Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems , RNA-dependent DNA cleavage systems , and RNA-dependent RNA cleavage systems . All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented. Genome editing is a conventional method that is often applied to alter the genotype and phenotype of organisms. In general, two methods are typically adopted for gene function analysis. One of these is the traditional forward genetics method  which enables the identification of new functional genes via T-DNA tag or map-based cloning. This method involves cultivation and screening for special mutants with key traits  which either promote random mutations via error-prone non-homologous end-joining  has been used to provide a rapid, low-cost, and high-throughput technology for the analysis of gene function , type V-A protein, and CRISPR-C2c1 (class 2 candidate 1), a type V-B protein, have also provided active RNA-guided DNA cleavage systems  has also been developed that is able to break down the limitation of selected target sequences, the secondary structure of single guided RNA, and the conformation of target DNA. Building on these developments, the aim of this paper is to emphasize an non-comprehensive review on different GETs that have been developed in recent years as well as their incomplete applications to plants.Very recently, a novel but controversial GET of GETs could be divided into two groups. The members of one group are mediated by protein-guided nuclease, such as ZFN and TALEN, while members of the other are mediated by special RNA/DNA-guided nuclease, including RNA-dependent DNA cleavage systems like CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1, and RNA-dependent RNA cleavage systems like RNAi and CRISPR-C2c2.Drosophila melanogaster to the cleavage domain of the FokI restriction endonuclease of a Flavobacterium okeanokoites , was discovered in the plant pathogenic bacteria Xanthomonas Hrp-type III secretion (T3S) system  motifs did result. In addition, research has shown that the cytotoxicity of targeted deaminase ZF-AID is less than is the case for ZFN  that can bind directly to the NmeCas9/sgRNA complex and prevent in vitro DNA cleavage. Pawluk et al.  three nts adjacent to the PAM, while its counterpart, the RuvC-like domain, cleaves strands of non-cDNA in a three-to-eight nt region adjacent to the PAM  that can only induce SSB when RuvC D10A or HNH H840A mutation is introduced Figure . The numOptimized CRISPR ‘base editor’-targeted deaminase technology-has also recently been developed, which enables efficient point mutations rather than just random insertions or deletions (indels). This approach also has the advantage that it does not generate DSBs by fusing targeted deaminase with dCas9 guided by sgRNA 6)2+ ion may play an important role in stabilizing the conformation of crRNA and further enhancing Cpf1 recognition  xxxH motifs, as is typical of the higher eukaryote and prokaryote nucleotide-binding domain  loss-of-function mutations were obtained in T2 bread wheat using RNAi  left/right homologous arm into a targeted region of this gene via CRISPR-Cas9-meditated replacement; they found that the frequency of 0.8% stably transformed T0 transgenic plants carried eGFP signal and expressed actively in both the leaves and roots of T1 generations (Zhao et al., N. benthamiana protoplasts (Li et al., Weinthal et al.  co-delivIn sum, GETs have great potential for the future analysis of gene function, including for the targeted therapy of human diseases and crop breeding. Stroud et al. , for exaKZ carried out the analysis and wrote the manuscript. NR and BZ made helpful suggestions to the manuscript. ML designed, led and coordinated the overall study.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.


Marchantia polymorpha, editing may have been lost during evolution. Most RNA editing events can restore the evolutionarily conserved amino acid residues in mRNAs or create translation start and stop codons. Therefore, RNA editing is an essential process to maintain genetic information at the RNA level. Individual RNA editing sites are recognized by plant-specific pentatricopeptide repeat (PPR) proteins that are encoded in the nuclear genome. These PPR proteins are characterized by repeat elements that bind specifically to RNA sequences upstream of target editing sites. In flowering plants, non-PPR proteins also participate in multiple RNA editing events as auxiliary factors. C-to-U editing can be explained by cytidine deamination. The proteins discovered to date are important factors for RNA editing but a bona fide RNA editing enzyme has yet to be identified.RNA editing by cytidine (C) to uridine (U) conversions is widespread in plant mitochondria and chloroplasts. In some plant taxa, “reverse” U-to-C editing also occurs. However, to date, no instance of RNA editing has yet been reported in green algae and the complex thalloid liverworts. RNA editing may have evolved in early land plants 450 million years ago. However, in some plant species, including the liverwort,  Trypanosoma brucei where uridines were inserted at specific sites in the mitochondrial (kinetoplast) cytochrome c oxidase II (coxII) transcript to restore the proper protein-coding sequence [coxIII mRNA [Physarum polycephalum are heavily edited by the insertion of mononucleotides and dinucleotides at specific sites [RNA editing is a posttranscriptional modification to nuclear, mitochondrial or chloroplast genome-encoded transcripts, and occurs in a wide range of organisms. It was discovered in 1986 in III mRNA . This prIII mRNA . Similaric sites . In addiic sites  and adentrans-acting factors involved in C-to-U RNA editing in chloroplasts and plant mitochondria.In the plant kingdom, RNA editing was first identified as a C-to-U exchange in mitochondrial transcripts in 1989 ,8,9, folAmborella trichopoda. In seed plants, all these editing events are of C-to-U type. Most of the sites in translated regions are efficiently edited, with 90%–100% efficiency, in green leaves. On the other hand, the efficiency of C-to-U editing events that create a translation initiation codon (by an ACG to AUG change) has been surprisingly low. For instance, the editing efficiency at the ndhD-1 site in the Arabidopsis chloroplast ndhD transcript is 45% [Physcomitrella patens chloroplast rps14 mRNA is 70% in filamentous protonemata, which reduces further to only 20% in leafy tissues [RNA editing sites in translated regions can be predicted by a comparison of amino acid sequences deduced from genomic DNA sequences from various plant species. Subsequently, RNA editing can be verified by cDNA sequence analysis. A number of editing sites identified in various land plant mitochondria and chloroplasts ,41,42,43t is 45%  and that tissues . This su tissues .Nicotiana tabacum mitochondria, five sites are in tRNAs and 73 in non-coding regions [Marchantia polymorpha to 3415 in the spike moss Selaginella uncinata [Recent high-throughput RNA-seq analyses have revealed minor RNA editing events in untranslated regions and intron sequences as well as in protein-coding regions. For instance, in addition to the 34 already known editing sites in Arabidopsis chloroplasts , nine no regions . Across uncinata   and 340 sites in Huperzia lucidula chloroplasts [Selaginella, one of the early vascular plant lycopods, seem to be most commonly edited.Out of 3415 sites identified in 74 chondria  and PREPchondria . Some 18P. patens, where there are only two identified C-to-U editing sites in chloroplasts [Anthoceros and Phaeoceros laevis undergo substantial RNA editing [Chara vulgaris (stonewort), suggesting that the process of RNA editing may have evolved only after the plants established themselves on the land.In case of the bryophyte (early non-vascular land plants) roplasts , there aroplasts ,43. On t editing ,48. Howecox3, evening primrose cob and cox2 and pea cox2 transcripts . While ences in  with an roplasts  and mitoroplasts  as well elmannii . The cDNelmannii . For insy absent . It woulPhe [His [I. engelmanni mitochondria, ten tRNAs are edited to improve base pairing in stem regions [RNA editing in plant organelles mostly affects mRNAs, thus providing the means to correct genetic information for proper protein function. In addition, editing affects some tRNAs and rRNAs encoded in the organellar genomes ,50,51,52 regions . Thus, eOenothera mitochondrial 26S rRNA 25 years ago [S. moellendorffii mitochondria [Exemplifying the rarity of editing in rRNAs in seed plants, there was no such report for a long time after an initial reporting of two potential sites in ears ago . Recentlchondria . Three ochondria .nad1 group II intron 3 from Oenothera mitochondria is modified by C-to-U editing to generate the typical domain VI