonic or emotional experiences of the sensory aspects of foods, and thus distinct from food preference or liking, which is thought to include more cognitive and behavioral components ,11,12. AOne of the practical implications of the present study for the domain of food business is that marketers should carefully manage the number of available options on their food/drink menus, in stores, or web-sites. As shown in a previous study, large options indeed attract customers’ attention at first, but customers in such a situation often hesitate to decide which one to buy, and end up buying nothing . Our finAnother implication is that affording customer multiple opportunities of choice might greatly increase the palatability perception of a food or beverage. For instance, a coffee shop where customers can customize their choice in a number of aspects, such as the type of beans, origin, roasting, grinding and so forth. Each aspect might consist of a manageable number of options . In this way, customers are afforded multiple opportunities of choice from a manageable number of options. Even if the outcome of the customization does not perfectly match the customers’ original preference, this system might greatly increase customers’ satisfaction with their choice, and can thus increase the palatability perception of their chosen coffee. It is meaningful for both researchers and marketers to examine this possibility in the future.It should be noted that the only two types of food products  were used in the experiments. Furthermore, in Experiment 2 the only one tea product was sampled by the participants, which was aimed to control the choice outcome and examine the choice effect more directly ,21. We dThe present study aimed to demonstrate the choice effect on the palatability perception of foods and beverages, using curry and tea as model products. The results indicate that the exercise of choice from an optimal number of options, even when the choice is ostensible, makes participants perceive their chosen curry and tea as being more palatable. Although the psychological mechanism underlying the choice effect still needs to be elucidated further, we believe that the present study sheds light on a new aspect of the effect of contextual factors on our hedonic responses to foods and beverages.


In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts. H. sapiens and M. musculus, according to the Ensembl and Mouse Genome Informatics (MGI) database respectively), global regulation of all the genome components  is not achieved by transcription factors (TFs) alone because of their low number  compare mammals ,8. The eThe genome of multicellular eukaryotes is mostly comprised of non-coding DNA  through a complementary oligonucleotide composed by a series of deoxy-thymines (oligod(T)). First, we will describe the lncRNA classification and known functions. Then, we will introduce single-cell analyses and discuss methods available for scRNA-seq and other approaches used for the analysis of lncRNAs at a single-cell level focusing on their pros and cons. We will present results obtained using these methods and discuss what new paradigms have been proposed. To conclude this review, we will present available databases that include information about lncRNAs and variations in the genome, transcriptome, and proteome of single-cells that could be useful for scientists that are planning studies in which lncRNA and single-cell information can be advantageous for the experimental design.The term “long non-coding RNA” (lncRNA) refers to a very heterogeneous group of genes/transcripts. In association with our poor knowledge about the biological functions of several lncRNAs they also do not conserve a primary structure among species, a feature that can be helpful to understand their role. For these reasons, there are different methods of classification, each of them focusing on a different feature of these genes.The name lncRNA is relatively recent. The first non-coding transcripts studied were classified only based on their role . In the early 2000s, when the abundance of non-coding transcripts became evident [It is known that lncRNAs represent the largest part of the transcriptome of mammalians ,34 and pLong intergenic non-coding RNAs are transcribed from genomic regions between coding genes; therefore, lincRNAs are not inserted in any protein-coding loci A. LincRNLncRNAs can also be localized in a genomic region that overlaps protein-coding genes. These transcripts are called genic lncRNAs. COLDAIR which is transcribed from the first intron of the coding gene Flowering Locus C (FLC) [Genic lncRNAs could be intronic when entirely overlap with an intron of a protein-coding gene or exonic when entirely or partially overlap with exons of protein-coding genes. In these cases, the transcriptional orientation of genic lncRNAs can be in sense or antisense compared to the transcriptional orientation of protein-coding genes. Genic antisense lncRNAs are transcribed from the opposite strand of the closest protein-coding gene and can overlap with a 5′ head-to-head divergent orientation  . It is rDifferent RNAs are transcribed by different RNA polymerases (RNA Pol): Transfer RNAs (tRNAs) are transcribed by RNA Pol III, ribosomal RNAs (rRNAs) are mostly transcribed by RNA Pol I and Pol III, while most RNAs are transcribed by RNA Pol II. The latter one synthesizes for messenger RNAs (mRNAs), microRNAs (miRNAs), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), and most lncRNAs ,44. SomeHistorically, first long non-coding transcripts described were rRNAs thanks to their abundance in cells. They are the major structural constituents of the ribosome and can interact with specific sequences of mRNAs D. ProkarIn the late 