We have shown previously that their folding properties  and conformation depend on their local sequence, and we have elucidated a series of rules for prediction of these properties from sequence. In this work, we have expanded the rules for prediction of folding properties, and then applied the full set to predict the folding and conformation of four probable k-turns we have identified amongst 224 structured RNA species found in bacterial intergenenic regions by the Breaker lab . We have The majority of RNA sequences transcribed in cells do not code for protein, and they have diverse functions. Many of these regulate gene expression in various ways, and riboregulation is important in all domains of life ,3. In hutrans sugar-Hoogsteen conformation, also known as sheared base pairs, and the overall structure is tightly kinked, with an included angle between the axes of the flanking helices of 50°. The conformation of the k-turn is determined by two properties;To a first approximation, RNA secondary structure can be reduced to rigid helical sections that are connected by junctions, the conformations of which will determine the trajectory of the helices. These will set up the formation of longer-range tertiary interactions that generate the overall three-dimensional fold. The kink-turn (k-turn) is a widespread helical junction in RNA. The standard k-turn comprises a duplex RNA containing a three-nucleotide bulge followed by tandem G:A and A:G base pairs  Figure . The G:AIn the absence of metal ions or binding proteins, the RNA adopts a conformation that is not tightly kinked, but rather bent in the manner of a simple three-nucleotide bulge. Many  k-turns adopt the k-turn conformation on addition of divalent metal ions, but some will not. The great majority of k-turns will fold on binding of proteins such as the L7Ae family, including those that do not undergo folding in metal ions .The conserved adenine nucleobases of the sheared G:A base pairs both form important cross-strand hydrogen bonds with 2′-hydroxyl groups at the L1 and −1n positions ,14. HoweWe have explored what sequences determine whether or not a given k-turn sequence folds in response to metal ions ,17, and 3b:3n = Watson−Crick base pairs lead to poor or non-existent folding in metal ions.3b = C leads to good folding in response to metal ions.k-turns with 3n = G also fold well in response to metal ions.6 of adenine 1n, thereby preventing the k-turn from folding as the first step of the snoRNP assembly  protection  and Cy3  were attached to the 5′ termini of the oligonucleotides as phosphoramidites in the final cycle of the synthesis, as required.RNA oligonucleotides were synthesized using hemistry  as descr , 25 mM NaCl by slowly cooling an equimolar mixture of bulged and unbulged strands from 80°C to 4°C, and purified by gel electrophoresis in 12% polyacrylamide in 45 mM Tris−borate (pH 8.3), 25 mM NaCl buffer at 4°C. The hybridized species were recovered by excising the bands and elution from the crushed fragments followed by precipitation in ethanol. The sequences are listed in full in RNA was purified by gel electrophoresis in 16% polyacrylamide in 90 mM Tris−borate (pH 8.3), 10 mM EDTA, 7 M urea at 23 W constant power at room temperature. RNA-containing bands were excised and recovered by elution from the crushed fragments. The eluted RNA was precipitated with ethanol and dissolved in 0.1 M TEAA. Fluorescent RNA strands were purified by reversed-phase chromatography on a CClostridium perfringens was synthesized and then purified by gel electrophoresis in polyacrylamide under denaturing conditions in the presence of 7 M urea. The full-length RNA product was visualized by UV shadowing. The band was excised and electroeluted using an Elutrap Electroelution System  into 45 mM Tris−borate (pH 8.5), 5 mM EDTA buffer for 12 h at 150 V at 4°C. The RNA was precipitated with isopropanol, washed once with 70% ethanol, and dissolved in water. The sequence is shown in duplex form in The oligonucleotide 5′-GUCUAUGAAGGCUGGAGAC-3′ from Thermoanaerobacter tengcongensis SAM-I riboswitch was changed by site-directed mutagenesis in order to convert the sequence of the k-turn region to that of the Actinomyces-I k-turn. The sequence of the modified plasmid was verified by DNA sequencing, and the sequence of the Actinomyces-I k-turn as inserted into the riboswitch is shown in A plasmid containing a gene for the A. fulgidus L7Ae cloned into a modified pET-Duet1 plasmid (Novagen) (Escherichia coli BL21-Gold (DE3) pLysS cells (Stratagene), and purified as reported previously (Novagen)  was expreviously .FRET efficiency was measured from a series of RNA duplex species , terminaEFRET) were measured using the acceptor normalization method  and absorption spectra were measured for each RNA between 400 and 600 nm using a Thermo Evolution 201 UV−visible spectrophotometer. Spectra were deconvoluted using a corresponding RNA species labeled only with Cy3, and fluorophore absorption ratios required for FRET analysis were calculated using a program implemented in MATLAB. Fluorescence spectra were recorded in 90 mM Tris-borate (pH 8.3) at 4°C using an SLM-Aminco 8100 fluorimeter. Spectra were corrected for lamp fluctuations and instrumental variations, and polarization artifacts were avoided by setting excitation and emission polarizers crossed at 54.7°. Values of FRET efficiency  as the search model.From crystal density considerations, five RNA molecules were expected to be present in the asymmetric unit. The structure was determined by molecular replacement using the program PHASER  with HmKP43212 were obtained. Diffraction data were collected on different beamlines, including BL17U, BL18U1 and BL19U1 at SSRF. Data were indexed, integrated and scaled using iMOSFLM and Scala from the CCP4 suite of programs . Crystals of space group programs . Structuprograms  or 5FK3 programs . A compoprograms .Crystallographic data and statistics are presented in H. marismortui Kt-7 (HmKt-7) is the best characterized k-turn structure (A. fulgidus L7Ae (AfL7Ae) protein. 17 independent crystal structures in a variety of environments reveal that when not in situ in the 50S ribosomal subunit HmKt-7 adopts the N3-conformation , C:G, U:A or A:U  measured using the acceptor ratio method (HmKt-7 (−1b:−1n = C:G) folds on addition of Mg2+ ions, changing from EFRET = 0.25 to 0.56 as the RNA kinking concomitant with k-turn conformation brings the fluorophores closer together, with a calculated apparent Kd = 70 μM. As observed previously, addition of AfL7Ae increased EFRET slightly further to 0.63. Reversing the −1b:−1n base pair to G:C resulted in an RNA that underwent no folding into the k-turn conformation, either with addition of Mg2+ ions or AfL7Ae protein. We have previously found that -1b:-1n = G:C prevents ion-induced folding in similar sequence backgrounds, and noted that natural k-turns have a strong preference for −1b:−1n = C:G  and 4b:4n = G:U. We have previously observed that an adjacent G:U base pair can have a major structural effect on an A:G base pair  measured using the acceptor ratio method  might exert some influence, but drum and HmKt-7 share a GA sequence. Given that placing a G:U base pair in the 4b:4n position of Kt-7 results in better ion-induced folding it is probable that this is similarly responsible for the superior folding of the RAGATH-18 and drum k-turns. The HOLDH sequence has 3b:3n = A:G, but −1b:−1n = U:A. As we have shown above, inclusion of the latter feature leads to weak ion-induced folding in HmKt-7. The folding of HOLDH induced by addition of Mg2+ ions is clearly weaker than that of drum and RAGATH-18, reaching a limiting EFRET = 0.4. Yet that is rather better than the folding of the variant of HmKt-7 with −1b:−1n = U:A, likely a consequence of the 4b:4n = G:U. The 4b:4n = G:U provides a partial rescue of the effect of the −1b:−1n = U:A.All four sequences fold into a state with elevated FRET efficiency on addition of Mg2+ ions. The RNA underwent folding up to a limiting EFRET of 0.52, similar to HmKt-7. The main distinction between the behavior of the Actinomyces-1 RNA and the other three k-turns (plus HmKt-7) is the high value of EFRET in the absence of added Mg2+ ions. This indicates that the structure of this RNA before folding into the k-turn conformation is different. All these molecules likely adopt the structure of a standard 3-nucleotide bulge under these conditions i.e. kinked, but not to the extent observed in a standard k-turn geometry. It appears that the Actinomyces sequence kinks to a greater extent prior to k-turn folding.The Actinomyces-1 sequence has −1b:−1n = C:G and 3b:3n = C:C, and was therefore predicted to undergo folding into the k-turn conformation on addition of MgAfL7Ae protein to each of the four k-turns led to similar extents of folding, in the range EFRET = 0.53 to 0.67, and similar to the value for HmKt-7 of 0.63. Each therefore binds AfL7Ae and probably adopts a very similar conformation.Addition of HmKt-7 in sequence. We therefore predicted that these three would adopt the N3 conformation. By contrast, Actinomyces-1 has 3b:3n = C:C which is predicted to confer the N1 conformation according to our conformational rules  and 7EAG (RAGATH-18 k-turn).gkab333_Supplemental_FileClick here for additional data file.