secondary structure. Self-splicing in vitro is observed only in the edited (A:U basepair) form, indicating that this editing event is a prerequisite for splicing [S. uncinata chloroplasts, a number of intron editing events have been identified, which could possibly improve the RNA secondary structure of group II introns, including the highly conserved domains V of the intron 3′ termini [Like some instances in tRNAs, perhaps the editing sites within group II introns are also of functional importance because editing can improve the base pairing required for splicing. Domain VI of splicing . In the  termini . Such edndhA mRNA is edited at two sites, one of which is located only 12 nucleotides downstream of the 3′ intron-exon splice site. To assess if RNA editing occured after or before splicing, short “spliced” and “unspliced” ndhA gene fragments were introduced and transcribed within tobacco chloroplasts. The subsequent cDNA analysis showed that only spliced ndhA mRNAs were edited [P. patens mitochondrial atp9 gene [atp9 gene is interrupted by three introns and an editing site lies within the third exon (only 8 nt long). This site is completely edited in fully spliced mRNA, while it remains unedited in the unspliced mRNA [Leu gene contains a group I intron between the first and second position of the UAA anticodon. In the moss Takakia lepidozioides, the CAA anticodon of tRNALeu is edited to create a canonical UAA codon [Leu is completely edited while unspliced tRNAs are partially edited. This suggests that the anticodon editing of tRNALeu may occur before RNA splicing.RNA editing in exons close to splice sites may also affect intron splicing or vice versa. For instance, the spinach chloroplast e edited . A similtp9 gene . This atced mRNA . These oAA codon . The splcis-acting elements adjacent to editing sites and discovering putative site-specific proteins that interact with these elements. In all such instances, the cis-elements comprise stretches of 20 to 25 nucleotides upstream of the editing sites. The identification of the first trans-acting factor, however, was not the outcome of any genetic and biochemical study for editing factors, but instead it resulted from a study on photosynthetic mutants in 2005 [chlororespiratory reduction 4 (crr4), showed defects in the accumulation of the plastidic NADH dehydrogenase (NDH) complex, which is a multi-subunit complex in the thylakoid membrane. The loss of NDH complex was correlated directly to the loss of a C-to-U editing event that otherwise creates the start codon AUG in ndhD mRNA. It was later found that the CRR4, a member of the pentatricopeptide repeat (PPR) protein family, binds to a 36 nucleotides (−25 to +10) region surrounding its target editing site [bona fide trans-acting factor essential for recognizing this RNA editing target site. Following this discovery, several other PPR proteins were identified as site recognition factors affecting editing in chloroplasts and mitochondria [cis-element sequences [To elucidate the molecular mechanism of RNA editing in plant organelles, in vitro, in vivo, and in organello studies have extensively been performed using flowering plants such as wheat, tobacco, pea, and cauliflower ,59,60,61 in 2005 . One of ing site . This suchondria ,65  motifs of 35 amino acids, while the PLS-class consists of canonical P motifs and their variants L  and S , which differ in sequence length and conservation ,70. At tRotifera ,73,74. T. patens ,16. All cis-element for editing. PPR crystal structure analyses have shown that a PPR in N-to-C terminus orientation interacts with RNA in the 5′ to 3′ orientation for the target RNA and confirms the RNA-binding code [cis-elements for editing in a one-PPR motif to one-nucleotide manner.Recently, a code for PPR-RNA recognition has been elucidated ,76,77. Ting code ,79,80,81Plant-specific E domains, which contain two PPR-like motifs, have been shown to be essential for editing ,82. OkudLike E-type PPR proteins, in most cases a single PPR-DYW editing protein is involved in RNA editing at target sites a. The DYPhyscomitrella, the DYW1 protein in Arabidopsis has been identified as an RNA editing factor acting specifically on the chloroplast ndhD-1 site [ndhD mRNA. Both proteins have been shown to be required for this editing event, suggesting that the DYW domain is essential but could be provided in trans if not present in cis on the PPR editing factor . For instance, RARE1 and VAC1 , both of which are DYW-type PPR proteins, are identified as editing PPR proteins targeting the same roplasts ,89. Muta editing  while th editing . VAC1 isith CRR4 c.P. patens) genome encodes 10 DYW-type PPR proteins but no E-type PPR protein. In this moss, the 13 C-to-U editing events are coordinated by nine DYW-type PPR proteins [Similarly, studies on moss PPR editing factors also support the importance of the DYW domain in RNA editing. The moss . Various transgenes for wild type PpPPR_56 (56comp), and mutant variants with the HxE(x)nCxxC motif changed to alanine, AxA(x)nCxxC (56M1) and HxE(x)nAxxA (56M2), respectively, were introduced into the PpPPR_56 knockout moss (∆56-22). In the 56comp moss line, editing of nad3-C230 and nad4-C272 sites was restored to wild type levels. Whereas, 56M1 and 56M2 constructs failed to complement the mutant editing phenotype, thereby suggesting that the zinc-binding motif of the DYW domain could also play an important role in the process of editing in the moss system as well.Recently, DYW domains have been shown to bind zinc ions and have been implicated in RNA editing in bidopsis ,94. Morebidopsis . This suArabidopsis. Seven of these target sites in mitochondria, two (MORF2 and 9) in chloroplasts and one (MORF8) acts on its targets in both the organelles. In contrast to PPR editing factors, mutants of either MORF2 or MORF9 gene are affected at almost all RNA editing sites in Arabidopsis chloroplasts [Besides RNA sequence-specific PPR editing factors, another group of proteins, known as multiple organellar RNA-editing factors (MORFs) have also been linked to RNA editing in flowering plants . MORFs aroplasts . This suroplasts . Similarroplasts ,98 and froplasts . These froplasts . Althougroplasts . In someArabidopsis chloroplasts [cp31a mutant [Arabidopsis and maize [Arabidopsis and nine sites in maize chloroplasts. ORRM2, ORRM3 and ORRM4, which have a single RRM domain and a glycine-rich domain, are likewise shown to be important for efficient editing at many mitochondrial sites [Other types of proteins involved in RNA editing belong to RNA recognition motif (RRM)-containing proteins: chloroplast ribonucleoproteins (cpRNPs) and organelle RRM proteins (ORRMs). CP31A, a member of the cpRNP family containing two RRMs, influences the efficiency of editing at 13 sites in roplasts . Howevera mutant . The ORRnd maize ,103,104.nd maize . The losArabidopsis [Arabidopsis of which three are predicted to be localized in chloroplasts while one is mitochondrial. The OZ1 was identified by co-immunoprecipitation with DYW-type PPR editing factor, RARE1 [OZ1 in Arabidopsis leads to an alteration in the level of editing of most sites in chloroplasts. OZ1 can interact with PPR editing factors, where it is assigned to the cognate sites, and ORRM1, but not with MORFs. This interaction supports the notion that OZ1 takes part in the editosome of chloroplasts.Two additional novel proteins, PPO1 (protoporphrinogen IX oxidase 1) and OZ (organelle zinc finger), have been characterized as general editing factors ,106. Notbidopsis . PPO1 inArabidopsis. PPR proteins and non-PPR editing factors are targeted to either chloroplasts or mitochondria, or both. This suggests that the basic machinery for a C-to-U editing event is perhaps conserved in both organelles. This editosome model has been drawn from molecular evidence found mostly in Arabidopsis. However, in the early land plants (mosses) and early vascular plants (lycophytes), the non-PPR editing factors described above are not encoded in their nuclear genomes. Unlike the complex editosome of seed plants, RNA editing may occur in a simpler editing complex, composed of a single PPR-DYW editing protein and a few other unidentified non-PPR editing factors, at least in mosses. The remaining central question is the nature of the RNA editing enzyme. Despite circumstantial evidence supporting the DYW editing enzyme [In plant organelles, PLS-type PPR proteins recognize and bind specifically to editing sites. The PPR–RNA complex is organized into the editosome with several additional non-PPR protein factors such as MORF and ORRM proteins. However, the order of addition/assembly of individual protein factors into the editosome has yet to be clarified. These non-PPR proteins might play a key role in editing as regulators of