1960s, James Bonner introduced and described a distinct class of RNAs capable of binding chromatin: chromosomal RNA or cRNA . LncRNAsPTENP1). The protein-coding gene Phosphatase And Tensin Homolog (PTEN) is regulated by miR-19b and -20a which can be sequestered by the pseudogene PTENP1 affecting the level of its cognate gene [LncRNAs can interact with miRNAs to act as post-transcriptional regulators of protein expression. miRNAs are short non-coding RNAs (18–28 nt)  that intate gene . Anotherate gene . LncRNAs transcribed from enhancers are called enhancer RNAs (eRNAs). Enhancers are short DNA regions (50–1500 bp), usually isolated from coding genes, that can influence gene transcription by binding specific protein activators . eRNAs cSINEUPs are modular antisense lncRNAs that have two sequence specificities: they contain an inverted SINEB2 sequence and a small complementarity sequence with the targeted mRNA. Due to these characteristics, SINEUPs can up-regulate the translation of mRNAs in a gene-specific manner without affecting gene expression. SINEUPs are coded by genes that overlap with the target protein-coding gene to allow a 5′ head-to-head binding ,78,79 FH. SINEUPAnalyzing the ability of ribosomes to associate with lncRNA molecules  researchSome chromatin interacting lncRNAs act by targeting the chromatin region from which they are transcribed. These are called cis-acting lncRNAs. The targeted chromatin is not necessarily restricted to the close proximity of the lncRNA, for example, the well-known lncRNA XIST acts by targeting the whole X chromosome in cis J.On the other hand, if the lncRNA target is not located in the proximity of its gene it is described as a trans-acting lncRNA J. HOTAIRFor a summary of functionally defined lncRNAs see The role of lncRNAs is strictly dependent on their subcellular localization. For example, chromatin interacting lncRNAs need to be localized in the nucleus while miRNA sponges are most commonly found in the cytoplasm.LncRNAs that are only found in the nucleus are classified as nuclear lncRNAs. These lncRNAs contain specific sequences to indicate that they have to be retained in the nucleus ,91. MostLncRNAs that localize into the cytoplasm are classified as cytoplasmic lncRNAs. The transport of lncRNAs such as mRNAs from the nucleus to the cytoplasm involves several different proteins localized on both sides of nuclear pore. The complex composed of the Tho complex, the RNA helicase UAP56  and Aly is loaded onto the mRNA or lncRNA in the nucleus producing the RNA protein complex (RNP). Then, the nuclear export receptor composed of Nuclear RNA Export Factor 1 (Nxf1) and Nuclear Transport Factor 2 Like Export Factor 1 (Nxt1)  associates with Aly and allows the transport at the nuclear pore of its cargo physically interacting with the phenylalanine (F) and glycine (G) repeats of proteins in the interior of nuclear pore. After the transport through the nuclear pore the RNP is remodeled to remove certain nuclear associated exported factors, recycled back into the nucleus, and to make the mRNA or the lncRNA stably located in the cytoplasm .Cytoplasmic lncRNAs are more commonly involved in post-transcriptional regulation and as previously described, competing endogenous RNAs can act as alternative targets for miRNAs to reduce the amount of RNA interference while SINEUPs, another family of cytoplasmic lncRNAs, can enhance the translation of target mRNAs without affecting mRNA transcription. Another example of cytoplasmic lncRNA is Tissue Differentiation-Inducing Non-Protein Coding RNA (TINCR) that binds to mRNA through a 25 nt sequence. This lncRNA has a strong affinity for the protein STAU1, the complex formed by this interaction is known to mediate the stabilization of mRNAs involved in epidermal differentiation . Eukaryotic cells have specific organelles for energy production (mitochondria) or photosynthesis (chloroplasts) in the case of cells of plants. Both organelles have endosymbiotic origins and present their own genome that during the evolution has lost most of the genes leaving to the nuclear genome the task of synthesizing for most of the proteins for organelles functions. Mitochondrial genome maintains the capacity of synthesizing for 13 proteins (required for part of OxPhos complexes), 22 tRNAs, and two ribosomal RNAs (12S and 16S). Mitochondrial DNA is a double-stranded circular molecule where it can be distinguished a heavy strand (rich in guanine) and a light strand. Heavy strand is reach in genes while light codes only for one subunit of nicotinamide adenine dinucleotide dehydrogenase (ND), 8 tRNAs, and 3 lncRNAs. Interestingly, these lncRNAs regulate ND5 (NADH dehydrogenase subunit 5), ND6 (NADH dehydrogenase subunit 6), and CYTB (cytochrome b) throughout complementary binding of respective RNAs . NuclearA. thaliana chloroplasts by RNAseq proposing a regulatory action by matching to the 5′-UTR, 3′-UTR, or coding regions of specific targets [The genome of chloroplasts, such as mitochondrial one, synthesizes for lncRNAs. Hotto and colleagues identified 107 non-coding RNAs from  targets . See [10 targets  for a reSince the first reports of single-cell RNA-sequencing in 2009 ,111, novTo obtain a cell suspension, the enzymatic digestion or mechanical disruption of tissues are commonly used as isolation methods. Depending on the biological question to be addressed and on the type of starting material, a pool of cells can be enriched based on its particular morphophysiological features . Different technologies can be used for enrichment, such as microfiltration, density gradient centrifugation, immunoaffinity, and magnetic-activated cell sorting . HoweverThe fluorescent activated cell sorting (FACS) has higher efficiency than the previous