The inhibitory effects of 87 derivatives of furoquinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furoquinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (−1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro −1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential −1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3- Recent outbreaks of zoonotic coronaviruses pose a global threat to public health. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an etiological agent of the newly emerged coronavirus disease-19 (COVID-19), has spread worldwide, causing enormous human casualties .Betacoronavirus, family Coronaviridae, order Nidovirales [These zoonotic viruses  belong to the genus ovirales . CoVs arovirales . The genovirales .ORF1a encodes polyprotein 1a (pp1a), while ORF1a and ORF1b together encode poly protein 1ab (pp1ab) through programmed −1 ribosomal frameshift (−1 PRF). The −1 PRF is an alternative mechanism for translation whereby the ribosome stalls on the mRNA secondary structure, shifts the ribosome reading frame back to one nucleotide, and continues the translation. As ORF1b lacks its own independent translation initiation site, ORF1b translation requires −1 PRF at the junction of the ORF1a/ORF1b overlap region ,8. AfterIn reverse genetics studies on CoVs, it has been reported that ORF1b-encoded proteins are essential for viral RNA replication. Work on the infectious bronchitis virus (IBV), a highly infectious avian CoV, demonstrated that point mutations in nsp13 in Vero cell-adapted IBV clones cause lethal viral infection in Vero cells . Hence, Other viruses utilize −1 PRF to control the gene expression involved in replication and viral pathogenesis. During human immunodeficiency virus 1 (HIV-1) infection, the pol gene, which is downstream of the gag gene, is translated by frameshifting and the gag to gag-pol ratio is strictly regulated for efficient viral replication, assembly, and infectivity ,16,17. IIn general, frameshifting signals of CoVs are composed of two elements: conserved heptameric slippery sequences (UUUAAAC), followed by RNA secondary structure (a pseudoknot RNA structure) ,8. TheseIn this study, we determined the frameshifting efficiencies of SARS-CoV, SARS-CoV-2, and MERS-CoV, and searched for small molecules that inhibit the −1 PRF of MERS-CoV. We discovered a novel compound that efficiently suppressed viral propagation via inhibition of −1 PRF, suggesting that the −1 PRF of coronaviruses is a potential target for antiviral therapy.Huh7 and A549 cells were purchased from American Type Culture Collection . Huh7, A549 and HEK293 cells were maintained in Dulbecco’s modified Eagle medium with high glucose (DMEM) with 10% fetal bovine serum  and 1% penicillin/streptomycin . The Middle East respiratory syndrome coronavirus (MERS-CoV) strain isolated from a Korean patient (GenBank accession number KT029139.1) was kindly provided by the Korea Centers for Disease Control and Prevention . All expRenilla luciferase and firefly luciferase genes in the pSGDluc vector. Further, −1 PRF sequences were inserted in a manner that firefly luciferase is only expressed with Renilla luciferase when −1 PRF occurs. Then, the −1 PRFs with reporter genes were subcloned into a lentiviral vector, specifically the pLenti CMV/TO Puro empty (w175-1) vector. Lentiviruses were generated using packaging plasmids (pMD2.G and psPAX2). A549 cells were transduced with lentiviruses, and the cells stably expressing reporter genes were selected using puromycin. Positive and negative controls that did not contain −1 PRF signals were also generated. In control constructs, Renilla and firefly luciferase genes were linked in-frame for a positive control or out of frame for a negative control. Thus, the positive control expresses both Renilla and firefly luciferases, whereas the negative control only expresses Renilla luciferase.Reporter plasmids containing the frameshifting signals were generated using the dual-luciferase vector system (pSGDluc), which was developed by Loughran et al. to avoid assay distortions from fused dual luciferase reporters . The −1 ® luciferase assay system  and a Synergy H1 microplate reader . Frameshift efficiency was determined by measuring the ratio of Renilla luciferase activity to firefly luciferase activity. The positive control was set to 100%, and the negative control was set to 0%. Relative frameshifting ratio was calculated by comparing relative frameshifting ratio (Renilla luciferase activity to firefly luciferase activity) of the indicated samples to the ratio of the DMSO-treated or untreated samples, which was set to 100%. IC50 values were determined by a sigmoidal dose-response (variable slope) curve using GraphPad Prism 8  software.Small molecule libraries (each dissolved in 100% DMSO) were kindly provided by the Korea Chemical Bank  at the KThe viability of cells grown on a 96-well plate was measured using a WST-1 reagent kit  and a Synergy H1 microplate reader (BioTek). Cells were treated with increasing concentrations of the chemical compounds for 2 days.For viral infections, Huh7 cells grown on a 96-well plate were incubated with 0.02 MOI of MERS-CoV for 1 h. The cells were washed twice with serum-free media and then grown in fresh media with or without chemical compounds for 2 days. Viral RNA was extracted from cell culture supernatants using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s protocol. To detect the MERS-CoV genome, primers targeting the MERS-CoV nucleocapsid (N) gene  were used: forward primer 5′- GGG TGT ACC TCT TAA TGC CAA TTC -3′ and reverse primer 5′- TCT GTC CTG TCT CCG CCA AT -3′. RNA transcribed from the MERS-CoV nucleocapsid gene using the MEGAscript™ T7 transcription kit was used as a standard for qRT-PCR. qRT-PCR was performed with the One Step PrimeScript III RT-qPCR mix (Takara) reagent using the QuantStudio 3 real-time PCR system (Applied Biosystems).p values of less than 0.05 were considered statistically significant. Data produced by qRT-PCR, luciferase assays, and cell viability assays are presented as mean values, with error bars showing the standard deviations from three independent experiments, unless otherwise specified.Statistical analyses were performed using GraphPad Prism 8 software . MERS-CoV ORF 1b contains a replicase gene cluster, which consists of key components required for viral replication, including RNA-dependent RNA polymerase (RdRp) and helicase. ORF1b is translated by −1 PRF, followed by ORF1a expressing polyprotein 1ab (pp1ab) A. PresenRenilla and firefly luciferase genes such that firefly luciferase genes were translated only when frameshifting occurred through the pseudoknot secondary RNA structures in mRNAs decoded in −1 PRF signals  were removed. Six compounds were selected, and their IC50 values and cell viabilities (WST assays) were determined with various concentrations of compounds (0.01–10 μM). Of that group, compound KCB261770 showed the most promising inhibitory effects  and was chosen for further study , we initially chose a representative library , a natural compound library , and a clinically applied library . Compounds that exhibited over 70% inhibition at a concentration 5 μM of were selected A. Among er study B,C. Compof 294.3 . Althougof 294.3 ,32, diveof 294.3 ,33 owingof 294.3 ,35, antiof 294.3 , and antof 294.3 . The inhRenilla luciferase activities were measured for 60 min. Both firefly and Renilla luciferase activities were not significantly affected by compound KCB261770 , and cycloheximide . 6-thioguanine, a well-studied purine analog, inhibits de novo purine synthesis and is incorporated into DNA and RNA, leading to disruption of DNA synthesis and cytotoxicity ,43. CHX Renilla luciferase activities were measured in cells harboring reporter plasmids containing the MERS-CoV −1 PRF. 6-thioguanine inhibited both firefly and Renilla luciferase activities in a dose-dependent manner without decreasing the frameshifting ratio, suggesting that it was not inhibited by the cytotoxicity induced by 6-thioguanine  in the media were detected. In contrast, compound KCB261770 treatment dramatically decreased viral RNA levels in the media in a dose-dependent manner (approximately 3.7 × 109 genome copies/mL at 5 μM), suggesting that the production of progeny viruses was suppressed by the treatment . We previously showed that the inhibition of the frameshift correlates well with the decrease in SARS-CoV replication using antisense peptide nucleic acids (PNAs) specifically targeting the −1 PRF signal . In this study, we found a novel small molecule that strongly inhibited the frameshifting of MERS-CoV, which was confirmed in the virus-infected cells. The compound was also effective in the frameshifting of SARS-CoV or SARS-CoV-2, due to the structural similarities between the PRF signals of MERS-CoV and SARS-CoV . Thus, iFor further development of antiviral agents targeting frameshifting, it is necessary to identify the underlying mechanisms by which the novel compound interferes with frameshifting. The frameshifting event occurs via interaction between the ribosomal complex and the pseudoknot structure of the −1 PRF signal . A recenAlthough some furo-quinoline-containing plant extracts have been used in traditional medicine ,37, a li


It has only recently been found that this epigenetic modification plays an important role in many physiological and pathological processes, such as cell fate commitment, immune response, obesity, tumorigenesis, and relevant for the present review, gametogenesis. Notably the RNA metabolism process mediated by m6A is controlled and regulated by a series of proteins termed writers, readers and erasers that are highly expressed in germ cells and somatic cells of gonads. Here, we review and discuss the expression and the functional emerging roles of m6A in gametogenesis and early embryogenesis of mammals. Besides updated references about such new topics, readers might find in the present work inspiration and clues to elucidate epigenetic molecular mechanisms of reproductive dysfunction and perspectives for future research.Emerging evidence shows that m In one ications . The m6A(lncRNA)  and micrs A/C/U) , which ireaders)   .6A detection methods have emerged. Among them, LC-MS (liquid chromatography-mass spectrometry) and ELISA (enzyme linked immunosorbent assay)/antibody-based colorimetry can detect the overall level of m6A on mRNA and conduct quantitative analysis. meRIP-seq (methylated RNA immunoprecipitation)  and miCLIP-seq  are high-throughput sequencing methods, and m6A-seq can conduct qualitative analysis on hypermethylated mRNA regions, miCLIP-seq can accurately analyze the single base of m6A. In addition, as the third generation sequencing technology, nanopore-seq is mainly used for de novo genome assembly and can quantify differences in RNA modifications and detect different RNA modifications with position accuracy in vitro  undergo self-renewal mitotic divisions and at the same time give rise to A-paired (Apr) and A-aligned  spermatogonia. Aal spermatogonia can divide