editing efficiency or as connectors of site-specific PPR proteins with other proteins or an unidentified editing enzyme. Presumably, approximately 200 PLS-type PPR proteins found in Arabidopsis might be involved in RNA editing. Henceforth, several of the editing PPR protein would have to recognize more than one editing sites to be able to recognize all 600 editing events in g enzyme ,86, a biA. phyllitidis, there are 53 C-to-U and 70 U-to-C editing events known in the 1 kb mitochondrial atp1 mRNA [cis-elements which are needed to be properly identified in their unedited, partially-edited or fully-edited states. Although C-to-U editing requires PLS type PPR proteins, it is unclear if the same were true also for the reverse (U-to-C) editing. However, the possibility of PLS-type PPR proteins being involved in reverse editing cannot also be ruled out either. About 6000 C-to-U (but no U-to-C) editing sites are present in some Selaginella species, suggesting that thousands of PPR editing factors could be involved in all editing events.In some plant species, the existence of both conventional C-to-U and reverse U-to-C editing events is highly evident, but the mechanism of target recognition and features of the editing factors involved are completely unknown. In the fern tp1 mRNA . Such a cis-elements for editing, an in vitro or in organello assay system must be developed from the plants in which reverse editing also occurs. However, so far it has been difficult to prepare purified organelles from these plants. To identify the editing factors, a forward and/or reverse genetic approach could be useful, as it has been in case of flowering plants. However, such approaches have yet to be applied. The development of breakthrough technologies for RNA editing studies in reverse-editing plant taxa needs to be established in the near future. As an alternative approach, candidate editing factors could possibly be identified from the enormous genomic data that have been accumulated for bryophytes, lycophytes, and ferns. Subsequently, a loss-of function of those candidates via genome editing might lead to the identification of novel editing factors.To determine the


Increasing studies have shown that mature spermatozoa contain many transcripts including mRNAs and miRNAs. However, the expression profile of long non-coding RNAs (lncRNAs) in mammalian sperm has not been systematically investigated. Here, we used highly purified RNA to investigate lncRNA expression profiles in mouse mature sperm by stranded-specific RNA-seq. We identified 20,907 known and 4,088 novel lncRNAs transcripts, and the existence of intact lncRNAs was confirmed by RT-PCR and fluorescence in situ hybridization on two representative lncRNAs. Compared to round spermatids, 1,794 upregulated and 165 downregulated lncRNAs and 4,435 upregulated and 3,920 downregulated mRNAs were identified in sperm. Based on the “Cis and Trans” RNA-RNA interaction principle, we found 14,259 targeted coding genes of differently expressed lncRNAs. In terms of Gene ontology (GO) analysis, differentially expressed lncRNAs targeted genes mainly related to nucleic acid metabolic, protein modification, chromatin and histone modification, heterocycle compound metabolic, sperm function, spermatogenesis and other processes. In contrast, differentially expressed transcripts of mRNAs were highly enriched for protein metabolic process and RNA metabolic, spermatogenesis, sperm motility, cell cycle, chromatin organization, heterocycle and aromatic compound metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the differentially expressed lncRNAs were involved in RNA transport, mRNA surveillance pathway, PI3K-Akt signaling pathway, AMPK signaling pathway, protein processing in endoplasmic reticulum. Metabolic pathways, mRNA surveillance pathway, AMPK signaling pathway, cell cycle, RNA transport splicesome and endocytosis incorporated with the differentially expressed mRNA. Furthermore, many lncRNAs were specifically expressed in testis/sperm, and 880 lncRNAs were conserved between human and mouse. In summary, this study provides a preliminary database valuable for identifying lncRNAs critical in the late stage of spermatogenesis or important for sperm function regulation, fertilization and early embryo development. Sperm are specialized cells generally considered transcriptionally and translationally inert. Early studies proposed that the lack of RNAs in sperm cells was a result of loss of most of the cytoplasm during processes in spermatogenesis. The RNAs detected in the paternal gamete were initially assumed to be either left behind after degradation and expulsion of the residual body from spermatogenesis or simply contaminants from other surrounding cells , 2. HoweLncRNAs, which are commonly defined as noncoding RNAs longer than 200 bp, are novel regulatory molecules that modulate a wide variety of functions and are involved in various pathophysiologic processes and human diseases. LncRNAs are highly enriched and specifically expressed in the testes and during spermatogenesis stages , 14, impThe aim of this study was to elucidate the lncRNA profile in mouse mature sperm with stranded-specific RNA-seq by Ilumina HiSeq 4000 technology. We also used bioinformatics methods and performed function predictions to screen lncRNAs candidates that are potentially involved in spermatogenesis and sperm function.The research involving animals was conducted with the approval of the Animal Research Ethics Committee of Medical School of Nanchang University.ad libitum) and water. Sixty mice (10–16 weeks old) were divided into four groups to provide four pools of sperm samples from 15 mice each. The testes harvested from above-mentioned mice were arranged into two biological replicates of testes samples (n = 2). Mice were sacrificed by cervical dislocation. Sperm were collected as previously described [2 incubator  at 37°C for 15 min. The samples were then filtered with a 500-mesh sieve to remove the tissue debris. The sperm were then treated with somatic cell lysis buffer  for 30 min on ice to eliminate somatic cell contamination. Non-sperm cell contamination (< 0.2%) was checked by microscopy. After centrifugation, the top layer was carefully removed and the sperm pellet was washed twice with 10 ml of somatic cell lysis buffer at 700 g for 5 min at 4°C. Finally, the purified sperm samples were used for RNA isolation.Eight-week-old healthy male C57BL/6J mice were purchased from Hunan SJA Laboratory Animal Co., Ltd. . All mice were maintained at a constant temperature (22 ± 2°C) and relative humidity (40–60%) with a 12 h light/dark schedule and free access to laboratory chow  according to the manufacturer’s instructions with some modifications. We added 1 ml of buffer RLT (a guanidine-thiocyanate lysis buffer supplemented with 10 mM DL-Dithiothreitol) to the sperm pellet and 10 mg testes tissues, and then homogenized the sample by passing the samples through a 20-G needle attached to a 1-ml syringe 10 to 15 times. After total RNA was bound to the membrane and other contaminants were efficiently washed away, on-column DNase digestion was performed to eliminate trace amounts DNA contamination. The concentration and integrity of the total RNA samples were evaluated using a NanoDrop ND-1000 spectrophotometer  and a 2100-Bioanalyzer with the RNA 6000 Nano Chip , respectively.Total RNAs were extracted from the purified sperm (n = 4) and testes (n = 2) samples using RNeasyTM 4000 with PE100 according to the manufacturer’s instructions developed by BGI . Briefly, the RNase H protocol was performed to remove rRNA as previously described [TM 4000.Strand-specific transcriptome sequencing was conducted using Illumina HiSeqescribed . The DNaThe raw data produced by HiSeqTM 4000 was subjected to quality control tests, including removing the adaptors as well as empty reads and then filtering the low-quality reads. The clean reads were aligned to reference sequences using the SOAPaligner/SOAP2 software. The distribution and coverage of reads aligned with the reference genome were counted. The gene expression level was calculated using FPKM method .FPKM = (fragments per kilobase transcriptome)/(million mapped reads)http://www.geneontology.org) allows functional association of differentially expressed lncRNA-targeted mRNAs and mRNAs using three structured networks of defined terms that describe gene-product attributes. GO terms with corrected p values of less than 0.05 were considered significantly enriched by differentially expressed genes.GO analysis (http://www.genome.jp/kegg/).Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, organism and ecosystem, from molecular level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies  of spermatozoal RNA. PCR products were subjected to fractionation on 1.5% agarose gels at 120 V, 30 min. After separation, the gels were stained with ethidium bromide and visualized using a UV transilluminator. Images were captured digitally.The RNA was reverse transcribed to cDNA using the PrimeScript RT reagent with gDNA Eraser Kit . RT-PCR was used to verify a subset of full-length transcripts  using the SYBR Premix DimerEraser Kit  according to the manufacturer’s instructions. The qPCR procedure was carried out with denaturation at 98°C for 30 s, followed by 40 cycles of denaturation at 98°C for 5 s, annealing at 60°C for 15 s, and extension at 72°C for 20 s. The expression levels of actin, We performed lncRNA FISH staining as previously described , 20. BriAAGACGAACGGCTCTCCGCACCGGACCCCGGTCCCGACGCCCGGCG GGGGGAGACCCCGCGCCGCCGCGGGGACGACGCGGGGACCCCGCCGAGCGGGGACGGACGGGGACCCGGCTATCCGGGGCCAACCGAGGCTCCTTCGGCGCTGCCGTATCGTTCCGCCTGGGCGGGATTCTGACTTAGAGGCGTTCAGTCATAATCCCACAGATGGTAGCTTCGCCCCATTGGCTCCTCAGCCAAGCACATACACCAAATGTLnc2: TTTCCCCTCGTTCTGCCAGACAACTACCTGGCTGAAGCCGTTTGGCTT GCTTCTCTGTGTCCTGCATTATTTTCTCGGCCCATTTCCAGTACCTGACACTCCTTTCATGGCTGCCAGAAGCACTTCCTCCGCCTCTGTCTTTGTAATGTAGGCTTTTGTCTTGTTCTGTCCCTCTCCTCTGTCCCAGCTCACACAGAGACAGACTCTCCGTCACCATCTTATCCAAGAGTTCATTTTTGTTGATGGTTATCCACAGTTTCLnc3: Data are represented as mean ± standard deviation (SD). All statistical analyses were performed using SPSS 19.0 . Statistical significance was determined by the t-tests. P < 0.05 was regarded as statistically significant and P < 0.01 was considered extremely statistically significant.http://www.ncbi.nlm.nih.gov/geo) under accession numbers GSE88732.The RNA-seq data sets generated in this study have been submitted to the NCBI Gene Expression Omnibus , testicular germ cells (c-kit) and epithelial cells  were unable to be amplified from the RNA extracted from our sperm sample, while positive markers of sperm  were easily detected . RNAs that are not intact transcripts might be degraded or cleaved to small fragments  lncRNA profiles abstained from noncode database, and screened the testes and/or sperm-specific expression lncRNA based on the FPKM. The results showed that 6,983 lncRNA  exclusive expressed in testis and sperm , 6 of whWe performed GO and KEGG enrichment analysis of the differentially expressed genes between sperm and round spermatid . The difP < 0.05).We compared the lncRNA and mRNA expression levels in the sperm of our data and round spermatid groups from GEO database (GSM1968532 and GSM1968533). We identified 4,040 upregulated lncRNAs and 4,261 downregulated lncRNAs  and 1,28To characterize the function of lncRNAs in the late stage of spermatogenesis or in mature sperm, we attempted to predict the cis- and trans-regulated target genes of differentially expressed lncRNAs. Based on the “Cis and Trans” principle, we identified 14,259 targeted genes of lncRNAs. In terms of GO terms, differentially expressed transcripts of mRNAs genes were highly enriched for protein metabolic process and RNA metabolic, spermatogenesis, sperm motility, cell cycle, chromatin organization, heterocycle and aromatic compound metabolic processes et al. For the differentially expressed lncRNA targeted genes, highly enriched GO terms included nucleic acid metabolic, protein modification, chromatin and histone modification, heterocycle compound metabolic, sperm function, and other processes.Based on our KEGG pathway analysis, the most enriched pathways for the differentially expressed mRNAs were mainly related to the metabolic pathways, mRNA surveillance pathway, cell cycle, RNA transport and endocytosis et al. For the differentially expressed lncRNA targets, the most significant pathways were those involved in RNA transport, PI3K-Akt signaling pathway, AMPK signaling pathway, protein processing in endoplasmic reticulum, and some other processes.To confirm the reliability of our sequencing data, we randomly selected four upregulated lncRNAs and mRNAs from the pool of mRNAs and lncRNAs whose fold changes were > 2.0, and analyzed their expression levels by qPCR in testes and sperm samples. As shown in The importance and potential function of lncRNAs in male sterility have been the subject of attention, as lncRNA expression levels are highly enriched and specific in testes and during spermatogenesis stages , 14, andCD4), testicular germ cells (c-kit), epithelial cells  and genomic DNA products of Prm2. In addition, RNA-seq in sperm is far more complex than that in somatic cells because the intrinsic characteristics of spermatozoal RNA were mainly fragmented [Sperm obtained from epididymis and vas deferens often contain some somatic cells like epithelial cells, testicular germ cells and leukocytes. Since sperm RNA content is about 1% of that in somatic cells and DNA quantity in sperm is very high with respect to RNA, the precaution of contamination caused by somatic cell RNAs and sperm DNA is very critical. In our study, we obtained high purity sperm RNA as indicated by the inability of PCR amplification of biomarkers of leukocytes  in sperm, with a distribution similar to that of testes. However, as mentioned above, many sperm RNAs are fragmented, and therefore we developed a computational approach to globally evaluate the integrity of lncRNAs. As expected, the integrity ratio of sperm lncRNAs was lower than that of testes. Furthermore, two mRNAs and two lncRNAs of interest were selected from the top 100 most intact profiles to test Testis-specific lncRNA might be important for male infertility . In the Sperm might retain certain transcriptional and translational activities , 33, whiIn our study, eight differently expressed genes that were identified by sequencing data were further corroborated by qPCR results, which suggested that sequencing data were reliable. It is likely that the dis-regulated RNAs in sperm, compared to round spermatid, are required earlier in spermatogenesis, and that RNAs associated with nuclear organization and the chromosome are no longer required in fully differentiated sperm and are simply residual. However, some abundant sperm transcripts may also function in the oocyte upon fertilization , 38 and In summary, the present study systematically identified and characterized many lncRNAs in mouse mature sperm. These lncRNAs were detected in sperm as fragmented and intact forms. The abundant, intact and testis/sperm specific lncRNAs related RNA metabolism, spermatogenesis, and sperm function represent good candidates to elucidate the functions of lncRNAs in mature sperm. Our results will contribute to the current understanding of the lncRNA complexity in mature sperm and provide a preliminary database of sperm lncRNAs for investigating their regulatory roles in sperm.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.


LOOP MBs), an architecture that elicits marginal levels of nonspecific signals in cells. We showed that MBs can detect single transcripts containing as few as 8 target repeat sequences with ~90% accuracy. In both the nucleus and the cytoplasm, mRNAs harboring 8 repeats moved faster than those with 32 repeats, suggesting that intracellular activities are less impeded by smaller engineered insertions. We then report the first MB-based imaging of intracellular dynamics and localization of single long noncoding RNAs (lncRNAs). We envision the proposed minimally-engineered, MB-based technology for live-cell single-molecule RNA imaging could facilitate new discoveries in RNA research.Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering. The MBs are composed of 2′-O-methyl RNAs with a fully phosphorothioate-modified loop domain (2Me/PS As individual fluorophores are difficult to detect when imaged under a widefield fluorescence microscope, one approach for achieving single-molecule sensitivity is to design fluorophore-tagged oligonucleotide probes complementary to unique sequences in the target RNA. When imaged, the multiple fluorophore-tagged probes hybridized to a single transcript are easily detected by conventional fluorescence microscopy as a bright spot representing a single RNA transcript. However, information pertaining to RNA dynamics cannot be easily acquired, as fixation is required during smFISH sample preparation.As the list of characterized RNA molecules and functions expands, visualizing the distribution and dynamics of various RNAs at the single-molecule level in living cells can add invaluable information regarding their physiological roles. Single-molecule fluorescence 4. In this way, specific RNAs are labeled by multiple FPs through the MS2 protein-RNA interaction. Another approach employs molecular beacons (MBs)5, which are single-stranded oligonucleotide probes capable of forming a stem-loop structure with a fluorophore and a quencher at the two termini. In the absence