methods and the principle of this technique is based on tagging cells with a fluorescent monoclonal antibody specific to surface antigens or intracellular molecules. This allows the instrument to separate positive cells from negative cells and debris  Figure C. BesideAfter the isolation step, single-cells have to be lysed and the RNAs must be reverse transcribed maintaining the ability to identify from which cell derives the RNA information. As previously discussed, the number of cells that can be analyzed with single-cell approaches is comprised between 100 and 10,000 cells . It is eOn the other hand, the linear amplification is based on the in vitro transcription (IVT) by the T7 RNA polymerase, that synthesizes multiple copies of RNA, followed by another round of reverse transcription . This ad6 reads per cell with the possibility to increase the sequencing depth (15–25 × 106 reads per cell) if interested in RNA alternative splicing. Differently, by sequencing 3′ end libraries researchers can use a lower depth sequencing (104–105 reads per cell) [Final steps of single-cell RNA (scRNA) analysis are the sequencing and bioinformatic analyses. Alternatives to RNA sequencing are qRT-PCR and microarrays. The former is laborious and has a limited number of targets, while microarrays demand previously established transcriptomic probes and might have some background noise interfering on low abundant transcripts ,139. Thuer cell) . It is ier cell) ,146. As lncRNAs keep getting increased attention, it has become clear how their localization, tissue, and cell specificity are key factors in their role as regulatory elements. Many studies have described the developmental and tissue-specific expression of these transcripts in different organisms  ,148,149.XIST and Sry) having sexually distinct patterns of expression in the early embryo. Female samples, that have two X chromosomes, always result in XIST amplification but no Sry amplification, while male embryos, that have one X and one Y chromosome, always generated equal numbers of XIST and Sry amplicons since the Sry gene is on the Y chromosome. In 2007 they developed a new method to quantify different genes whose expression is not functionally guided by sex. Using the Linear-After-The-Exponential-PCR technique  they deLATE-PCR  . By combining RNA single-cell profiling and functional screenings Zhou and colleagues  showed tIn 2015 Kim and colleagues published a research where, using single-cell RNA sequencing with the SMART-seq method (Illumina), they described the dynamic expression of 437 lncRNAs during the reprogramming of fibroblasts to pluripotent cells. The authors showed that the activation of many lncRNA is an early molecular event in early stage iPSCs. Moreover, they showed that some lncRNAs are functionally relevant for reprogramming  Figure C. In addRather than using pluripotent cells, several studies have instead focused on specialized/differentiated cells. In 2015 Cabili and colleagues developed single-molecule RNA-FISH to quantify the subcellular localization of lncRNAs in three different cell types: hFFs, hLFs, and HeLa cells . They weLINC01314) was specifically expressed in astrocytes, the long intergenic non-coding 00152 (LINC00152) in endothelial cells, the long intergenic non-coding 00520 (LINC00520) in microglia, the Maternally Expressed 3 (MEG3) in neurons, the long intergenic non-coding 01105 (LINC01105) in oligodendrocytes, the Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) was highly expressed in astrocytes and endothelial cells while the Small Nucleolar RNA Host Gene 6 (SNHG6) was expressed in all cells analyzed [Even if the manually recovering of a single cell is laborious, it can be a good alternative to analyze cells with particular features and identify functionally lncRNAs. In 2017, RNAseq on manually selected neuronal cells was used by Spaethling and colleagues to analyze the expression of single cells purified from the human brain. The authors evidenced that among 935 identified lncRNAs, 113 were differentially expressed throughout the cell types and associated the expression of lncRNAs with marker-based cell types. For example, the long intergenic non-coding 01314 , fungi (1 for S. cerevisiae) and protists (2 for P. berghei). The Institute of Physical and Chemical Research in Japan (RIKEN) also developed a single-cell centric database [H. sapiens and M. musculus single cells. Broad institute developed its own database for single-cell datasets [After the development of different techniques to physically separate single cells, single-cell genomic and/or transcriptomic profiles have been accumulated. This huge amount of data is available, usually as raw data, in the Sequence Read Archive (SRA)  or in thdatabase  to enabldatabase . The RIKdatasets  containiS. scrofa can use Ensembl or Pig lncRNA net [As previously described, lncRNA classification is complex and defined according to their genomic position, function, and subcellular localization. Several databases integrated the knowledge about lncRNAs in different species. All these databases may be functional for researchers that approach this newly discussed RNA molecules. The most known databases for lncRNAs are Ensembl , Refseq cRNA net . CANTATAcRNA net  is a datUntil now we described databases including gene loci and expression data about lncRNAs, but as previously discussed, to understand the function of a lncRNA it is important to know its subcellular localization. The database LncATLAS  displaysNon-coding RNAs appear to be the preponderant part of transcribed genomes in eukaryotes with a regulatory capacity in different physiopathological processes that were described in several manuscripts  of their ambient (extracellular matrix) ,222,223.


