Oral squamous cell carcinoma (OSCC) is a common malignant tumor worldwide. Metastasis is the main cause of the death of OSCC patients. Long noncoding RNAs (lncRNAs), one of the key factors affecting OSCC metastasis, are a subtype of RNA with a length of more than 200 nucleotides that has little or no coding potential. In recent years, the important role played by lncRNAs in biological processes, such as chromatin modification, transcription regulation, RNA stability regulation, and mRNA translation, has been gradually revealed. More and more studies have shown that lncRNAs can regulate the metastasis of various tumors including OSCC at epigenetic, transcriptional, and post-transcriptional levels. In this review, we mainly discussed the role and possible mechanisms of lncRNAs in OSCC metastasis. Most lncRNAs act as oncogenes and only a few lncRNAs have been shown to inhibit OSCC metastasis. Besides, we briefly introduced the research status of cancer-associated fibroblasts-related lncRNAs in OSCC metastasis. Finally, we discussed the research prospects of lncRNAs-mediated crosstalk between OSCC cells and the tumor microenvironment in OSCC metastasis, especially the potential research value of exosomes and lymphangiogenesis. In general, lncRNAs are expected to be used for screening, treatment, and prognosis monitoring of OSCC metastasis, but more work is still required to better understand the biological function of lncRNAs. Oral squamous cell carcinoma (OSCC) is the most common type of head and neck squamous cell carcinoma (HNSCC) which ranks sixth among cancers worldwide . The iniThe metastasis of OSCC has two characteristics , 8: on tLong noncoding RNAs (lncRNAs) are a subtype of RNA with a length of more than 200 nucleotides that has little or no coding potential . There aStudies have shown that lncRNAs can affect tumor metastasis by regulating a variety of biological processes, such as chromatin modification , transcrvia competitively binding to miR-16-5p, lncRNA AGAP2-AS1 (AGAP2 antisense RNA 1) could weaken the inhibitory effect of miR-16-5p on annexin A11, thereby promoting the migration and invasion of liver cancer cells by enhancing the activation of AKT (protein kinase B) signal pathway  to sponge miRNAs, blocking their regulation on the expression of their downstream genes, and then inducing various biological effects . Moreove pathway . Similar pathway .via (but not limited to) the above mechanisms, thereby inhibiting or promoting the metastasis of OSCC  is a process of epithelial phenotype cells differentiating into mesenchymal phenotype cells. In this process, epithelial cells lose polarity and intercellular adhesion and instead acquire the characteristics of migration and invasion . FurtherIn addition to HOTAIR, lncRNA SNHG16  is also overexpressed in OSCC tissues and can promote the migration and invasion of OSCC cells. Mechanism studies showed that silencing SNHG16 will increase the expression of E-cadherin, and also downregulate the expression of N-cadherin and Snail . InteresWnt/β-catenin signaling pathway is one of the key pathways regulating tumor development. To date, activation of the Wnt/β-catenin signaling pathway has been proved to contribute to the metastasis of lung cancer , CRC 8282, breasAKT signaling pathway is another pathway that has been widely studied and plays an important role in the metastasis of pancreatic cancer , gastricThe key role of the NF-κB signaling pathway in tumor metastasis has been proved by several studies , 88. BesIn vitro and in vivo experiments showed that UICC could facilitate the expression of IL-6 by combining with its promoter and directly interact with the phospho-STAT3 to improve its protein stability, thus promoting tumor growth and metastasis  is highly expressed in cervical cancer tissues, and its high expression is also related to lymph node metastasis. tastasis . There htastasis , 92, butvia directly binding to miR-1914-3p, MALAT1 is capable of enhancing the stability of YAP (Yes-associated protein) mRNA, thus promoting the invasion and metastasis of non-small cell lung cancer (in vivo and in vitro by enhancing the expression of JAG1 (jagged 1) through the miR-124/JAG1 axis , transcribed from the region between protein-coding genes PLGLB2 (plasminogen like B2) and PLGLB1 (plasminogen like B1), is also known as CYTOR (cytoskeleton regulator RNA). Through interfering with cell signaling pathways such as PI3K/AKT , FSCN1 , and WntR-139-5p . Beyond in vitro . HoweverUCA1 was first discovered in bladder cancer  and has via upregulating the expression of Twist1  and thereby exerting its pro-metastasis effect , miR-876/SP1 (specificity protein 1), and miR-101-3p/EZH2 signaling pathways respectively (LncRNA HOXA11-AS (homeobox A11 antisense RNA) was found to be highly expressed in OSCC tissues and cells. Moreover, HOXA11-AS was demonstrated to trigger the expression of YBX2 (Y box binding protein 2) by targeting miR-98-5p, thus promoting the migration, invasion, and EMT of OSCC cells . In contCC cells . SimilarCC cells . Wang etCC cells . AdditioCC cells , LINC011CC cells , SNHG17 CC cells , and RC3CC cells  have beeectively Figure 1The probability of distant metastasis occurring in patients with OSCC is relatively low, only about 10% , 120. HoCurrently, there are few studies on the role of lncRNAs in the distant metastasis of OSCC. LncRNA HOTTIP  was reported to be overexpressed in TSCC tissues, and its high expression is associated with distant metastasis . Mu et aTo evaluate the influence of HOTAIR gene polymorphism and environmental factors on the susceptibility of OSCC, Su et al. detected four