of complementary target RNA, the complementary sequences flanking the loop domain anneal to form a stable stem, bringing the fluorophore and quencher together. Hybridization of the loop domain to target RNA disrupts the stem configuration, causing separation of the fluorophore from the quencher and restoration of its fluorescence. Currently, MBs are predominantly used in applications where detection of specific RNAs is based on ensemble fluorescence measurements. It has been demonstrated that MBs can also be used to image RNAs with single-molecule sensitivity when target RNAs are engineered with multiple repeats of a known MB target sequence6.Live-cell single-molecule RNA dynamics has been studied predominantly using engineered RNA molecules with multiple tandem repeats that are bound by specific protein or oligonucleotide probes. The most common approach is the MS2 system, in which a fluorescent protein (FP) fused to the coat protein of bacterial phage MS2 is co-expressed with an engineered RNA construct containing multiple tandem repeats of the MS2 binding sequence8. False-positive signals are primarily detected in the nucleus, arising as a result of nuclease degradation and/or nonspecific binding to endogenous biomolecules8. Strategies to reduce false-positive signals include conjugating MBs to macromolecules to inhibit nuclear entry10, and synthesizing MBs with chemically-modified backbones to increase biostability14. Based on the latter approach, we recently showed that the 2Me/PSLOOP architecture, which incorporates phosphorothioate (PS) linkages throughout the loop domain of a 2Me MB backbone, enables accurate imaging of single mRNAs harboring 32 tandem repeats of a target sequence with minimal nonspecific signal11.Although combining MBs with multiple repeats of an MB target sequence could be a widely utilized approach to image RNA dynamics, and compared with the MS2-FP system may offer the added benefits of smaller probe size, incorporation of a wider variety of fluorophores, and improved signal-to-background due to quenching when not bound to target, the tendency of MBs synthesized with conventional DNA or 2′-O-methyl RNA (2Me) backbones to generate false-positive signals in cells limits their utility in RNA researchLOOP MBs, we demonstrated that an RNA engineered with 8 target repeats could be detected in cells with high accuracy, while showing reduced interference with normal RNA trafficking as compared with RNAs engineered with larger target inserts. We further demonstrated the first report of MB-based imaging of dynamics and localization of long noncoding RNAs (lncRNAs) in living cells at the single-molecule level. We anticipate the MB-based approach for imaging single RNAs with minimal target engineering developed in this study can be useful in furthering our understanding of the role of RNA trafficking and localization in health and disease.Because RNAs engineered with large sequence insertions could potentially exhibit altered functions or activities, in this study we investigated the minimal target engineering necessary for MB-based imaging of single RNAs using conventional widefield fluorescence microscopy. Using 2Me/PSCAGGAGTTGTGTTTGTGGACGAAGAGCACCAGCCAGCTGATCGACCTCGA-3′ (the underlined sequence is the unique MB target site) were kind gifts of Dr. Sanjay Tyagi, Rutgers University, NJ, USA. The derivative constructs, pEGFP-N1-1x, pEGFP-N1-2x, pEGFP-N1-4x, pEGFP-N1-8x and pEGFP-N1-16x, were generated using the same procedure to create pEGFP-N1-32x as described previously11. In brief, they were constructed by inserting the EcoRI-BamHI digested fragments of the parental pGEM constructs into pEGFP-N1 (Clontech). The pBFP-N1-8x construct was generated by inserting the EcoRI-BamHI digested fragment of pEGFP-N1-8x into pmTagBFP2-N1 . The pNEAT1-8x construct was created by PCR amplification of the MS2-NEAT1 plasmid  with forward primer 5′-ACCTGCGGATCCGGAGTTAGCGACAGGGAGGGATGCGCGC-3′ and reverse primer 5′-ACTGCTGCGGCCGCTTGTGCTGTAAAGGGGAAGAAA-3′. The PCR product was then inserted into pEGFP-N1-8x backbone digested with BamHI and NotI to excise EGFP. The pHOTAIR-8x construct was created by PCR amplification of cDNA from HEK293 cells with forward primer 5′-AGAACCGGTGACTCGCCTGTGCTCTGGAGCTTGATCCGA-3′ and reverse primer 5′-ATAAGAATGCGGCCGCTTTTTTTTTTGAAAATGCATCCAGATATTA-3′. The PCR product was then inserted into the AgeI- and NotI-digested pEGFP-N1-8x. The EYFP-PSP1α construct was a kind gift of Dr. Archa H. Fox, University of Western Australia, AUS and Dr. Angus I. Lamond, University of Dundee, UK.pGEM-1x, pGEM-2x, pGEM-4x, pGEM-8x, pGEM-16x, which encode transcripts containing 1, 2, 4, 8, and 16 tandem repeats of the 50-base sequence 5′-LOOP anti-repeat MB (anti-repeat MB) is labeled with an ATTO647NN reporter dye at the 5′ end and an Iowa Black RQ-Sp quencher at the 3′ end and has the sequence: 5′-mCmUmUmCmG*mU*mC*mC*mA*mC*mA*mA*mA*mC*mA*mC*mA*mA*mC*mU*mC*mC*mU*mGmAmAmG-3′ . The control MB is labeled with an ATTO647NN reporter dye at the 5′end and an Iowa Black RQ quencher at the 3′end and has the sequence: 5′-mCmUmCmAmG*mC*mG*mU*mA*mA*mG*mU*mG*mA*mU*mG*mU*mC*mG*mU*mG*mA*mCmUmGmAmG-3′. The MB sequences are designed to avoid hybridization with endogenous RNAs in mammalian cells. All MBs were synthesized by Integrated DNA Technologies .The 2Me/PS2, and 90% relative humidity.HEK293, NIH3T3, COS7, HeLa cells (American Type Culture Collection) and HeLa cells stably expressing the tandem repeat constructs were cultured in Dulbecco’s Modified Eagle’s Medium without phenol red , supplemented with 10% (vol/vol) FBS (PAN Biotech), 1xGlutaMAX (Thermo Fisher) at 37 °C, 5% (vol/vol) CO11. In brief, HeLa cells were transfected with plasmids expressing EGFP mRNA harboring various numbers of tandem repeats of the MB target sequence using Fugene HD (Promega). 24 h following the transfection, cells were cultured in media containing 0.8–3 mg/mL geneticin for 3 weeks. Single colonies were isolated and maintained in the absence of any antibiotics. The presence of the engineered repeats was confirmed by smFISH against the MB target sequence. Clones that showed individual RNA punctae easily distinguishable from one another were used for further studies. The same procedure was used to create HeLa cells stably expressing pNEAT1-8x (HeLa-NEAT1-8x), pHOTAIR-8x (HeLa-HOTAIR-8x) and pBFP-N1-8x (HeLa-BFP-N1-8x).HeLa cell lines stably expressing pEGFP-N1 (0x), pEGFP-N1-1x, pEGFP-N1-2x, pEGFP-N1-4x, pEGFP-N1-8x, and pEGFP-N1-16x, denoted as HeLa-N1-0x, HeLa-N1-1x, HeLa-N1-2x, HeLa-N1-4x, HeLa-N1-8x, and HeLa-N1-16x, respectively, were generated according to procedures previously used to generate HeLa-N1-32x cells stably expressing pEGFP-N1-32x15. In brief, after cells were trypsinized, washed with 1xPBS and pelleted, they were then resuspended in 11 µL resuspension buffer R (Thermo Fisher) so that the final cell concentration was 5,000 cells per µL and the MB concentration was 1 or 5 µM. A Neon transfection system (Thermo Fisher) was used with parameters set at 1005 V, 35 ms pulse width and 2 pulses total for HeLa, 1150 V with a 20 ms pulse width and 2 pulses total for HEK293 cells, 1050 V with a 30 ms pulse width and 2 pulses total for COS7 cells and 1200 V with a 20 ms pulse width and 3 pulses total for NIH3T3 cells. Following microporation and three washes in culture medium to remove free probes, cells were seeded on fibronectin-coated 8-well Lab-Tek Chambered Coverglass  for imaging at the indicated time points.Microporation was used in this study as the delivery method for MBs because it enables efficient delivery of MBs into a large number of cells with high viability, as shown previouslyAll microinjection experiments were performed using a Femtojet and Injectman NI2 (Eppendorf) microinjection system fitted with Femtotips I (Eppendorf). Cells were incubated in DMEM with no phenol red, supplemented with 10% FBS, in glass bottom dishes (Mattek) for all injection experiments.22, should be capable of delivering the MBs used in this study as well.In addition to microporation and microinjection, we anticipate that other cellular delivery methods previously used in MB research, including lipofectamine, nanoparticles, TAT-peptide, and streptolysin-O23 or custom-written MATLAB  programs.All microscopy experiments were performed on an Olympus IX 83 motorized inverted fluorescence microscope equipped with a 100x UPlanSApo 1.4NA or a 60x PlanApo N 1.42NA objective lens, back-illuminated EMCCD camera (Andor), Sutter excitation and emission filter wheels and an MT-20E excitation source (Olympus), controlled by CellSens Dimension software. Images were acquired using the Olympus MT20 filter set for DAPI, EGFP and TAMRA and a Chroma filter set for Cy5 . Three-dimensional image stacks were