and proliferate to form A1 spermatogonia which generate type B spermatogonia. These enter into meiosis as primary spermatocytes after some mitotic divisions; at the end of the first meiotic division, each primary spermatocyte forms two secondary spermatocytes which in turn at the end of the second meiotic division, generate two haploid spermatids. Finally, these, through a complex process termed spermiogenesis, develop into spermatozoa which are released inside the lumen of seminiferous tubules (spermiation) . m6A modogenesis .6A plays in the regulation of spermatogonia mitosis, meiosis and spermiogenesis in mammals has been recently reviewed  and METTL14, two of the methyltransferase “writers” of mr tissue . Transcr factors . Combine factors . METTL3 bidopsis , while ibidopsis . In humamotility .6A is involved in spermatogenesis by affecting the function of testicular somatic cells. The loss in Sertoli cells of WTAP, the regulatory subunit of the METTL3/METTL4 complex, impairs their ability to sustain the SSC niche  and ALKBH5 are highly expressed in testis. Ablation of ALKBH5 in mice resulted in spermatogenesis defects, but the mice showed no other significant phenotypic changes , YTHDF1, YTHDF2, YTHDF3, and YTHDC1 work in concert to quickly process mRNAs in the nucleus, control the translation of mRNAs, and then participate in the rapid degradation of translated mRNAs , promote the targeting of the exon of the transcriptional product mRNA and block the binding activity of srsf10 (SRp38) mRNA, which plays an important role in maintaining the development of mouse spermatogonia  after knockout of YTHDF2 showed down-regulation of the expression of MMPs , affecting cell adhesion and proliferation . miRNA is a non-coding single stranded small RNA molecule with a length of about 21–23 nucleotides  and processed by DGCR8 . As an Rexosomes . In addiocessing . Therebyocessing .6A exon to back-splicing. m6A modification regulates its translation through the identification of YTHDF3 and eIF4G2 , are rich in testis and seminal plasma. During the process of spermatogenesis, circRNAs gradually become a regulator of sperm quality, particularly when late pachytene spermatocytes evolve into round and elongated sperm cells. It compensates for linear RNA degradation in late spermatogenesis and maintains protein levels during chromatin condensation . In addid eIF4G2 . At the ic cells .Oocyte development includes oogenesis, folliculogenesis and ovulation. Briefly, oogenesis mainly refers to differentiation of oogonia from primordial germ cells (PGCs), oogonia proliferation and entering into meiosis as primary oocytes, growth and cytoplasmic/nuclear maturation of oocytes. As primary oocytes reach the diplotene stage of meiotic prophase I, their inclusion within a primordial follicle imposes meiotic arrest and marks the beginning of folliculogenesis culminating in ovulation.6A-RNA is a crucial part of these regulatory processes.For mammalian oocytes, the germinal vesicles (GV) in the nucleus of oocytes gradually become inactive after a period of active transcription in the rapid growth stage . Under t6A modification plays an important role in the development of oocytes and follicles , a component of the RNA m6A methyltransferase complex, plays critical roles in folliculogenesis and the maintenance of oocyte competence. The specific deletion of KIAA1429 in oocytes leads to female infertility, and fully grown GV  oocytes fail to undergo GVBD , so they lose the ability to resume meiosis. In addition, the loss of KIAA1429 could also lead to abnormal RNA metabolism in GV oocytes and the conditional depletion of KIAA1429 decreased the m6A levels in oocytes and mainly affected the alternative splicing of genes associated with oogenesis  effectively reduced the m6A modification level, significantly reduced the ratio of 4-cell embryos to blastocysts, and disturbed the normal embryonic cell differentiation  activity plays an important role in pluripotent stem cells. Intriguingly, GSK-3 deletion or inhibition by 2i medium containing inhibitors PD0325901 and CHIR99021  can signem cells .6A modification  retrotransposons was interrupted, thereby increasing transcriptional activity , writers (methyltransferases) and erasers (demethylases). It is now clear that expression and activities of these proteins are essential for the correct regulation of most if not all reproductive processes. Strong evidence has emerged on the various functions of these proteins and the corresponding functions of targeted RNA in oocyte, sperm and embryonic development. The mTORC1 signaling pathway is activated in granulosa cells and oocytes during the process of follicular activation and m6A RNA modification is closely related to the mTORC1 signaling pathway of cancer cells  and sex differentiation of the germ cells and somatic cells of the gonads. Interestingly, in this regard, in Drosophila, IME4 (a homologous of METTL3)-null mutants show a sex bias towards males.N- methyladenosine (mA) is a large number of modifications to mRNA and DNA. It was first discovered and characterized in the 1970s. Among them, mer cells . These a6A modification in development and reprogramming during MZT and ZGA. In addition, the wrong m6A RNA modification during embryonic development impacts the differentiation process, which is worthy of further study. With the advancement of technology, such as gene-editing technology and the continuous updating of various detection technologies, it is likely that the understanding of the regulation of m6A in reproductive and developmental processes will continue to improve in the years to come.Of particular interest is the role of m


MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research. MicroRNAs (miRNAs) represent a class of short (~22 nucleotides) non-coding RNA molecules that are well conserved across various species. They are known mainly for their post-transcriptional regulation of gene expression via degradation and translational repression . HoweverMiRNA genes are located both within defined transcription units as well as in intergenic regions, where they create clusters transcribed as polycistronic RNA . Their bSince the discovery of the first miRNA lin-4 in nematodes, more than 38,000 miRNAs have been detected in 271 organisms, including 1917 human miRNAs (miRBase v22) . As a siMicroarrays, reverse transcription-quantitative PCR (RT-qPCR) and RNA-sequencing (RNA-seq) are the three most common approaches for miRNA profiling. For a long time, microarray analysis represented the most commonly used high-throughput technology for miRNA profiling . HoweverArabidopsis thaliana [Caenorhabditis elegans [RNA-seq has undoubtedly revolutionized the characterization of the small transcriptome, enabling high-throughput profiling and discovery of novel forms of short non-coding RNAs  . The firthaliana . A year  elegans . Since t elegans . With th elegans .The small RNA-seq workflow involves three main steps: (i) isolation of RNA; (ii) cDNA library construction; and (iii) sequencing . Small RDuring the past years, the major technical development in the small RNA-seq was focused on the reduction of the ligation and PCR bias. At present, there are 15 commercial protocols available, which cover three distinct approaches: (i) original two-adaptor ligation; (ii) improved two-adaptor ligation; and (iii) ligation-free (polyadenylation-based) approach . BesidesThe traditional two-adaptor ligation approach utilizes the ligation of the pre-adenylated 3′-end adaptor followed by the ligation of the 5′-end adaptor . AlthougFirst, the ligation reaction is a major source of bias that negatively affects miRNA quantification in traditional two-adaptor small RNA-seq protocols ,43,44. CSecondly, as the method utilizes T4 RNA ligases that recognize the hydroxyl group on the 3′-end and the phosphate group on the 5′-end of miRNAs, it enables profiling of all RNA species with such modifications. This includes long non-coding RNAs, which need to be removed by bead-based size selection or polyacrylamide gel electrophoresis (PAGE) purification. Other small RNAs with ligase-compatible 5′- and 3′-end modifications and similar lengths, such as piRNAs, are retained in the library and analyzed , whereasThe final problematic aspect of the traditional two-adaptor approach is the formation of adaptor dimers, which may consume a substantial proportion of the sequenced reads when not removed properly. Especially in low input samples, where libraries are prepared using a higher number of PCR cyclers, this may cause complete loss of lowly abundant small RNAs. There are multiple approaches how to remove or limit the formation of adaptor dimers. These include: (i) bead-based or PAGE removal of adaptors from libraries ; (ii) enThe improved two-adaptor ligation approaches aim to prevent or mitigate the ligation or PCR bias. Currently, there are three approaches available: (i) ligation of two adaptors with randomized nucleotides A; (ii) l8), which caused over-deduplication and over-estimation of PCR bias [The superior performance of methods utilizing randomized adaptors over the traditional two-adaptor approach is frequently reported in the literature ,59,60, iPCR bias . TherefoThe ligation of a single 3′-end adaptor and subsequent circularization represents another promising approach that has been recently introduced to mitigate ligation bias . The decThe last group of methods focused on the mitigation of PCR bias. This is achieved via the utilization of UMIs that may be introduced in one or both adaptor sequences, alternatively in the RT primer. There is an ongoing debate on the importance of PCR bias. Where early reports highlighted a minor role of PCR bias ,43,67, rLigation-free methods include protocols based on polyadenylation with template switching  and probHistorically, the polyadenylation method was introduced together with the original two-adaptor ligation approach and contributed to the discovery of various mammalian miRNAs . The firThe application of small RNA-seq methods is especially advantageous where novel forms of small RNA/miRNAs are of interest. However, these methods suffer from protocol-specific biases that do not allow for the direct cross-study comparison and complicate meta-analysis. Moreover, the complex workflow negatively affects their applicability for clinical diagnostics where rapid turnaround time and robust protocols are desirable. To answer these requests, several modern hybridization methods have been recently developed, including Nanostring nCounter, Abcam FirePlex and EdgeSeq from HTG Molecular Diagnostics. Nanostring nCounter utilizes direct molecular barcoding and digital detection of targeted molecules by color-coded probes ,71. UniqOver the past five years, several new approaches for small RNA profiling have been introduced and the majority of them transformed into commercial products . This naWe conducted a thorough literature search and identified twelve comparative studies that compared at least two different approaches for small RNA-analysis. The results are summarized in To sum up, the approach using randomized adaptors seems to be the most robust approach currently available. The status of this well-established method will probably even increase in the future, as some of the research consortia (ERRC) selected its application as a standard for small RNA-Seq studies ,77. For In the majority of comparative studies, none of the protocols outperformed others across all tested metrics. The results rather documented their variable performance and highlighted their different applications in miRNA research. When selecting a small RNA-seq protocol for the new study, the choice of the protocol should be therefore considered with respect to target-, sample-, and protocol-specific features, requirements on the analysis and expected outcomes. Here, we provide a simplified decision diagram for protocol selection  and summ(i) First, the choice of a protocol is given by the molecule of interest. miRNAs are enriched in the majority of ligation-based protocols, whereas other small RNAs along with miRNAs are captured by polyadenylation methods. As polyadenylation suffers from a low mapping rate and inability to distinguish isomiRs, protocol adjustments  might be applied in the ligation-based protocols to capture also other small RNA classes ,80.