Gastric cancer is the third most common cause of death from cancer in the world and it remains difficult to cure in Western countries, primarily because most patients present with advanced disease. Currently, CEA, CA50 and CA72-4 are commonly used as tumor markers for gastric cancer by immunoassays. However, the drawback and conundrum of immunoassay are the unceasing problem in standardization of quality of antibodies and time/effort for the intensive production. Therefore, there is an urgent need for the development of a standardized assay to detect gastric cancer at the early stage. Aptamers are DNA or RNA oligonucleotides with structural domain which recognize ligands such as proteins with superior affinity and specificity when compared to antibodies. In this study, SELEX  technique was adopted to screen a random 30mer RNA library for aptamers targeting CEA, CA50 and CA72-4 respectively. Combined with high-throughput sequencing, we identified 6 aptamers which specifically target for these three biomarkers of gastrointestinal cancer. Intriguingly, the predicted secondary structures of RNA aptamers from each antigen showed significant structural similarity, suggesting the structural recognition between the aptamers and the antigens. Moreover, we determined the dissociation constants of all the aptamers to their corresponding antigens by fluorescence spectroscopy, which further demonstrated high affinities between the aptamers and the antigens. In addition, immunostaining of gastric adenocarcinoma cell line AGS using CEA Aptamer probe showed positive fluorescent signal which proves the potential of the aptamer as a detection tool for gastric cancer. Furthermore, substantially decreased cell viability and growth were observed when human colorectal cell line LS-174T was transfected with each individual aptamers. Taking together, these novel RNA aptamers targeting gastrointestinal cancer biomarker CEA, CA50 and CA72-4 will aid further development and standardization of clinical diagnostic method with better sensitivity and specificity, and potentially future therapeutics development of gastric cancer. According to the statistics from World Health Organization 2015, Gastric cancer is the third most common cause of cancer-related death in the world . Cancer Currently, enzyme immunoassay (EIA) and radio-immunoassay (RIA) are commonly employed for the detection of tumor markers . Althougin vitro selection' technique allows a simultaneous screening of individual nucleic acid molecules up to 1015 per selection for a particular target [Aptamers which are either DNA or RNA oligonucleotides, are capable of binding different targets with high affinity and selectivity. The method for screening specific aptamers is known as 'SELEX' , which was first developed in 1990. This 'r target , 7. Althr target , 9 such Over the last decade, as the rapid progress of SELEX, developing aptamers for commercial and clinical interesting targets is becoming more and more efficient , 12. SELAn increasing number of DNA/RNA aptamers targeting gastrointestinal cancer biomarkers and cancer cells have been discovered in recent years. RNA aptamer YJ-1 binds specifically to CEA-positive cells and inhibited homotypic aggregation, migration and invasion by CEA-positive cells in mice . MoreoveIn this study, we set out to screen the library of aptamer by SELEX for three biomarkers of gastrointestinal cancer, CEA, CA50 and CA72-4, respectively. We identified 6 novel RNA aptamers (two aptamers for each biomarker) using high-throughput sequencing technology and then measured their affinity by fluorescence spectroscopy. Their dissociation constants ranged from 16.5 to 156nM which implicates high affinities between the aptamers and the antigens. Intriguingly, the predicted secondary structures of RNA aptamers from each antigen showed significant structural similarity and immunostaining using CEA aptamers demonstrated positive fluorescent signal on gastric carcinomas AGS cells, suggesting the structural recognition between the aptamers and the antigens. Moreover, we found that the cell viability and growth rate of human colorectal cell line LS-174T were substantially decreased after transfected with the aptamers which may demonstrate a functional role in cancer development. These results suggest the potential clinical applications of these aptamers as a diagnostic or therapeutic tool for gastric cancer in the future since the nature of aptamers can overcome the limitations of immunoassays with better sensitivity and specificity.BamH I and a Hind III restriction site flanking as the boundary, and a barcode sequence at the 3’-end for discrimination in sequencing. Gastrointestinal cancer biomarkers  were respectively purchased: CEA (Abcam), CA50 (BIO-RAD), and CA72-4 (BIO-RAD).An initial DNA library that had a random 30-nucleotide sequence (N30) in between as the viable region, were deliberately designed and ordered (Life Technologies) as the template . The len(5’-ATCGTTACCTTAGAAGCTT-N30-GGATCCGACACGACCCTATAGTGAGTCGTATTA-3’) was first transcribed into a starting RNA pool by pairing