acquired with 0.25 µm increments in the z-direction. All images were analyzed with Fijiin situ hybridization was performed as previously described with modifications24. In brief, cells previously microporated with MBs cultured in 8-well chambered coverglass (50–70% confluency) were fixed in 1xPBS solution containing 4% (wt/vol) paraformaldehyde for 30 min at room temperature, washed with 1xPBS, and permeabilized at 4 °C in 70% (vol/vol) ethanol overnight. On the following day, cells were washed thrice with wash buffer  and then incubated in hybridization buffer  containing a pool of singly-TAMRA-labeled oligonucleotides complementary to different regions of the EGFP coding sequence11, the BFP coding sequence  was used on all 3D images, followed by identification of particles using the 3D Laplacian of Gaussian plug-in available for Fiji23. After low-intensity spots were removed, a region of interest (ROI) was drawn around individual cells and applied to the filtered stack. The Find Stack Maxima macro plug-in  was used to identify all local maxima in each slice of the z-stack. To identify which 2D local maxima were 3D local maxima, a custom MATLAB program compared the intensity of each local maximum in each slice with the intensity of the neighboring pixels in the current slice and the two adjacent slices . Each 3D maximum was considered to be an individual RNA.Single RNA transcripts in HeLa cells stably expressing different numbers of tandem repeats of the MB target sequence were identified as described previously25. In brief, an MB 3D local maximum was considered to be a colocalization event if an smFISH 3D local maximum was found within a 3 × 3 × 3 voxel cube centered around the MB maximum. The colocalization percentage was calculated by dividing the number of colocalization events by the total number of smFISH local maxima.After determining the 3D coordinates of RNA transcripts in smFISH and MB images using the methods described above, a custom MATLAB program was used to identify the extent of colocalization in three-dimensions as previously described11. In brief, Laplacian of Gaussian followed by Differences of Gaussian filters were used to identify individual peaks and their coordinates26. Peaks that belong to the same track were determined by simple Linear Assignment Problem tracker . The assigned tracks were then imported into @msdanalyzer written in MATLAB27. Tracks containing at least 15 time lags (Δτ) were selected for calculating the Mean Square Displacement (MSD). For simplicity, the 2-dimensional diffusion coefficient Deff was obtained from a linear fitting of MSD vs. Δτ, using the first 25% of total time lags, with a minimum fitting threshold of R2 > 0.9. The minimum Deff threshold for mobile fractions was set at 0.0006 μm2/s, using Deff calculated for Tetraspek beads (Thermo Fisher) immobilized on a coverslip as a control for xy-drift.All images were acquired at 100 ms per frame to identify and localize single engineered RNA transcripts using the TrackMate plugin for Fiji, as shown previouslyTo quantify fluorescence intensity of each particle, all images were acquired for 100 ms and analyzed in Fiji. In brief, particulate objects were first enhanced by applying rolling-ball background subtraction (background = 2) to all images. Appropriate thresholds were then set to filter out background noise. The total integrated intensity of each particle was analyzed using the “analyze particles” tool, with the size of particles set to be at least 3 × 3 pixels.All experiments were repeated at least three times and statistics were performed using t-test or one-way ANOVA with post hoc testing of pairwise comparisons using Fisher’s protected least significant difference unless otherwise stated. Kolmogorov-Smirnov test was used to further analyze distributions in Figs LOOP MBs complementary to a repeat target sequence   Fig. . Since b11. Similarly, discrete MB and smFISH signals were visible in the nucleus and the cytoplasm of HeLa-N1-16x, -8x, and -4x cells. In cells expressing EGFP transcripts harboring fewer than 4 repeats   revealed that in HeLa-N1-32x cells, 90% ± 5% of the MB spots colocalized with smFISH spots at 8 h post-microporation Fig. , in agreµM) Fig. . FurtherµM) Fig.  and 93% µM) Fig.  expressiµM) Fig. . FinallyµM) Fig.  and S8. One concern when engineering RNAs with large sequence insertions is the potential to alter RNA trafficking, causing changes in their functions and activities. Based on the above finding that anti-repeat MBs can detect both pEGFP-N1-32x and pEGFP-N1-8x transcripts with ~90% accuracy, we next performed single-particle tracking analysis to determine whether the pEGFP-N1-32x and pEGFP-N1-8x transcripts, which are ~2300 nucleotides (nt) long and ~1100 nt long, respectively, exhibit a difference in intracellular mobility. To investigate this, HeLa-N1-32x and HeLa-N1-8x cells were microporated with anti-repeat MBs and time-lapse images were acquired at 8 h post-microporation Movies  and S2. 2/s in the nucleus and 0.0199 ± 0.0033 µm2/s in the cytoplasm  and (HOX Transcript Antisense RNA) HOTAIR, lncRNAs that have emerging roles in the regulation of cellular behavior and disease progression37.Long noncoding RNAs (lncRNAs), a class of transcripts greater than 200 nucleotides long, are emerging as crucial regulators of diverse biological functions at epigenetic, transcriptional and post-transcriptional levels35. Consistent with its nuclear localization property, time-lapse images, acquired at 8 h following microporation of anti-repeat MBs into HeLa cells stably expressing pNEAT1-8x, revealed individual bright spots indicating single pNEAT1-8x transcripts (~4.1 kb in length) localized predominantly in the nucleus (Movie eff < 0.0006 µm2/s), with the remaining mobile particles moving only at 0.0019 µm2/s ± 0.0004 µm2/s  to form paraspecklesnal Fig. , resembl37. Consistent with these reported functions, following microporation of the anti-repeat MBs into HeLa cells transfected with pHOTAIR-8x transcripts (~2.6 kb) (HeLa-HOTAIR-8x), bright spots, indicative of single pHOTAIR-8x transcripts, were observed in both the nucleus and in the cytoplasm  in the nucleus were immobile, with the remaining mobile fraction moving at 0.0026 ± 0.0002 µm2/s Fig. . In the pts Figs  and S11.To our knowledge, this is the first study that visualizes NEAT1 and HOTAIR lncRNAs in living cells at the single molecule level. With potential for minimized impact on RNA biology, we envision the MB-based imaging approach can be applied to study other RNA molecules, elucidating their functions with previously unattainable detail and resolution.LOOP MBs), we showed that single RNA transcripts engineered with as few as 8 tandem repeats of an MB target sequence can be detected at the single-molecule level with high accuracy under standard widefield fluorescence microscopy. Shortening the engineered insertion was also shown to reduce the impact on the mobility of intracellular RNAs. Additional studies showed for the first time that MBs can be used to image lncRNAs and monitor their activities at the single-molecule level in living cells.With the growing interest in deciphering the role of RNAs in health and disease, it has become essential to be able to visualize single RNA transcripts in living cells with high spatial and temporal resolutions. Using MBs that elicit a marginal level of nonspecific signals  is smaller than an MS2-tag that typically contains 24 tandem repeats of MS2-binding sites , so engineered RNAs with the MB tag are expected exhibit less disruption of RNA trafficking, localization and functionality. Finally, MBs (~11 kDa) are smaller than MS2-FP fusion proteins (~44 kDa), and therefore may move more freely in the intracellular environment and may cause less disruption of target RNA activity when bound. This could be crucial in studies of RNAs that localize in the highly crowded environment of the nucleus or are tightly associated with proteins to form ribonucleoprotein complexes. With the increasing need in visualizing RNA molecules with high resolution, accuracy and sensitivity, we envision that the MB-based imaging approach presented here can aid discoveries of new RNA biology.Supplementary MaterialsRepresentative movie of mRNA transcripts in a HeLa-N1-32x cellRepresentative movie of mRNA transcripts in a HeLa-N1-8x cellRepresentative movie of mRNA transcripts in a HeLa-N1-1x cellRepresentative movie of mRNA transcripts in a HeLa-N1-2x cellRepresentative movie of mRNA transcripts in a HeLa-N1-4x cellRepresentative movie of NEAT1 lncRNA transcripts in a HeLa-NEAT-8x cellRepresentative movie of HOTAIR lncRNA transcripts in a HeLa-HOTAIR-8x cell


Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. With the help of different guide RNAs, the dyskerin complex pseudouridylates ribosomal RNA, small nuclear RNA and selective messenger RNAs. To monitor Ψ levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total Ψ levels in different cellular RNA fractions. Our method was validated to be accurate and precise within the linear range of 0.06–15.36 pmol/μL and to have absolute Ψ quantification levels as low as 3.07 pmol. Using our optimized HPLC assay, we found that 1.20% and 1.94% of all ribonucleosides in nuclear-enriched RNA and small non-coding RNA pools from the HEK293 cell line, and 1.77% and 0.98% of ribonucleosides in 18S and 28S rRNA isolated from the HeLa cell line, were pseudouridylated. Upon knockdown of dyskerin expression, a consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization. Of the 166 known RNA nucleoside modifications, pseudouridine (Ψ) is the most abundant post-transcriptional modification and was the first to be discovered . The isoKnown as the fifth ribonucleoside, Ψ is widely distributed in almost all RNA-containing species and constitutes over 1% of total nucleosides and over 7% of all uridines in ribosomal RNA (rRNA) fractions. Ψ has been shown to enhance transfer RNA (tRNA) binding to ribosomes and translational fidelity of rRNAs ,5. It isRecently, with the advent of massive parallel sequencing of the transcriptome, hundreds of naturally modified Ψ sites in mRNA were discovered in yeast and human cells ,8,9. SpeIn humans, there are 13 known Ψ synthases (PUSs) that can catalyze the isomerization of uridine to Ψ . Based oThe H/ACA RNP complex is comprised of one box H/ACA small nucleolar/Cajal body RNA (snoRNA or scaRNA) and two copies of a protein catalytic subunit comprised of dyskerin, GAR1, NHP2, and NOP10. The catalytic core, formed by the dyskerin/NHP2/NOP10 trimers complexed with a single guide RNA, was assembled through sequential binding with biogenesis factors including SHQ1 and NAF1 . GAR1 brO-methyl  modifications. H/ACA-scaRNAs are found localized to the Cajal body and are involved in the nucleoside modifications of selected small nuclear RNAs involved in the spliceosome complex [While the dyskerin/NHP2/NOP10 complex is the only known RNA-dependent PUS, it is responsible for the modification of various targets by virtue of its ability to switch RNA guides. All box H/ACA guide RNAs are non-coding small RNAs with a hairpin–hinge–hairpin structure and the signature ACA residues at the 3′ terminus at maturation . The mos complex .N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide (CMC) p-tosylate [p-tosylate reacts with all guanosine, uridine, guanosine-like, and uridine-like residues to form adducts. A subsequent cleavage step hydrolyzes all adducts except those formed at the Ψ bases [Since Ψ’s discovery, multiple methods have been developed to map the location and quantify the levels of Ψ. The most popular method for studying Ψ in RNA is based on chemical derivatization of RNA by tosylate . The CMC Ψ bases ,18. As tIn contrast, the traditional physicochemical property-based chromatography method provides an alternative way to quantify relative Ψ levels by simple enzymatic digestion of cellular RNA into single nucleosides . Upon seFollowing liquid chromatography, mass spectrometry (MS) is also a popular detection choice for Ψ level measurements in recent years . The fraIn the current study, a previously reported HPLC-UV method  was modiThe chemical reference compounds Ψ and 7-methylguanosine (7-metG) were obtained from Santa Cruz Biotechnology  and Sigma-Aldrich , respectively. Nucleoside test mix from Sigma was used as a reference to identify major nucleosides  in RNA pools. The aqueous mobile phase was prepared with ammonium dihydrophosphate from Sigma-Aldrich . Methanol (HPLC grade) was obtained from Fisher Scientific . Ultrapure water was obtained from a MilliQ water purification system . A previously described small hairpin (sh) RNA construct was applied to reduce dyskerin expression level by RNA interference .2. Cells were trypsinized, quantified using a Coulter counter , and seeded at a density of 3 million cells per 100 mm plate. Cells were allowed to recover overnight until they were 40–60% confluent at the time of transfection.The human embryonic kidney cell line HEK 293 was obtained from American Type Culture Collection . The human cervical adenocarcinoma cell line HeLa was a generous gift from Dr. Eric Jan’s laboratory. Cells were cultured in 1X DMEM high glucose media  supplemented with 5% fetal bovine serum and maintained at 37 °C with 5% COStandard calcium phosphate mediated transfection was performed. The cells were maintained under puromycin selection to inhibit the growth of cells that did not express the shRNA plasmid. Cells were harvested 96, 120, and 144 h after the transfection.2, 0.2 mM EGTA, 10% glycerol, 1 mM DTT, 0.1 mM PMSF) and subjected to four consecutive freeze–thaw cycles in liquid nitrogen and in a 37 °C water bath. Following centrifugation at 1000× g for 15 min, the supernatant containing hypotonic lysis buffer and cytoplasmic components was removed. The remaining pellet was washed once with hypotonic lysis buffer. The nuclear RNA pool was then extracted with Trizol .For collection of nuclear-enriched RNA pool, cells were resuspended in hypotonic lysis buffer  gel electrophoresis and the gel fraction between the region with xylene cyanol dye migration (~75 nt) and 5S rRNA (121 nt) was harvested. The excised band was sliced into small pieces with a clean razor and moved to a 15 mL conical tube. To the sliced gel pieces, 3× vol. of 0.3 M NaOAc (pH 5.2) and 0.1× vol. of phenol-chloroform were added. The tube was kept shaken at 37 °C overnight. Nucleic acid in the supernatant was extracted and precipitated with ethanol. Similarly, 18S and 28S rRNA were separated by size using a denaturing formaldehyde agarose gel, gel purified by the Ultrafree-DA centrifugal filter unit , and precipitated with ethanol.2, and 1 mg/mL BSA) following two overnight incubations at 37 °C. The final volume of each digested RNA sample was adjusted to 200 μL and 210 pmol of 7-metG was added to each sample as an internal control.RNA levels were quantified using Nanodrop , and the concentrations of selected RNA samples were verified with the Ribogreen assay . Five micrograms of each RNA sample were sequentially hydrolyzed by five units of RNase T2  with 2× RNase T2 buffer  and dephosphorylated by 5 units of Shrimp Alkaline Phosphatase  with 10× SAP buffer . Chromatographic separation was performed with a 4 μm Waters Nova-Pak C18 column 3.9 mm × 150 mm . The column was kept at room temperature, and the detection wavelength was set at 254 nm. The two mobile phase components were adapted from a previous study . Mobile r2) of data points on the plotted curves. The absolute Ψ amount was expressed as picomoles per microliter (pmol/μL) and converted to picomoles later.Stock solutions of Ψ were made by dissolving the appropriate amount of pure Ψ in water to yield a final concentration of 200 ng/μL. Working solutions with concentrations of 0.10, 0.26, 0.64, 1.6, 4, 10, and 25 ng/μL was prepared every month by a 2.5-fold serial dilution from the stock. Calibration curves for Ψ levels were created on each analysis day by diluting 30 μL of working solution with water and digestion enzyme buffers into 200 μL . The analyte to internal standard (I.S.) peak area ratios were plotted against matched amounts of Ψ added in the injected blank sample. The calibration curves were calculated by the least squares method. Linearity was assessed by determining the coefficient of correlation  between the nominal and measured concentrations was expressed as accuracy of the method, whereas relative standard deviation (RSD) of repeated measurements was expressed as precision.A = kσ/S, where A was LOD or LOQ, k was the coefficient for the two parameters (k = 3.3 for LOD and k = 10 for LOQ), σ was the standard deviation of the response , and S was the slope of the curve.Sensitivity of the method was estimated using the calibration curve method. The limit of detection (LOD) and limit of quantitation (LOQ) of the present method were calculated by the following formula: Specificity of the nucleosides in RNA pools was tested by injecting a nucleoside test mix and referring to the relative retention time in the standard.The Western blot protocol was the same as described in . Whole cp < 0.05.Data were analyzed using GraphPad Prism software . Error bars denote SEM. The one-way ANOVA with post-hoc Bonferroni correction was applied to adjust for multiple comparisons. Differences were considered significant at Representative chromatograms of a calibration standard sample and a digested RNA sample are shown in r2) of all calibration curves were greater than 0.999.  