single-nucleotide polymorphisms (SNPs) of HOTAIR, namely rs920778, rs1899663, rs4759314, and rs12427129, in 1200 control participants and 907 OSCC patients. They found that rs920778 is associated with a high rate of lymph node metastasis. In addition, a motif search on the HOTAIR enhancer region showed that rs920778 is located in the putative binding motif of PRDM14 (PRDI-BF1 and RIZ domain containing 14). In particular, PRDM14 is a transcription factor that plays an anti-cancer role in HPV-positive OSCC cells . MoreoveStudies have validated that in a hypoxic environment, some lncRNAs could accelerate the metastasis of OSCC. Shih et al. reported that the expression of lncRNA MIR31HG (miR-31 host gene) in OSCC tissues is increased and can be induced by hypoxia. In addition, MIR31HG overexpression is also closely related to lymph node metastasis. Furthermore, MIR31HG in the hypoxic environment has a more obvious promotion effect on the migration, invasion, and metastasis of OSCC cells, compared with that in the normoxic environment. Mechanism studies confirmed that MIR31HG is capable of promoting the transcription of HIF-1α and bind to it, thereby assisting its binding to HIF-1β (hypoxia inducible factor 1 subunit beta) and histone acetylase p300, thus forming the HIF-1 complex. Apart from that, MIR31HG is also able to recruit the HIF-1 complex to the promoter region of the metastasis-promoting genes, such as VEGF , LICAM(L1 cell adhesion molecule), and LOXL2 (lysyloxidase homologe2), to activate their transcription . SimilarLncRNA SNHG3  was found to be overexpressed in OSCC cells. Silencing SNHG3 will inhibit the migration ability of OSCC cells. Further research showed that by recruiting ELAVL1 (ELAV like RNA-binding protein 1) to bind with NFYC (nuclear transcription factor Y subunit gamma) mRNA and then enhance its stability, SNHG3 could increase the expression of NFYC and thereby induce the migration of OSCC cells . As anotIn recent years, the tumor microenvironment composed of cells  and extracellular components  has become a hot spot in tumor research . StudiesCurrently, only one study has explored the role of lncRNAs in the regulation of CAFs on the metastasis of OSCC. Jin et al. observed that lncRNA TIRY is highly expressed in CAFs derived from OSCC tissues. Furthermore, after co-cultivation of OSCC cells with conditioned medium of CAFs treated with TIRY overexpression plasmids, the capabilities of migration and invasion of OSCC cells were significantly improved. Moreover, knocking down TIRY caused increased expression of miR-14 in CAFs and their exosomes, whose mechanism is the direct binding of TIRY to miR-14. Further research showed that the overexpression of TIRY inhibits the transmission of miR-14 from CAFs to tumor cells through the transport of exosomes, thus enhancing the activation of the Wnt/β-catenin signaling pathway in tumor cells and promoting EMT. This may be the mechanism by which TIRY in CAFs regulates the migration and invasion of OSCC cells. Interestingly, the expression of miR-14 in OSCC cells was substantially increased after treatment with conditioned medium of TIRY-silenced CAFs . HoweverResearch in the past decade has partially revealed the key role played by lncRNAs in the development of OSCC. In particular, abnormally expressed lncRNAs are also important factors affecting OSCC metastasis. LncRNAs can regulate the metastasis of OSCC through different mechanisms at epigenetic , transcrin vivo study demonstrated that RPPH1 is able to promote the metastasis of breast cancer. Mechanistically, through exosomes transport into macrophages, RPPH1 derived from breast cancer cells could induce M2 polarization of macrophages. In turn, M2-polarized macrophages will further accelerate breast cancer metastasis . Subsequently, the exosomal LNMAT2 will be internalized by HLEC, and then promote the expression of PROX1 (prospero homeobox 1) at the epigenetic level, thus leading to lymphangiogenesis and lymph node metastasis of the tumor. Therefore, LNMAT2 has the potential to serve as a therapeutic target for lymph node metastasis of bladder cancer. However, to date, there has been no research on the regulation of lncRNAs on OSCC lymphangiogenesis. Subsequent research should pay more attention to this direction.The high rate of lymph node metastasis is the main cause of death in patients with OSCC . Not onlIn addition to the crosstalk between tumor cells and stromal cells, the crosstalk between tumor cells and the extracellular matrix (ECM) also plays a critical role in the metastasis of tumors. Huang et al. reported that integrin alpha 2 (ITGA2), upregulated in omental metastases of patients with ovarian cancer, can trigger tumor cell adhesion to collagen, and then facilitates the migration and peritoneal metastasis of tumor cells. The speculated mechanism of which is that ITGA2 activates the phosphorylation of focal adhesion kinase and the mitogen-activated protein kinase pathway . In anotIn summary, lncRNAs are expected to serve as indicators for screening, treatment, and prognostic monitoring of OSCC metastasis. However, the current understanding of the role of lncRNAs in OSCC metastasis is still in its infancy. Therefore, further research on the role of lncRNAs in the crosstalk between OSCC cells and tumor microenvironment will help enrich the blueprint of the mechanisms of OSCC metastasis.YX and EJ discussed and designed the study. YX performed the research and analyzed the data. ZS and ZJS revised the article. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China 81772897, 81672666.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.