the T7 Initial-F primer (5’-AATACGACTCACTATAGGGTC-3’) and using in vitro transcription with T7 MEGAscript T7 Kit (Ambion) at 37°C for 6 hours. RNA product was subsequently purified by flowing through a Centri-Spin 20 column (PRINCETON). The starting RNA pool (~1014 sequences) was then incubated with a cancer antigen (~1 μg) in RSB-100 buffer  at room temperature for 30 min. Filter binding assay was performed to retain RNA-bound proteins on a PVDF membrane . Bound RNAs were released after the antigen was digested by proteinase K (Promega), and recovered through acid:phenol chloroform extraction and isopropanol precipitation. Recovered RNAs were subsequently amplified by RT-PCR using Superscript III First-Strand Synthesis System (Life Technologies) and iProof DNA polymerase (BIO-RAD) for 16–20 PCR cycles, followed by ethanol precipitation and in vitro transcription to transcribe dsDNA library to RNA library again to complete a SELEX cycle. Generally, the enrichment process was iteratively conducted for 10–15 cycles for selection of aptamers with elevated stringency through lowering antigen concentration, shortening incubation time, and increasing salt concentration of wash buffer. The final round of SELEX was stopped after RT-PCR. The corresponding purified DNA product was subsequently subjected to TA cloning with pGEM-T easy vector (Promega) and SANGER sequencing (BGI), as well as high-throughput sequencing (HTS) by ion Torrent sequencing platform (Life Technologies) when the RNA pool was enriched to a certain level. The quality and quantity of the same pool of DNA for ion Torrent was first verified by Agilent 2100 Bioanalyzer (Agilent Technologies). Such DNA pool was then applied to construct an amplicon library with the Ion Plus Fragment Library Kit (Ambion) according to manufacturer’s instructions, followed by sequencing on Ion 314 Chips (Life Technologies). Through a systematic bioinformatics analysis on the reads by Cygwin (Redhat), sequences were extracted and listed. Top ranking aptamers for each cancer biomarker were chosen for following biochemical characterization.SELEX, workflow shown in in vitro transcription using T7 MEGAscript Transcription Kit (Ambion), and purified on a 6% denaturing urea/polyacrylamide gel. The corresponding bands were visualized under UV, excised and chopped. RNA aptamers were eluted with 400 μl RNA elution buffer  at 42°C overnight. Purified RNA aptamers were recovered through acid:phenol chloroform extraction, isopropanol precipitation, and dissolving in appropriate volume of RNase-free distilled water.To characterize top ranking aptamers, specific full length oligos, with replacement of N30 in the initial DNA library by identified sequences, were chemically synthesized for further study. Similarly, RNA aptamers were first generated through d values were calculated using SigmaPlot v11.0 software (Systat). The error associated with the Kd values was calculated by the experiments in biological triplicate and each measurement was done in technical triplicate.RNA aptamers were fluorescence labeled with Ulysis Alexa Fluor 488 nucleic acid labeling Kit (Life Technologies) following manufacturer’s instructions. Briefly, 1 μg ethanol precipitated gel purified RNA aptamers were mixed with 1 μl ULS reagent in DMSO and added up to 25 μl with labeling buffer. The mix was incubated at 90°C for 10 min, followed by stopping the reaction on ice. A Centri-Spin 20 column (PRINCETON) was applied to purify fluorescence labeled RNA. Next, samples for RNA-antigen interaction were prepared by incubating 1 pmol fluorescence labeled RNA and titrated with various concentrations of antigen  in RSB-100 buffer in the presence of non-specific competitor yeast tRNA (1ug/ul) at room temperature for 10 min respectively. Excitation fluorescence values at 516 nm were measured using Fluormax-4 Spectrofluorometer (Horiba), and fluorescence changes were obtained by subtracting the value in the absence of antigen . Binding2 environment. For passage, cells were detached by trypsin, spun down, and resuspended in fresh DMEM with FBS and PS, followed by a 1/10 subculture in flask. AGS and Hela cells were grown and maintained in RPMI-1640 medium 10% FBS and 1% penicillin/streptomycin (PS), incubating at 37°C in a 5% CO2 environment.The human colon adenocarcinoma cell line LS-174T, gastric adenocarcinoma cell line AGS and uterine cervical cancer cell line Hela used in this study were purchased from American type Culture Collection (ATCC). LS-174T cells were grown and maintained in culture flask with Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS), incubating at 37°C in a 5% CO5 cells were seeded in a 6-well plate to reach 50% confluence overnight, and transfected with 1 μg gel purified selected RNA aptamer in 10 μl Lipofectamine 2000 (Life Technologies) following the manufacturer’s instructions. Antibiotic-free DMEM for transfection was replaced by 2 ml DMEM with FBS and PS after 5-hour incubation. The 48-hour post-transfected cells were detached by trypsin for following assays.Cell density of LS-174T was determined by trypan blue (Life