(ii) Sample input requirement is another protocol-specific aspect for consideration. Although the majority of current methods supports low-input applications, including analysis of liquid biopsies, their performance varies broadly ,75,76,78(iii) After considering target- and sample-related factors, the accuracy of measurement is a key aspect in small RNA-seq analysis. As discussed, ligation and PCR biases might significantly confound the analysis and negatively influence the overall performance. To mitigate the bias while preserving good performance, hybridization-based approaches  or randomized adaptors-based approaches are the methods of choice . On rich(iv) A common goal of small RNA-seq studies, is finding differential abundant miRNAs, the reproducibility of measurement needs to be considered as well. From this perspective, all available small RNA-seq methods demonstrated low within-protocol variability, allowing for precise differential expression analysis ,76. Alth(v) Lastly, the practical aspects of protocol selection are represented by the level of multiplexing, the complexity of library preparation or costs of measurement. The majority of protocols offers 96 indexes allowing the pooling of the same number of samples and their analysis in a single sequencing run. However, some protocols still offer a limiting level of multiplexing (48 or 24), which requests multiple sequencing runs and increases total costs in the case of analysis of large sample cohorts. From the view of the complexity of library preparation, EdgeSeq automated platform stands out over competitors, making the process of library preparation easy, fast and robust. This is however accompanied by expensive instrumentation and a higher cost of reagents.In addition to existing protocols and platforms, there is ongoing research introducing novel improvements to small RNA-seq. When incorporated into current protocols, these advances possess a good promise to further increase the performance and applicability of small RNA-seq analysis. For example, the combination of optimized ligation temperature, the addition of polyethylene glycol (PEG) and 12bp-long randomized adaptors usable as UMIs have been demonstrated to achieve higher performance than commercially available NEXTflex . SimilarHere, we reviewed the current state of small RNA-seq technology for analysis of miRNAs. Undoubtedly, small RNA-seq technology underwent a dramatic development in recent years. The major changes were targeted on the reduction of technical bias and simplification of the analysis workflow. The success rate of these efforts varies which is reflected by the diverse performance of small RNA-seq protocols in comparative studies. Overall, none of the protocols are superior to others and experiment-specific factors need to be carefully considered when selecting a protocol for a new study. To guide the selection, we summarized all major advancements in the field and provided our perspective on their applicability for miRNA profiling. We believe that small RNA-seq holds the promise to become a leading technology for the analysis of miRNAs and once standardized, its applicability will grow even more in popularity.


Our results suggest that poliovirus genome release is a complex process that consists of multiple rate-limiting steps. Interestingly, we found that the addition of exogenous wild-type capsid protein VP4, but not mutant VP4, enhanced the efficiency of genome translocation. These results, together with prior structural analysis, suggest that VP4 interacts with RNA directly and forms a protective, membrane-spanning channel during genome translocation. Furthermore, our data indicate that VP4 dynamically interacts with RNA, rather than forming a static tube for RNA translocation. This study provides new insights into poliovirus genome translocation and offers a cell-free assay that can be utilized broadly to investigate genome release processes in other nonenveloped viruses.Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood. Although cellular assays provide useful information about cell entry and genome release, it is difficult to obtain detailed mechanistic insights due both to the inherent technical difficulties associated with direct visualization of these processes and to the prevalence of nonproductive events in cellular assays performed at a very high multiplicity of infection. To overcome these issues, we developed an  The replication of viruses critically depends on their ability to deliver their genomes into host cells . In cont2–5–8–Enterovirus of the family Picornaviridae. The virus consists of a positive-sense single-stranded RNA genome  encapsulated by an icosahedral capsid that contains 60 copies of the four capsid proteins  (1517Poliovirus (PV) is an ideal model to study genome translocation in nonenveloped viruses because it is biochemically, genetically and structurally well characterized and it is closely related to a number of other important viral pathogens, including coxsackievirus A16 (CAV16) and enterovirus 71 (EV71) . PV, theand VP4) , 12. Poland VP4) . The binand VP4) , 15. Thid VP4) 1517. TheseVP4) 151719. Live-VP4) 1517.Biochemical and genetic characterizations, together with cryo-electron microscopy (cryo-EM) structures of key poliovirus entry intermediates, provide strong evidence that externalized peptides form membrane-spanning channels to transport the RNA genome into the cytoplasm , 21–27. 21–A thorough understanding of the mechanism of infection requires a detailed characterization of the behavior of individual virus particles that are involved in genome translocation. Previous cellular assays were able to provide insights into the endocytic pathways used for internalization, and the location and average timing of genome release . HoweverCryo-electron tomographic (cryo-ET) reconstructions of the model virus-membrane complexes, produced by elevating the temperature of virus-receptor-liposome complexes to 37°C, were previously obtained by averaging subtomograms of individual complexes . RemarkaTo probe the mechanism of RNA translocation across a membrane, we have developed a liposome-based fluorescence assay by capturing YoPro-1 (a nucleic acid-binding dye whose fluorescence increases upon binding RNA) in the lumens of RDLs . Our iniin vitro. However, the addition of exogenous N-terminally myristoylated VP4 enhanced the efficiency and extent of this process, which further supports the idea that VP4 plays a vital role in genome translocation. Together, the biochemical and structural results suggest that VP4 promotes genome translocation by interacting with RNA to form a protective connection between the virus surface and the membrane, forming a membrane-spanning channel, and perhaps by facilitating the unfolding of the secondary structure of the RNA. The ability of exogenously added VP4 to facilitate RNA translocation together with the high particle-to-PFU ratio of poliovirus and other enteroviruses further suggests that VP4 from multiple virions may participate in productive RNA release and raises the exciting possibility that this process may be dynamic, with VP4 repeatedly attaching to, releasing, and reattaching to the viral RNA in transit, rather than forming a static tube that the RNA slides through. Finally, we believe that our newly developed ability to obtain that kind of mechanistic detail, using a tethered-liposome model, represents a valuable technological advance that will be broadly applicable to other nonenveloped viruses.In the present study, we characterized the mechanism of viral genome translocation across a membrane by monitoring the genome release of individual viruses in real time using surface-tethered RDLs that had bound exactly one virus per liposome. These single-particle imaging results suggest that genome release is a complex process that may include multiple rate-limiting steps. We found that the efficiency of genome translocation across the model membrane was comparatively low at the single-particle level RDLs were constructed using a mixture of phospholipids and cholesterol to mimic the biological membranes in cells. The ectodomain of the poliovirus receptor (PVR) containing a C-terminal six-histidine tag was anchored to the surface of the liposome through a small fraction of lipids containing nickel-nitrilotriacetic acid (Ni-NTA) head groups as previously described . To deteWe confirmed that PV is able to inject its genome into these RDLs using a bulk fluorescence assay . The chaTo reveal the mechanism of viral genome translocation across a membrane, it is essential to monitor the genome translocation process in real time using one virus per liposome. As the first step to achieve this goal, purified poliovirus was fluorescently labeled with amine-reactive Cy3 fluorophore  to visuaIn order to monitor individual viral RNA translocation events in real time using time-lapse imaging, TOTO-3-containing RDLs were immobilized on a streptavidin-coated slide via biotin-streptavidin linkages . After w10.1128/mBio.03695-20.1FIG S1FIG S1, JPG file, 0.2 MB.Receptor-decorated liposomes entrap TOTO-3 for single-particle imaging of RNA translocation. (A) Fluorescence images of TOTO-3  containing liposomes labeled with a small fraction of lipids containing rhodamine  and merged image (right). Bars, 5 μm. (B) Average fold change in intensity of TOTO-3 containing RDLs observed with time in the absence of poliovirus at 37°C. Shading indicates the standard deviations of the data points. (C) Distribution of fold change in intensity of liposomes observed after 10 min. The error bar indicates the standard deviation. Download Copyright © 2021 Karunatilaka et al.2021Karunatilaka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To assess the viral genome translocation in