was calculated to be 44 pmol. Since 1.25 μg of the starting material, i.e., intact nuclear RNA is equal to 3.68 nmol, we concluded that approximately 1.20% of nucleosides in nuclear-enriched RNA are Ψs. This is in agreement with previous reports [Using the same method, the amount of Ψ in 18S and 28S rRNA from HeLa cells in log-phase growth were calculated to be 1.77% and 0.98%, respectively . It is kIt was also found in our study that 1.94% of gel-purified small RNA populations are pseudouridylated. The average Ψ concentration measured by our assay, and the equivalent amount and levels in different cellular RNA pools are listed in Next, we applied our assay to quantify changes in Ψ levels upon the reduction of dyskerin expression. Only 3 out of 13 known PUSs can catalyze pseudouridylation in rRNAs . Out of We reasoned that as rRNA are known to have long half-lives , p = 0.0188). As expected, there was also a consistent and significant reduction in Ψ levels in nuclear RNA fractions 96, 120, and 144 h post-shRNA transfection (p = 0.0291). The reduction was not directly proportional to the reduction in dyskerin expression. Although HEK293 cells showed widespread cell death (data not shown), suggesting that they were under severe stress with the reduction in dyskerin expression, the average Ψ levels in these shRNA-treated cells were only ~10% lower than the control group (no dyskerin shRNA transfection).The HEK293 cells were directed to express a shRNA duplex against dyskerin and kept under antibiotic (puromycin) selection for at least 48 h. Antibiotic selection inhibited the growth of cells that were negative for the expression of the shRNA construct. With this approach, we confirmed that dyskerin expression in HEK293 cells was reduced to ~45% of regular levels . This is an easily collected sample amount from biochemical experiments, as well as from clinical/patient sources.The LOD and LOQ for our assay were estimated to be 0.01 pmol/μL and 0.02 pmol/μL, respectively. These values equaled 0.31 and 0.92 pmol of Ψ or 10 and 31 ng of RNA input (assuming the percentage of pseudouridylation in the RNA sample for detection is around 1%). As well, the intra-day and inter-day coefficients of variation for precision and accuracy were below 10% for concentrations tested within the quantification range. In other words, minimal changes in Ψ levels as low as 0.31 pmol in RNA samples may be accurately and precisely analyzed by the current assay. Therefore, our assay provides a sufficient window for measuring changes in pseudouridylation under different conditions.DKC1 gene [DKC1 gene, but not all disease-associating mutations, led to reproducible but modest loss of steady-state Ψ levels in rRNA. We contended that, while these moderate changes in rRNA Ψ levels were not enough to drive the disease, they could nonetheless modulate the rate of disease onset and/or severity by a mechanism that involves reduced stress tolerance conferred by optimal rRNA Ψ modifications.Based on the observed retention times, the major nucleosides were well separated. Importantly, Ψ had a stable retention time of 2.0 min that allowed it to be easily identified. Our method has already been applied to quantify the steady-state Ψ levels in rRNA samples from XDC patients with mutations in the dyskerin-encoding KC1 gene ,28. We fDKC1 is an essential gene in metazoan, and knockout of DKC1 leads to embryonic lethality in mouse models [The observed non-proportional relationship between Ψ levels and dyskerin expression may be explained by the existence of different mechanisms for pseudouridylation. There are at least 12 additional PUSs known in humans, on top of the H/ACA RNP complex. Specifically, pseudouridylations in rRNA by PUS7 and PUS7L are carried out in a guide-RNA independent manner and are unaffected upon the knockdown of dyskerin expression [e models . It is tIt is worth nothing that the nuclear-enriched RNA pool we collected from our shRNA experiments is comprised of almost all organelles, including the nucleus, ribosomes, endoplasmic reticulum, Golgi body, cell membrane, and mitochondria. This RNA collection contains not only nascent rRNA, but also includes mRNA, tRNA, and other small RNAs. As dyskerin is not known to be substantially involved in the modifications of these other RNA populations, with the notable exceptions of snRNA and a small percentage of mRNAs, Ψ levels in these RNA fractions will not be affected by dyskerin knockdown. Even though rRNAs constitute over 80% of total cellular RNA , the nasPreviously, functional studies of Ψ focused on the different pseudouridylation levels at specific Ψ residues in rRNA, tRNA, and mRNA ,8,36. WhWith only the digestion step prior to analysis, our method provides accurate quantification of Ψ levels without any additional sample derivatization or manipulation. It has been used to measure Ψ levels in vivo and to measure changes in pseudouridylation over time. Our fast and economical HPLC-UV method can also be applied to detect and determine low levels of Ψ in different RNA fractions with small amounts of RNA input.


Organelles without membranes are found in all types of cells and typically contain RNA and protein. RNA and protein are the constituents of ribosomes, one of the most ancient cellular structures. It is reasonable to propose that organelles without membranes preceded protocells and other membrane-bound structures at the origins of life. Such membraneless organelles would be well sheltered in the spaces between mica sheets, which have many advantages as a site for the origins of life. Paramecium .,7,8.6,7,Mica provides another big advantage over other porous rocks: mechanical energy  6,10]. ,10. 6,10Mechanical energy is the product of the forces acting on the mica sheets and the distances of the open-and-shut motions of the mica sheets. Moving mica sheets can generate a wide range of energies. The mechanical energy from moving mica sheets depends on the spring constant of the mica spring, which is proportional to the thickness of the mica spring. The thickness of the mica spring varies by 1-nm increments, because single mica sheets are 1 nm thick.Mechanical energy may have preceded chemical energy at the origins of life. Moving mica sheets may have functioned as enzymes function today, with oscillating motions that push and pull substrate molecules to cause chemical reactions. There are at least three lines of evidence that enzymatic reactions involve protein motion. First, hexokinase is a glycolytic enzyme with no mechanical function. When it binds glucose and ATP, however, a large cleft closes, bringing two domains of the enzyme closer by almost 1 nm . Second,Membraneless organelles form by liquid–liquid phase separation under favorable conditions ,19. For Nucleoli  are probMembraneless organelles are typically composed of RNA and protein and are known as ribonucleoprotein particles (RNPs) . Some ofThe proteins in RNPs typically have Low Sequence Complexity (LSC) and Intrinsically Disordered Regions (IDR) . PrebiotMany proteins in RNPs are RNA-binding proteins. Therefore, RNA can nucleate RNP assemblies. Base-paired RNA hairpins partition into membraneless organelles more effectively than unstructured RNA of the same length. Double-stranded nucleic acids are destabilized in RNPs ,25. DNA Membraneless organelles in living cells maintain their shape and their separation from each other partly because of cellular or nuclear forces acting on them. Crowding and force fluctuations facilitate the formation of these organelles . A smallSizes of membraneless organelles can scale with the amount of cytoplasm. A single large membraneless organelle is thermodynamically favored over multiple smaller droplets because of the surface tension of the interface. Therefore, membraneless organelles can fuse with time, in a process known as Ostwald ripening . Some RNIf RNA and proteins now self-assemble into nucleoli and other organelles without membranes, did they start doing this at life’s origins? Proto-ribosomes might have formed this way. Spaces between mica sheets would have provided a protected environment where this could have happened.The origin of life from non-living materials is an example of chemical emergence. As David Deamer says, “Emergence is now being used in science to connote the process by which a physical or chemical system becomes more complex under the influence of energy… The emergent property is typically unexpected and cannot be predicted” .Another example of chemical emergence can be found in Madagascar’s Tsingy Rouge . The TsiMembraneless organelles are found in both Eukaryotes and Prokaryotes today. Their primary components, RNA and protein, are the two essential components of ribosomes, which contain some of the most ancient RNAs . Therefo