Technologies) staining. Around 4×10Cell viability was determined by trypan blue exclusion. Briefly, 10 μl detached LS-174T cells were stained with 10 μl 0.4% trypan blue solution, and 10 μl mix was then loaded onto a hemocytometer. Numbers of viable cells (transparent) and dead cells (blue) were counted respectively under a light microscope at low magnification. Cell viability was calculated as the number of viable cells divided by the total number of cells within each 4×4 grid on the hemocytometer.570 nm on Day N divided by A570 nm of Day 0. The error was calculated by the experiments in biological triplicate and each measurement was done in technical triplicate.Cell growth rate was determined by MTT tetrazolium reduction assay. LS-174T cells treated with various RNA aptamers were seeded as 3000 cells/well in 96-well plates respectively. After 24, 48, 72 and 96-hour  incubation, the plates were taken out one by one, and assayed with 10 μl MTT reagent mixture (5 mg/ml in PBS) incubating at 37°C for 4 hours. Absorbance at 570 nm was then measured by a Powerwave XS microplate reader (BioTek). Relative cell viability was calculated as A2Cl 5mM, glucose 4.5g/L, Salmon Sperm DNA 0.1 mg/ml and yeast tRNA 0.1 mg/ml) for 30 min, followed by incubation of 1:100 RNA aptamer targeting CEA or 1:100 anti CEA, anti CA-50 and TAG72 antibodies (Abcam) in binding buffer for 1 hr. For secondarty antibody incubation, cells were washed with PBS for three times and incubated with 1:1000 rabbit anti-mouse/FITC antibody (Abcam) for 30 min. The cells were then washed three time with buffer, mounted, sealed and observed by fluorescence microscopy. All images were captured under a fluorescent microscopy with 400X magnification.AGS were grown on 10mm coverslip, harvested by washing with PBS for three times and fixed with cold methanol for 10 min. After washing three times with PBS, cells were incubated with binding buffer , Mgin vivo transcription was set as approximately 1014, which means 1014 different RNA molecules in the starting RNA pool. This number of RNA copies which is equivalent to 2–5 μg covers the majority of the possible combinations of N30. In order to eliminate the non-specificity of the RNA to the PVDF membrane, a pre-clearing step was performed by immersing the membrane into the initially transcribed RNA pools for 15 min. The recovered RNA pool was then incubated with a cancer antigen to initiate SELEX.A random library of N equal to 30 was used to screen RNA aptamers. The initial number of DNA template for Enrichment of RNA aptamers specific to cancer antigens was monitored by the binding ratio calculated before and after the binding experiment (amount of recovered RNA versus amount of input RNA). Through multiple rounds of SELEX, binding ratios of 18.1% for CEA at Round 14, 15.6% for CA50 at Round 12, and 24.6% for CA72-4 at Round 15 were recorded, indicating that specific RNA aptamers for cancer antigens were accumulated to a certain level, respectively.In order to assess the sequences of enriched RNA aptamers after selection, the purified aptamers were reverse transcribed, amplified, subjected to overhang addition at 3' end and subcloned into the TA vector. For each antigen, 15–20 single colonies were randomly picked for sequencing. As shown in Since SANGER sequencing could only provide limited data, we adopted the high-throughput sequencing (HTS) to reveal the entire population of enriched RNA aptamers for the three antigens. To do this, RNA aptamers of CEA at Round 15, CA50 at Round 12, and CA72-4 at Round 15 were subjected to HTS using Ion Torrent system. The libraries were constructed and sequenced for each antigens according to the manufacturer protocols. The sequences were sorted and ranked based on their number of reads as shown in mfold RNA Folding program [in vitro transcribed, purified and labeled with fluorescence probe Alexa 488. The fluorescence labeled RNA aptamers were titrated with increasing amount of cancer antigens and the enhancement of fluorescence intensity was used to plot the binding isotherms. The curves were then fitted with the nonlinear regression—one site saturation ligand binding equation to obtain dissociation constant Kd as shown in d values of CEA A01, A02 were 16.5 and 156 nM respectively; Kd values of CA50 A01, A02 was 38.0 and 30.7 nM respectively; Kd values for CA72 A01, A02 were 52.7 and 71.2 nM respectively  and Hela (antigen negative/control) were performed. AGS cells incubated with CEA aptamers showed positive fluorescent signal similarly to CEA, CA-50 and TAG72 antibodies whereas Hela cells demonstrated no signal  for RNA SELEX. HTS techniques were developed and mature in recent years, which enabled researchers to screen efficiently and effectively, and profile nucleotide sequences within days. The compatibility of HTS in DNA SELEX was confirmed by several previous studies , 35, butThe advantage of employing the human colorectal carcinoma cell line LS-174T as a model to study the potential role of RNA aptamers is that this cell line carries all three biomarkers of CEA , CA50 33 and CA7Furthermore, the