a population of virus particles, intensity trajectories obtained from 130 PV-RDL complexes, each containing one PV per liposome, were synchronized as shown in the heat map of −1 at 37°C.Mechanistic insights into genome release can be obtained by analyzing the genome translocation events that are measured from individual virus particles. Analysis of 130 translocation events showed a broad distribution of translocation times, with an average of 3.7 ± 0.1 min at 37°C . To extrTo further characterize the genome translocation process, we measured the lag times that preceded the observed genome translocation , L. If vin vivo  Wild-type VP4-N45. Since the full-length VP4 was poorly soluble, we tested the effect of adding exogenous VP4 on RNA translocation using a myristoylated synthetic peptide that corresponded to the N-terminal 45 amino acids of VP4 (VP4-N45). The first 45 amino acids of VP4 form an intricate stable-looking pentameric complex on the inner surface of mature 160S poliovirus and other related viruses, including rhinoviruses  absence or (B) presence of exogenous wild-type VP4-N45 peptide under physiological conditions. Download Copyright © 2021 Karunatilaka et al.2021Karunatilaka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03695-20.3FIG S3n = 200). Shading indicates the standard deviations of the data points. (B) Distribution of maximum fold change in intensities of individual liposomes observed after 10 min in the presence of exogenous VP4-N45 with  Average fold change in intensity of TOTO-3-containing receptor-decorated liposomes with time in the presence of VP4-N45 without poliovirus at 37°C . Note, however (−1 (−1) (P = 0.133).Not only did exogenous VP4-N45 increase the amount of RNA delivered into the liposome, it also increased the translocation time. Specifically, the addition of wild-type VP4 45-mer peptide to PV-RDLs increased the average translocation time from 3.7 ± 0.1 min to 6.8 ± 0.2 min  . The dis(−1 (−1) . Interes(ii) T28G mutant VP4-N45. To test the specificity of the observed effect of VP4 on viral RNA translocation, we characterized RNA release in the presence of the T28G mutant VP4 N-terminal 45-mer (VP4-T28G), which substitutes glycine for threonine-28 of VP4. Previous studies have shown that while the T28G mutant is lethal, its virions can be produced by transfection with viral RNA that contains the mutation. The mutant virions bind to and are internalized into cells, but the internalized virions fail to release their genomes into the cytoplasm  or with exogenous wild-type VP4 peptide added . Although PV-RDLs in the presence of exogenous VP4-T28G showed final intensities that were similar to those seen using PV-RDL alone, mutant VP4-T28G had a distinctly negative effect on the rate and extent of intensity increase  needs further investigation.Despite the fact that PV-RNA release is highly efficient in live cells , and in in vitro  Electron micrograph showing that VP4 45-mer peptide contains a mixture of oligomers and fibrous aggregates. (B) Even in the absence of RNA, VP4-N45 forms stacked, stick-like structures at 37°C that can be visualized after staining with uranyl acetate by transmission electron microscopy. (C) Electron micrograph of polio-on-a-stick (POAS) structures. The poliovirus sample was heated at 50°C for 10 min and visualized after staining with uranyl acetate by electron microscopy. (D) POAS nucleoprotein fibers and attached virus particles remained intact, even after incubation for 1 h at 37°C in the presence of RNase A. (E) Electron micrograph of POAS nucleoprotein fibers and virus particles labeled with an antibody against VP4 protein. Gold-conjugated secondary antibodies are only evident where the virus is associated with a filamentous structure indicating that VP4 is one of the main components in POAS structures. (F) Electron micrograph showing stacked, stick-like structures in POAS fibers. Yellow arrows indicate the positions of stacked structures. Download Copyright © 2021 Karunatilaka et al.2021Karunatilaka et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro, in the absence of specific cellular factors or membrane components (which future experiments may identify). Future research is also needed to clarify the roles of both the N-terminal and C-terminal portions of VP4 and the effect of the T28G mutation on umbilicus formation and RNA translocation.Since the number of RNA nucleotides to be translocated during genome release vastly exceeds the limited VP4 supply, it requires either that RNA be threaded through a channel formed by VP4 and shift along the channel or that VP4 repeatedly dissociate from RNA at (or beyond) the membrane and then recycle, reattaching to newly emerging RNA close to the virus. The observed PV-RNA translocation time courses were indeed affected by the presence of exogenous VP4 45-mer  and 4F, In summary, single-particle imaging of virus-bound receptor-decorated liposomes provides a useful cell-free system to investigate viral genome translocation. Based on our single-particle imaging studies, we propose that PV genome translocation is a complex, multistep process that involves an initial translocation burst with a series of RNA unwinding and propagation steps. Our real-time imaging results together with previous structural and genetic analysis support a model in which VP4 promotes RNA translocation by providing a protective channel during genome transfer. Besides the protection from RNases, interactions between the RNA genome and VP4 might also help maintain a single-stranded RNA structure for efficient translocation.Poliovirus serotype I (Mahoney strain) was grown in suspension HeLa cells and purified as previously described . BrieflyN-terminally myristoylated wild-type (VP4-N45) and T28G mutant (VP4-T28G) VP4 N-terminal 45-mer peptides were synthesized with >95% purity by GenScript. To improve the solubility of both peptides, six lysines were added at the C terminus of the VP4 45-mer . The pepUnlabeled and fluorophore-labeled PV titers were determined using plaque assays with adherent HeLa (S3) or Vero cells. Serial 10-fold dilutions of PV samples were added to cell monolayers in 6-well plates and incubated for 45 min at 37°C with rocking every 10 min. Growth medium containing 0.6% Noble agar (Sigma) was added to infected monolayers, and plates were kept for 30 min at room temperature. After incubation of plates for ∼48 h at 37°C, cells were stained with 0.2% crystal violet, and plaques were counted. Infectivity of fluorophore-labeled PV was expressed as a percentage of the control (unlabeled PV).sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA-Ni) and 1% (wt/wt) ammonium salt of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG 2000 biotin) (Avanti Polar Lipids) were added to the lipid mix for binding of the His-tagged receptor to the liposomes and for surface immobilizing the RDLs for single-particle imaging experiments, respectively. Chloroform in the mixture was evaporated with a stream of argon to produce a thin lipid film, which was dried further under vacuum for >4 h. Dried lipid film was rehydrated to a final lipid concentration of 4 mg/ml using rehydration buffer  containing 30 μM RNA binding dye TOTO-3 (Life Technology). TOTO-3 (excitation/emission wavelengths = 642/660 nm) was selected to monitor the viral RNA translocation for single-particle experiments with Cy3-labeled poliovirus, since it is a bright, far-red fluorescent dye that can be easily distinguished from Cy3 (excitation/emission wavelengths = 550/570 nm), and it is among the highest-sensitivity probes for nucleic acid detection. Liposomes were obtained by extruding the lipid mixture through a polycarbonate membrane with a 0.6- to 0.8-μm pore size (Avanti Polar Lipids).Liposomes were prepared by mixing phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, cholesterol, and phosphatidic acid in chloroform (Avanti Polar Lipids) in a molar ratio of 1:1:1:1.5:0.3, respectively. Final concentrations of 8% (wt/wt) nickel salt of 1,2-dioleoyl-The soluble ectodomain  of PVR with a six-histidine tag at the C terminus (sPVRHis) was a gift from the L. Shapiro lab . RDLs were made by incubating DGS-NTA-Ni-containing liposomes (∼4 mg/ml) with sPVRHis (0.5 mg/ml) at a 1:10 ratio (vol/vol) for 15 min at room temperature.20-Cy3 3′], which is complementary to the 3′ poly(A) tail of viral RNA, was used to immobilize the PV-RNA on a slide for imaging experiments. The PV-RNA–oligo(dT)-Cy3 complex was formed by heat-annealing a 2:1 mixture of PV-RNA and oligo(dT)-Cy3 at 85°C for 2 to 3 min and cooled for 15 to 20 min at room temperature. Preformed complex was added onto a streptavidin-coated slide, incubated for 15 min at room temperature, and washed with rehydration buffer to remove the unbound complexes. Excess TOTO-3 (30 μM) was added to the chamber and incubated in dark for 15 min at room temperature. Finally, the excess dye was removed by washing the slide with rehydration buffer containing an oxygen scavenger system , and the binding of TOTO-3 on full-length viral RNA was monitored by imaging the slide with both 561- and 642-nm lasers using a fluorescence microscope.Viral RNA was extracted and purified from the poliovirus solution using a QIAamp viral RNA minikit (Qiagen) according to the manufacturer’s instructions (without adding carrier RNA). Fluorophore-labeled oligo(dT) containing biotin at the 5′ end [5′ biotin-(dT)VP4 45-mer peptide was incubated for 15 to 30 min at 37°C in buffer containing 50 mM HEPES (pH 7.3) and 50 mM NaCl, adsorbed onto glow-discharged grids, and stained with 1% uranyl acetate to visualize the mixture of oligomers and fibrous aggregates. Polio-on-a-stick (POAS) structures were formed by heating the purified poliovirus sample at 50°C for 10 min and cooling on ice. POAS nucleoprotein fibers and virus particles were visualized by electron microscopy after adsorbing the diluted sample onto glow-discharged grids and stained with 1% uranyl acetate. The RNase-resistant nature of POAS fibers was determined after incubating the heated PV sample for 1 h at 37°C in the presence of RNase A, as previously described , 40. TheTo monitor the viral RNA translocation into the lumen of the TOTO-3 containing liposomes in bulk, a liposome-sPVRHis-PV (PV-RDL) complex was formed by mixing 4 μl of RDLs (4 mg/ml) and 2 μl of PV (0.5 mg/ml) in the presence of RNase A (50 μg/ml) in rehydration buffer. The reaction mixture (50 μl) was excited at 642 nm, and emission was recorded at 660 nm with a 5-nm slit width on a Varian Cary Eclipse fluorescence spectrophotometer under different reaction conditions, as mentioned above.For single-particle imaging, TOTO-3-containing biotinylated RDLs (30 μl of 20 μg/ml) were added to a streptavidin-coated Ibidi μ-slide channel  and incubated for 15 min at room temperature to immobilize liposomes on a surface via a biotin-streptavidin linkage. After removing the unbound RDLs by washing with rehydration buffer, Cy3-labeled PV (36 ng) was added to the slide and incubated for 20 min at room temperature with rocking every 5 to 10 