potential applications of fluorescently-labelled aptamers are being widely studied and proven to be applicable on cancer therapy and diagnosis, such as intraoperative surgery and cancer-type specific in vivo non-invasive imaging . In partTaken together, this study demonstrates that SELEX incorporated with HTS is a promising and powerful tool to screen aptamers for various targets. To obtain qualified aptamers with high specificity against cancer antigens, SELEX refinement and post-SELEX development of aptamers are yet indispensable. In order to enhance the sensitivity and specificity of aptamers for future diagnostic and clinical applications, several modifications could be considered: to truncate ribonucleic acids in block to simplify the aptamer and specify the core region for RNA-protein interactions; to individually substitute / delete / insert a particular ribonucleic acid fragment in the core region of aptamer to improve its specificity; to chemically modify particular ribonucleic acid(s) of aptamer with locked nucleic acid (LNA) to decrease its susceptibility to nucleases and increase its stability for long-term storage. Indeed, it is possible that such aptamers would become applicable in clinical applications or even therapeutics in the near future with better sensitivity and specificity, and without the limitations of immunoassays.


Over the past decade, transcriptome‐wide studies combined with advanced biochemistry and the discovery of methylation writers, readers, and erasers revealed roles for mRNA methylation in the regulation of nearly every aspect of the mRNA life cycle and in diverse cellular, developmental, and disease processes. Although large parts of mRNA function are linked to its cytoplasmic stability and regulation of its translation, a number of studies have begun to provide evidence for methylation‐regulated nuclear processes. In this review, we summarize the recent advances in RNA methylation research and highlight how these new findings have contributed to our understanding of methylation‐dependent RNA processing in the nucleus.Eukaryotic RNA can carry more than 100 different types of chemical modifications. Early studies have been focused on modifications of highly abundant RNA, such as ribosomal RNA and transfer RNA, but recent technical advances have made it possible to also study messenger RNA (mRNA). Subsequently, mRNA modifications, namely methylation, have emerged as key players in eukaryotic gene expression regulation. The most abundant and widely studied internal mRNA modification is NRNA Processing > RNA Editing and ModificationRNA Processing > Splicing Regulation/Alternative SplicingRNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional ImplicationsThis article is categorized under: The 5′ capping and 3′ polyadenylation modifications were discovered almost 50 years ago and have remained as one of the hallmarks of eukaryotic mRNA processing  cap at the 5′ end. It also unexpectedly detected internal methylated nucleotides  was the most abundant modification . In other cases, such as N1‐methyladenosine (m1A) or m6A, the detection methods either rely on interference with reverse transcriptase or utilization of specific antibodies  constitute yet another layer of gene expression regulation. In diverse eukaryotic lineages, dynamic RNA methylation plays a key role in such elementary processes as germline maturation  differentiation into specific cell types  in (KO) mouse ESCs is viable, but they failed to differentiate into specific lineages. Mettl3 KO in mouse is embryonically lethal  was required for mouse spermatogenesis  and YTH Domain Containing 2 (YTHDC2) were needed for early zygote development in mammals and zebrafish and successful meiotic program in the mammalian germline, respectively   found next to the cap will be discussed later in this review. Most modifications found in mRNA, such as m1A, N3‐methylcytosine (m3C), N5‐methylcytosine (m5C), and m6A, were first observed in noncoding RNA (ncRNA), mainly in the abundant and stable transfer RNA (tRNA) and ribosomal RNA (rRNA).m1A and m6A, were identified by the 1970s; however, more detailed molecular and biochemical studies began only recently. Both modifications are reversible, meaning that they can be added and subsequently erased from mRNA by specific enzymes.Both adenosine modifications, m1A was first identified on rRNA and tRNA, where it is important for the formation of tertiary structure conformations  and around start codons of several hundreds to several thousands of different mRNA transcripts  and linked m1A to translational repression and developmental regulation. Since this field is very young, it needs further biochemical and chemical validations to accompany the high‐throughput sequencing studies. For instance, it is important to test the relevance of 5′ terminal m1As as well as to study the molecular mechanism that underlies the role of m1A at structured elements. Notably, m1A has been detected on MALAT1‐associated small cytoplasmic RNA (mascRNA), which is a small tRNA‐like RNA processed from MALAT1 long noncoding RNA (lncRNA) by an unusual mechanism that involves RNase P and RNase Z  were recently also reported in mRNA , 5‐azacytidine‐mediated RIP (Aza‐IP), and methylation at individual nucleotide resolution crosslinking and immunoprecipitation (miCLIP)  + RNA, with even higher m5C levels in nuclear fractions   is a multistep process that generates two intermediates, N6‐hydroxymethyladenosine (hm6A) and N6‐formyladenosine (f6A), with expected lifetimes of approximately 3 hr  , a technique that provided the first data on m6A distribution within (pre‐)mRNA molecules , around the stop codons, and at the beginning of the last exons  ‐dependent methyltransferases. Mammals express at least two enzymes that have the potential to introduce m6A in mRNA, the heterodimer METTL3/METTL14 (METTL3/14) and METTL16. METTL3/14  revealed that METTL3 and METTL14 preferentially occupy regions that contain the RRACH motif , m6A is a reversible mark. It can be readily removed in mammals by two currently known demethylases, FTO and ALKBH5  in single‐stranded DNA (ssDNA)  led to the identification of several m6A RNA binders  revealed decreased folding of methylated RNA sequences comparing to unmethylated; the finding underscored the role of m6A in regulating mRNA folding , nucleoplasmic, and cytoplasmic mRNA did not reveal any significant differences; this finding implied that final m6A decoration is completed while the mRNA is still associated with the transcription locus. All the other m6A players, METTL16, FTO, and ALKBH5 target pre‐mRNA  of RNAPII  to the first 5′ terminal nucleotide of the mRNA  to create 2′‐O‐methylation (Nm). If just the first nucleotide after m7G is methylated, it forms the so‐called cap 1 structure (m7GpppNm). In mammals, this methylation is performed by the enzyme CMTR1 . These two additional methylations serve as a mark to distinguish self and nonself mRNA and to avoid the recognition of cellular mRNA by the innate immune system , and they promote mRNA translation under certain stress conditions  . It would be interesting to determine whether additional methyl groups are present on the cap and how the cellular machinery interprets this complex modification at the beginning of mRNA transcripts.In summary, the 5′ cap structure of mammalian mRNA can contain up to four methyl groups: the stable m4.26A has been thought to play a critical role in constitutive splicing  changes  and promote or repress recruitment of trans‐acting splicing factors. This mechanism occurred for YTHDC1, which bound m6A‐modified regions in the vicinity of the 5′ and 3′ SS and recruited the splicing factor SRSF3 while inhibiting the binding of SRSF10  were required to mediate gene silencing of the X chromosome, although it is unknown how YTHDC1 binding contributes to gene silencing , regulated the female‐specific AS of sxl, a key regulator of dosage compensation , FTO‐mediated demethylation was proposed to affect splicing of a group of exons that rely on the splicing factor SRSF2 ), endonucleolytic cleavage, and polyadenylation. It is mediated by a large multisubunit complex. Cleavage position is dictated by several sequence elements in the 3′ UTR. The best characterized example is the so‐called poly(A) site (PA). In humans, it is estimated that approximately 50% of protein coding genes contain more than one PA, so‐called alternative polyadenylation (APA) sites  probably due to their resistance to DCP2‐mediated decapping , they observed that m6A levels positively correlated with 3′ UTR length, in accordance with the findings by Ke et al. that m6A accumulated in long last exons  site selection  in HeLa cells resulted in delayed nuclear export of mature mRNA  and development of new approaches that allow direct, site‐specific detection of methyl marks without the use of indirect tools . Most importantly, further elaboration of robust and available bioinformatics analyses tools should be a key step in comparative analyses of data obtained from various groups. Currently, attempts are being made to interconnect the published data in databases  pattern. m6A and its writers have been located to RRACH motifs, but only a very small portion of such motifs at specific mRNA locations are methylated. This phenomenon could be regulated by adaptor factors, including RNA binding proteins, METTL3/14 auxiliary factors, or even other RNAs   deposit(s) methyl groups on purine rings in the 5′ terminal m6Am. Based on a recent report from Meyer et al., FTO prefers m6Am rather than m6A (Mauer et al., 6A.The expanding interest in the field has led to the identification and characterization of the key factors of the mTaken together, a number of studies have increased significantly our awareness about the importance of methyl marks on coding RNA and have given rise to the new field of mRNA epitranscriptome. The field is still in its infancy, and there are exciting opportunities for new fundamental discoveries.The authors have declared no conflicts of interest for this article.https://doi.org/10.1002/wrna.1166https://doi.org/10.1002/wrna.79https://doi.org/10.1002/wrna.1380https://doi.org/10.1002/wrna.1450