min. Unattached PV-Cy3 was removed by washing the channel with rehydration buffer containing an OSS  to reduce photobleaching of fluorophores. Single-particle experiments with exogenous VP4 were performed by adding 1 μg of wild-type (VP4-N45) or mutant (VP4-T28G) VP4 N-terminal 45-mer peptide (GenScript) per channel for a final concentration of ∼3.4 μM. This represents an ∼650-fold molar excess relative to the total amount of VP4 in the virions added to the system prior to the washing step before peptide is added. However, this ignores the fact that the VP4 from virions is tethered and therefore present at very high local concentrations, whereas the exogenously added VP4 is freely diffusing. If we attempt to correct for the difference between constrained and freely diffusing contributions by assuming that all of the exogenously added VP4 within a sphere with twice the diameter of the average liposome is accessible to facilitate RNA translocation, this would correspond to 30× more added VP4 in the vicinity of a virus-liposome complex than that contributed by the individual virion in the complex. Note that both of these estimates are subject to assumptions that make their meaning questionable and that they are intended only to provide very rough estimates. Note also that because the added VP4 is a mix of fibers and smaller aggregates and it is not clear that all of the aggregates can participate in genome translocation, these are likely to represent overestimates of the fold excess.Viral RNA translocation across the liposome membrane was monitored by time-lapse imaging of TOTO-3 encapsulated liposomes on a Nikon Ti motorized inverted microscope with a Perfect Focus System using a 1.49-numerical aperture (NA), 100× oil immersion Apo TIRF objective . The binding of PV-Cy3 on RDLs and binding of TOTO-3 on translocating RNA were monitored after exciting fluorophores with AOTF (acousto-optic tunable filter)-controlled 561-nm and 642-nm lasers , respectively. Fluorescence intensities were detected on a 512- by 512-pixel Hamamatsu ImagEM electron-multiplying charge-coupled device (EM-CCD) camera at 12 frames per min for 30 min with a 200-ms exposure time using appropriate filters . All imaging experiments were performed at 37°C using a microscope incubation enclosure (Okolab).Single-particle movies were captured using Metamorph software, which saved individual frames as TIFF images. These TIFF images were imported into ImageJ and MATLAB for data analysis. First, movies were stabilized in ImageJ using the “align slices in stack” function. Next, the locations of the vesicles were identified from the red channel using a standard MATLAB spot finder tool. Aggregates of vesicles and vesicles that appeared to be smaller than two pixels in diameter were omitted from further analysis. The sums of the intensities of the pixels covering the area of each vesicle were calculated for each frame to generate a trace for each vesicle. These traces were imported into a custom MATLAB GUI, which used a hidden Markov model to determine when the viral genomic RNA translocated into the vesicle based on an increase in intensity in the red channel. The start and end times of the translocation were calculated by fitting the traces to a sigmoidal function (a logistic function in MATLAB). The start times for each trace were synchronized computationally, and the intensity values were binned as two-dimensional (2D) matrices. The matrices of intensity values were represented as colors to generate the synchronized heat maps. Translocation rates of individual virus particles were obtained from linear fitting the slope of the intensity trajectories. Histograms and their fits were prepared in MATLAB using standard built-in functions. Average translocation times and lag times were calculated by fitting the histograms with gamma distribution functions, and the error in the mean was obtained by bootstrapping.The percentage of virus particles showing genome translocation was calculated by dividing the number of PV-RDL complexes showing an increase in fluorescence by the total number of PV-RDL complexes containing one virus per liposome under a given condition and multiplying the quotient by 100.


Comprehensive molecular and cytogenetic profiling of acute lymphoblastic leukemia (ALL) is important and critical to the current standard of care for patients with B‐acute lymphoblastic leukemia (B‐ALL). Here we propose a rapid process for detecting gene fusions whereby FusionPlex RNA next‐generation sequencing (NGS) and DNA chromosome genomic array testing (CGAT) are combined for a more efficient approach in the management of patients with B‐ALL.We performed RNA NGS and CGAT on 28 B‐ALL samples and, in four patients, compared fixed cell pellets to paired cryo‐preserved samples as a starting material to further assess the utility of cytogenetic fixed pellets for gene expression analysis.Among the fixed specimens, when using alternative techniques as references, including karyotype, fluorescence in situ hybridization, CGAT, and RT‐qPCR, fusions were detected by RNA NGS with 100% sensitivity and specificity. In the four paired fixed versus fresh cryopreserved samples, fusions were also 100% concordant. Four of the 28 patients showed mutations that were detected by RNA sequencing and three of four of these mutations had well‐known drug resistance implications.We conclude that FusionPlex is a robust and reliable anchored multiplex RNA sequencing platform for use in the detection of fusions in both fresh cryopreserved and cytogenetic fixed pellets. Gene expression data could only be obtained from fresh samples and although limited variant data are available, critical hotspot variants can be determined in conjunction with the fusions. This study describes a novel RNA next‐generation sequencing assay used in tandem with chromosomal genomic array testing upon fixed cell pellets typically used for cytogenetic studies to detect the molecular profiles of B‐acute lymphoblastic leukemia. Although B‐ALL is more common in children (~80%), pediatric patients with B‐ALL have a very high remission rate of around 98%. B‐ALL in adults, however, carries a much worse prognosis and most deaths from ALL are seen in adult patients. Comprehensive molecular and cytogenetic profiling of ALL is critical to the current standard of care for both adult and pediatric ALL, as these genetic aberrations drive prognostication, risk‐stratification, treatment options and can identify markers used for tracking the minimal residual disease.CRLF2 rearrangements and inactivating mutations of the JAK/STAT  or RAS signaling pathways . Recurrent gene fusions are seen in 93% of CRLF2‐overexpressed Ph‐like ALL patients,IKZF1 alterations (including both point mutations and deleterious deletions) is the most common mutation seen in Ph‐like ALL, with other cases showing mutations and/or copy number alterations (CNA) in ETV6, EBF1, PAX5, TCF3, ERG, RAG1/2, BLNK, BCL11A, IKZF2, LEF1, MEF2C, SOX4, and SPI1TP53, RB1, and IKZF2. Near haploid ALL displays a high frequency of IKZF3 mutations and alterations targeting the RTK and RAS signaling pathways, as well as deletions of a histone cluster at chromosome 6p22.Classification of ALL starting with the 2008 World Health Organization (WHO) was based on recurrent genetic abnormalities,As the relevant genetic diagnostics needed for the risk assessment of ALL have broadened to include cytogenetics, gene fusions, mutations, and gene expression, the challenge to rapidly perform these diagnostics has become apparent. A comprehensive testing algorithm is difficult to develop and commonly requires multiple high complexity testing strategies with long turnaround times. Here we propose a rapid process by which anchored multiplex fusion detection by RNA NGS and CGAT can be combined for a more efficient approach in the management of patients with B‐ALL.22.1Acute lymphoblastic leukemia samples for which CGAT was performed between 2011 and 2017 in our institution were identified for the current study. Those with at least two vials of fixed cells as a leftover from clinical testing were used. RNA was extracted using ReliaPrepTM RNA Cell Miniprep System (Promega) from 32 specimens representing 28 ALL samples . Hundred nanograms of RNA were used for each of the 32 RNA samples from 28 patients. RNA quality was assessed with PreSeq RNA QC Assay following procedures recommended by ArcherDX (CO). Briefly, complementary DNA (cDNA) was diluted 1:10 and amplified with iTaq SYBR Green Supermix (Bio‐Rad Laboratories) and VCP primer mix (ArcherDX). The reaction assesses the amount of RNA fragments greater than 100bp and the cycle threshold (Ct) values are intended to be tracked over time for each lab to establish rejection criteria regarding RNA quality.Complementary DNA quality was assessed with a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and visualized on a 1% agarose gel with ethidium bromide to detect/exclude degradation. The criteria for acceptable cDNA quality included visible bands by 1% agarose gel and spectrophotometer reading by A260 nm/A280 nm ratio of 1.4 to 2.2.2BCR‐ABL1, KMT2A (MLL), and IgH. Genes where FISH probes were not available to include IKZF1, STIL, TAL1, P2RY8, and CRLF2.Marrow aspirate or peripheral blood samples from patients were tested for cytogenetic abnormalities using clinically validated protocols at Seattle Cancer Care Alliance (SCCA) for standard culturing, G‐banding karyotyping, and FISH. Karyotype designation was based on the International System for Human Cytogenetic Nomenclature (ISCN 2016).2.3P2RY8‐CRLF2 fusion RNA assay were reverse transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher) and PCR amplified using primers targeting P2RY8 (NM_178129.4 Exon 1: 5′‐TTAAGCGTTGCATCCTGTTA‐3′) and CRLF2 (NM_022148.3 Exon 2: 5′‐TCAGGTTGGTCCTGGAGTAT‐3′). The PCR product was then Sanger sequenced at the Genomics core of Fred Hutchinson Cancer Research Center.The reverse‐transcription polymerase chain reaction (RT‐PCR) and Sanger sequencing validation for the 2.4Libraries were prepared using the FusionPlex ALL kit (ArcherDX) targeting 44 fusion genes with target regions detailed in Supporting Information 2.5Chromosome genomic array testing was performed for the detection of DNA CNA or copy‐neutral loss of heterozygosity (cnLOH) by SNP genotyping along with non‐SNP copy number probes using CytoScan HD (Affymetrix) according to the manufacturer's protocol. In general, CGAT was performed only on specimens with 20% or more tumor cells. Specimens with low tumor content  were flow‐sorted to enrich the abnormal lymphoblast population to greater than 50% prior to CGAT. The size filter for an abnormal call was 100 kb for CNA and 10 Mb  for cnLOH.33.1The characterizations of patients and samples are shown in Table 3.2Comparison of the different diagnostic platforms for the range of tumor burdens where abnormal results could be detected was reviewed for the limit of detection Figure . The tum3.3BCR‐ABL1 (n = 7), IKZF1 Δ4‐7 (n = 9), KMT2A‐AFF1 , EBF1‐PDGFRB, P2RY8‐CRLF2, and STIL‐TAL1 (n = 1 each for the last three). Among all samples, CGAT results were available in all, karyotype data in 26 samples, and FISH data in 22 samples. One patient had neither karyotype nor FISH data available for review. Based on the comparison between fusion calls by NGS and the existing clinical techniques in our lab, we present the following data groups:BCR‐ABL1 (n = 7), KMT2A‐AFF1 , and EBF1‐PDGFRB (n = 1). . The STIL‐TAL1 fusion in Patient 13 was minimally visible by CGAT , including BCR‐ABL1 (n = 7 by karyotype and/or FISH), KMT2A , EBF1‐PDGFRB (n = 1 by CGAT and FISH), P2RY8‐CRLF2 (n = 1 by RT‐qPCR), and STIL‐TAL1 (n = 1 by CGAT), corresponding fusion transcripts were successfully identified by RNA NGS. Among all the samples where no gene fusion was indicated, none had fusion transcripts reportable by RNA NGS. Hence, RNA NGS detected gene fusions with 100% sensitivity and specificity. These calculations exclude data from the IKZF1 oncogenic isoform because CGAT does not distinguish between deletions that lead to oncogenic isoform versus haploinsufficiency and is, therefore, not an appropriate reference for RNA NGS assay performance evaluation in this regard.Taken together, among all samples where an alternative testing method revealed gene fusions  was increased in three of the four fresh cases used within the RNA expression assay. Also, hotspot mutations were identified in 26/28 cases with the earlier‐described filtration criteria. However, the FusionPlex Heme v2 RNA sequencing assay was described by the manufacturer to reliably detect mutations in RNA at the hotspots outlined in Supporting Information JAK2, ABL1, and NOTCH1.The expression of Ph‐like genes  in the workup of ALL, providing additional information that aids the risk stratification and understanding of ALL pathobiology. The anchored multiplex technology allowed for the detection of novel fusions, hotspot mutations, and expression changes which indicated potential Ph‐like ALL. RNA sequencing with FusionPlex has a library preparation time of 28 hours , a software analysis time of <8 h per run, and reporting potentially performed in 1 day. Therefore, a total projected turnaround time of 5–7 working days to obtain fusion gene results and some common mutations validated on the platform, whereas karyotype analysis typically takes 5–10 working days . However, further advantages afforded by implementing RNA sequencing with FusionPlex chemistry include the ability to identify novel fusions without the need for a priori knowledge of fusion genes and their partners.IKZF1 oncogenic isoform in Patient 15, which had merely 2.6% abnormal blasts by flow cytometry at clinical evaluation. Nevertheless, the difference between the gene expression profile of the cryo‐preserved and the paired methanol/acetic acid‐based fixed pellets is seen. We recommend that cytogenetically fixed pellets shall not be used for expression analysis.The feasibility of using fixed cytogenetic cell pellets in an anchored multiplex PCR NGS assay is novel. Our success in utilizing methanol/acetic acid preserved specimens for RNA‐NGS fusion detection enables the use of archived cytogenetic cell pellets when fresh/frozen tissue is not available. Even though an acidic environment has been described previously to cause RNA degradation,KMT2A (MLL) rearrangements; (b) RNA‐NGS allows for the detection of cryptic rearrangements or fusion genes which are in close proximity to each other thus rendering the inability to visualize by karyotype, such as STIL‐TAL1 and P2RY8‐CRLF2; (c) IKZF1 oncogenic isoforms were discordantly detected by RNA‐NGS versus CGAT. RNA‐NGS and CGAT are complementary in detecting IKZF1 deletion, such that RNA‐NGS is more sensitive in detecting deletions that lead to oncogenic isoforms, namely IKZF1 Δ4‐7, while CGAT had missed several cases in our study with relatively low tumor content. Conversely, CGAT can detect deletions that lead to haploinsufficiency, but NGS cannot detect haploinsufficiency.Ribonucleic acid (RNA) fusion NGS is complementary with current methods of genetic profiling in the workup of ALL. A significant subset of samples was discordant (8/28 patients) between RNA‐NGS and our current clinical testing techniques such as karyotype, FISH, and CGAT. In studying the discordancy, we revealed several advantages of RNA‐NGS: (a) The anchored multiplex PCR enables the detection of fusions involving promiscuous genes that tend to have multiple known or novel partners as exemplified via CRLF2, IKZF1, RUNX1) was increased in three out of four fresh cases that were tested, indicating the possibility that gene expression signatures can be confirmed within Ph‐like ALL. However, a much more extensive validation with a larger series of fresh tissues would be required to confirm the utility of expression profiling. Patients 7 and 14 (leftmost and rightmost, respectively in Figure BCR‐ABL1 fusion, carry an oncogenic IKZF1 deletion (Table CRLF2 despite the absence of fusions or point mutations in those genes. This is consistent with prior research delineating IKZF1’s role as a repressor for CRLF2 expression.CRLF2‐overexpressed patients whose overexpression arises from other mechanisms of gene regulation,The utility of expression and variant data from RNA fusion NGS may be useful but requires more extensive validation prior to clinical implementation. To our knowledge, our study is among the first clinical implementations of an RNA‐based NGS assay that uses fixed cytogenetic preparations as its starting material. This is also the first study that was able to reliably detect fusion gene RNA transcripts by NGS on archival cytogenetic material. The RNA expression and variant data could support the identification of Ph‐like B‐ALL's diverse genetic signature. The expression of Ph‐like genes (such as Analysis of variant data from fusion NGS proved to be challenging due to calls being based on RNA. Inherent errors are a known problem with variant calls based on RNA sequencing,BCR‐ABL1, we recommend concurrent karyotype, CGAT, and targeted RNA sequencing studies to identify key diagnostic and prognostic markers as well as aberrations that guide therapy. Furthermore, this targeted RNA sequencing platform tested in this study is a good technology for detecting novel fusions and isogenic isoforms. While SNV can be detected, another more rigorous validation for specific variant analysis is recommended to detect specific RNA reads and the significance of these variants.We conclude that anchored multiplex PCR RNA sequencing is a good technology for detecting novel oncogenic fusions and isogenic isoforms. Our study demonstrated that, among karyotype, CGAT and targeted RNA sequencing, the abnormal result of one test does not exclude the necessity of the other tests but rather augment the diagnostic workup as a whole. Therefore, for clinical diagnosis of ALL patients, in addition to standard single‐target tests such as RT‐PCR for The Institutional Review Board at the Fred Hutchinson Cancer Research Center approved this study.Written/signed consent in the form of the IRB‐approved consent documents 999.209.4 Consent and Authorization for the Use of Medical Information in Research and 999.209.5 Consent and Authorization for the Use of Leftover Specimens for Research was obtained for all patients in this study.Inception of idea and funding support—Min Fang, Cecilia Yeung, Jerry Radich; Data collection and analysis—Cecilia Yeung, Xiaoyu Qu, Olga Sala‐Torra, David Woolston, Min Fang; Statistical analysis—Xiaoyu Qu; Drafting the manuscript—Cecilia Yeung, Xiaoyu Qu, Olga Sala‐Torra, Min Fang; Editing and revising of the manuscript: Cecilia Yeung, Xiaoyu Qu, Olga Sala‐Torra, David Woolston, Jerry Radich, Min Fang.The authors disclose no conflict of interest.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Some of the amazing contributions brought to the scientific community by the Protein Data Bank (PDB) are described. The focus is on nucleic acid structures with a bias toward RNA. The evolution and key roles in science of the PDB and other structural databases for nucleic acids illustrate how small initial ideas can become huge and indispensable resources with the unflinching willingness of scientists to cooperate globally. The progress in the understanding of the molecular interactions driving RNA architectures followed the rapid increase in RNA structures in the PDB. That increase was consecutive to improvements in chemical synthesis and purification of RNA molecules, as well as in biophysical methods for structure determination and computer technology. The RNA modeling efforts from the early beginnings are also described together with their links to the state of structural knowledge and technological development. Structures of RNA and of its assemblies are physical objects, which, together with genomic data, allow us to integrate present-day biological functions and the historical evolution in all living species on earth. Protein Data Bank (PDB) is an icon for structural biologists. In its virtual vaults, the PDB stores the structures of biological macromolecules obtained painstakingly by thousands of crystallographers, electron microscopists, and nuclear magnetic resonance spectroscopists over the last 50 years or so. This note is primarily written to honor and acknowledge those who set up the PDB and who have strived throughout all these years to improve and maintain the database. We thank the Journal of Biological Chemistry for organizing the publication of such pieces that provide a mixture of personal recollections, historical records about nucleic acids structure, and some general comments about databases and their future.The Cambridge Structural Database, CSD . PDB originally served as a repository system where crystallographers could mail in their data on punch cards, then computer tapes, for archiving and distribution. The announcement of the founding of the PDB in Nature New Biology was a simple little insert of 256 words . Only then could one deduce or check atomic distances, angles, stereochemistry, contacts and visualize the 3D structure using programs developed in-house. This was a tedious job, but very instructive to students and postdoctoral fellows. In those days, atomic coordinates were part of the paper and presented with temperature factors in tables . For example, can one really be confident in RNA structures at resolutions above 3.3 Å with average B-factors around 200 Årategies . In thisrategies , 23, 24.rategies .Escherichia coli or yeast . The ch78, 4TNA , 38. In 78, 4TNA  and 2TMV78, 4TNA ), contaiFollowing novelty and progress in chemical synthesis and purification of RNA oligonucleotides , the firPDB amounted to only about 1% of the present content of total RNA and RNA–protein complex (RNP) structures. By 1995, the number of X-ray RNA structures  in the Tetrahymena group I intron, a structure twice the size of tRNA, featuring striking compact folds and novel types of RNA tertiary contacts . And, after the Nobel Prize in Physiology or Medicine for RNA interference (RNAi) in 2006 to Andrew Fire and Craig Mello, the RNA community is celebrating the 2020 Nobel Prize in Chemistry awarded to Jennifer Doudna and Emmanuelle Charpentier for the structure-based design of the splendidly efficient RNA-programmed Crispr-cas9 system , short-range and long-range interactions (C), the central roles of non-Watson–Crick base pairs in mediating tertiary contacts (A and B), structured hairpin loops (with the famous U-turn) and their propensities to intimately interact, and base–base intercalative stacking interactions (C) that contribute to long-range loop–loop interactions.The accumulation of RNA structures, each one bringing either a new insight key to folding or additional confirmation and sequence variants, led to deep understanding of the main physicochemical features underlying RNA architecture. Some of these features, gained from structures of ractions C, the cecontacts , base–phcontacts , A and Bractions C that coTetrahymena thermophila . Inrmophila , unveileThe quote from Wainwright  capturesOne should always keep in mind the famous quote by T. Dobzhansky: “Nothing in Biology Makes Sense except in the Light of Evolution” . The acci.e., the ensemble of base-paired RNA helices) of a set of homologous RNA molecules. The third kingdom of life, the Archaea, were discovered by Carl Woese by sequencing ribosomal RNA and aligning them  recently launched the Biological Structure Model Archive (BSM-Arc)  .A–C). Interestingly, contacts between the D- and T-loops are proposed as well as base pairs between residues 8 and 14 as well as 15 and 48 , but both in the usual Watson–Crick configurations instead of the observed trans configuration (D and E). Sequence analysis coupled with model building led to the suggestion that some RNAs can form pseudoknots, for example, between a hairpin loop and a single strand (meaning that the single strand does not go through the loop) (https://www.rnasociety.org/), is present in a large number of functional structured RNAs. In 1987, Kim and Cech  . This foand Cech  publisheand Cech , 146 somand Cech . Afterwaand Cech .Figure 7ab initio modeling, the molecular objects are built without the constraints of crystallographic data. The model building can be driven by geometric and physicochemical energetic terms in (semi-) automatic fashion by various computing tools . SRNA-Puzzles computational contests were created in 2011  has been doing for protein modeling (https://github.com/RNA-Puzzles) . Toward in 2011 , 158. Thmodeling . All thePuzzles) .Research Collaboratory for Structural Bioinformatics (RCSB) in the United States, the Macromolecular Structure Database (MSD) at EBI, and the PDBj in Osaka, created the worldwide PDB  with the goal of maintaining a single archive of structure data of macromolecules that is freely and publicly available to the global community  aims “to stabilize and ensure sustainable financial support for the global biodata infrastructure and in particular to identify for prioritized long-term support a set of Global Core Data Resources that are crucial for sustaining the broader biodata infrastructure.”First, the sustainability and maintenance of databases for the use of a now internationalized community of scientists raise the issue of how the associated costs shall be equitably apportioned; these are recurrent costs to support trained and competent personnel, together with the maintenance and constant upgrading of the extensive infrastructure required to keep up with the growth in the size and complexity of the data. Short-term grants with a dedicated focus are not adapted to maintain large international resources . New busin situ, will open up visualization of macromolecules in their cellular environment. Maybe one day we will also include digital descriptions of phenotypes, including bones, skeletons, or wings in order to relate their forms and colors to the underlying genes and genetic networks. Launched by the RNAcentral Consortium in 2014 at the EMBL-EBI, Wellcome Genome Campus , RNAcentral  integrates and unifies access to all types of noncoding RNA sequences from all organisms. This is a major enterprise that goes in the right direction for the integration and interoperability of data. A bridge between sequences and standard secondary structures has been achieved recently (And, secondly, databases fundamentally comprise well-organized archives of chosen sets of objects. In structural biology, the objects are mainly molecular sequences and structures. However, as these two types of data attach to the same natural entities, they need to be integrated to grasp biological function and evolution more holistically. New developments in cryo-electron tomography , which arecently . In the It would take a whole book with several dozens of figures to convey the amazing knowledge accumulated on RNA structures and architectures since the beginning of RNA structural biology and the advent of the PDB. Only glimpses of RNA structural biology are presented here, and we would like to apologize for not mentioning the contributions of many structural biologists, especially those using NMR spectroscopy, a field that contributed several seminal structures, for example, on RNPs and telomerase , 172, wiTechnological improvements and breakthroughs are at the heart of discovery and scientific progress. One may recall the famous quote from Sydney Brenner “progress in science depends on new techniques, new discoveries and new ideas, probably in that order” . As alluNeocles B. Leontis died in a car accident (During the writing of this work, on December 8, 2020, accident . TogetheThe authors declare that they have no conflicts of interest with the contents of this article.


The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, based on a number of observations. One key observation was the presence of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin specifically of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA were highly enriched for host exonic, rather than intronic or intergenic sequences and often involved the same, highly expressed host genes. Although these findings do not rule out SARS-CoV-2 somatic integration, they nevertheless suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription. Viruses hijack the host cell to replicate their RNA or DNA genomes and create progeny virions. An extreme form of viral parasitism is the integration of a viral genome DNA copy into the host cell DNA . AlthougAlu elements, rely on long interspersed nuclear elements (LINEs) for their reverse transcription and integration -retrotransposons (MaLRs) rely on endogenous retroviruses (ERVs) for their reverse-transcription and integration. Similarly, short interspersed nuclear elements (SINEs), including egration .The reverse transcriptase and endonuclease activity of LINEs, carried out by the ORF2p protein, can also mediate reverse transcription and integration of unrelated viral and non-viral RNAs . Indeed,Recent studies reported a high frequency of reverse transcription and integration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in infected cells , with imPublic RNA-seq datasets under the accession numbers GSE147507 , GSE1503Chimeric reads between human and SARS-CoV-2 RNA have been identified in RNA-seq data from infected cells in two recent studies , presumeACE2, encoding the cellular receptor for SARS-CoV-2, and A549 cells overexpressing ACE2 from an adenoviral vector, and Calu3 cells that naturally express ACE2 (GSE147507). These cells were infected with SARS-CoV-2 or with other respiratory viruses, including human parainfluenza virus type 3 (HPIV3), influenza A virus (IAV), IAV lacking the antiviral NS1 gene (IAVdNS1), and respiratory syncytial virus (RSV), or were left uninfected, and were subsequently subjected to RNA-seq  cells, A549 cells that do not normally express  RNA-seq . In addi RNA-seq . The lat RNA-seq . Lastly, RNA-seq .ACE2 (A549 ACE2 cells) and Calu3 cells showed the highest number of viral reads, with parental A549 cells and NHBE cells showing lower read numbers , whereas chimeric reads aligning to introns or intergenic regions were far fewer . Notablyvia consecutive reverse transcription reactions is indirectly supported by the presence of mtRNA-nRNA fusions in public expression sequence tags (ESTs) databases. The spatial separation of mitochondrial and nuclear DNAs negates transcriptional slippage or trans-splicing, leaving consecutive reverse transcription reactions through SHS-mediated priming as a possible cause.In addition to reverse transcription and integration of viral RNA, followed transcription of the integrated copy, several alternative mechanisms might explain formation of chimeric RNA, such as genomic rearrangements, trans-splicing, or transcriptional slippage . Howevervia SHS-mediated priming during RNA-seq library construction, we first searched for mtRNA-nRNA chimeric reads, in order to assess the frequency of SHS at the junction of artifactual chimeric reads. Between 16 and 28% of analyzed mtRNA-nRNA junctions exhibited an overlap of three or more nucleotides between mitochondrial and nuclear sequences  in the murine host can provide a source of persistent antigen for the immune system . SimilarACE2, in target cells (Detection of chimeric reads between SARS-CoV-2 RNA and human RNA could also be indicative of somatic SARS-CoV-2 integration. Since detection of such chimeric reads in RNA-seq data would require transcription of the somatic integration, it would likely underestimate the total number of integrations. The high frequency of expressed somatic SARS-CoV-2 integrations reported  was, theet cells , which wChimeric reads between nuclear DNA-transcribed RNA and SARS-CoV-2 RNA involved host genes expressed at higher than average level. This correlation may have resulted from more probable detection of the higher expressed, than lower expressed genuine chimeric fragments. Alternatively, it could result from more frequent fortuitous joining, such as during RNA-seq library preparation for example, of SARS-CoV-2 RNA reads with the most abundant host gene RNA reads in the library. In support of the latter possibility, a substantial proportion of chimeric reads displayed complementarity, often over 10 nucleotides, in the joining region. Moreover, the substantially higher contribution of exonic than intronic or intergenic host sequences to human-SARS-CoV-2 chimeric reads is consistent with formation during RNA-seq library preparation, where exonic sequences are overrepresented relative to intronic or intergenic sequences.Detection of chimeric reads between SARS-CoV-2 RNA and human RNA is one of several distinct methods previously employed to estimate somatic SARS-CoV-2 integration . Given ihttps://www.ncbi.nlm.nih.gov/geo/ under the series GSE147507, GSE150316, and GSE151803.Publicly available datasets were analyzed in this study. This data can be found at: AK analyzed the data. AK and GK wrote the manuscript. Both the authors contributed to the article and approved the submitted version.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.