Advancement in the RNA sequencing techniques has discovered hundreds of thousands of circular RNAs (circRNAs) in humans. However, the physiological function of most of the identified circRNAs remains unexplored. Recent studies have established that spliceosomal machinery and RNA-binding proteins modulate circRNA biogenesis. Furthermore, circRNAs have been implicated in regulating crucial cellular processes by interacting with various proteins and microRNAs. However, there are several challenges in understanding the mechanism of circRNA biogenesis, transport, and their interaction with cellular factors to regulate cellular events because of their low abundance and sequence similarity with linear RNA. Addressing these challenges requires systematic studies that directly visualize the circRNAs in cells at single-molecule resolution along with the molecular regulators. In this review, we present the design, benefits, and weaknesses of RNA imaging techniques such as single-molecule RNA fluorescence in situ hybridization and BaseScope in fixed cells and fluorescent RNA aptamers in live-cell imaging of circRNAs. Furthermore, we propose the potential use of molecular beacons, multiply labeled tetravalent RNA imaging probes, and Cas-derived systems to visualize circRNAs. RNA molecules are conventionally known for the synthesis of proteins coded in the genome. However, this decade has seen an exploding number and type of RNA molecules in eukaryotic cells with the advent of next-generation sequencing. Interestingly, the noncoding (nc)RNAs contribute to more than 95% of the total RNA in the cell . The ncRHundreds of thousands of circRNAs have been identified in humans using high-throughput sequencing coupled with various bioinformatics tools (reviewed in ). HoweveIncreasing evidence suggests that circRNAs are aberrantly expressed in disease conditions and body fluids. Many recent studies have established that circRNAs may serve as a biomarker and therapeutic targets in various diseases, including cancer, diabetes, muscular atrophy, and aging  or RNA seq of poly-A enriched samples. Additionally, the circRNA sequence is the same as the parental mRNA, making it harder to detect and modulate their expression precisely. Intriguingly, the backsplice junction of circRNA provides a unique opportunity for their detection and manipulation. Several molecular biology approaches have been developed to identify and understand the molecular mechanism of circRNAs in cell physiology .The last decade has seen significant advancements in genomic sequencing technologies, including ribosomal RNA depletion strategies, longer sequencing reads, greater sequencing depth, and novel algorithms to map the reads to the genome. These developments have led to the identification of splice variants of mRNAs, non-polyadenylated RNAs, including circRNAs. CircRNAs are identified from RNA-seq reads mapped to the same gene but in the opposite order compared to the linear genomic sequence ,3,29. HoSince both the high-throughput methods can identify false-positive circRNAs, other molecular methods like northern blotting, RT-PCR, and Sanger sequencing of PCR products are recommended for further validation and quantification of identified circRNAs. Due to the speed and convenience, PCR-based identification and quantification of circRNAs have been widely used. The divergent primers across backsplice junctions are used for PCR amplification of specific circRNAs . Since dHundreds of thousands of circRNAs have been identified in human RNA-seq data using various circRNA annotation algorithms . SeveralThe function of circRNA is studied with silencing or overexpression in the target cells or tissues. Given that backsplice junction is the unique sequence for circRNA, the siRNAs or GapmeRs are designed to target the junction sequence. GapmeRs and siRNAs targeting the junction can be manually designed and checked for the specific knockdown of target circRNA without affecting the counterpart linear RNA. Since siRNAs may act as miRNAs for the counterpart linear RNA due to similar sequences, GapmeR may be better for specific silencing of target circRNAs. Since backsplicing of circRNA is known to be enhanced by intronic inverted repeats, vectors have been developed with inverted repeat sequences where the circRNA of interest can be inserted for overexpression in the cells . Since oseeing is believing”. The ability to visualize a circRNA in situ not only confirms the existence of circRNA in a cell but also gives information about circRNA quantity, localization, and association with other biomolecules, which are critical to finding the function of circRNA in cell physiology. Interestingly, a recent study showed that the very well-studied oncogene circRNA CDR1as (ciRS-7) is not present in colon cancer cells, but abundantly expressed in stromal cells of the tumor microenvironment [They say “ironment . This stironment . Unlike ironment . Given tImaging techniques are broadly divided into the fixed-cell and live-cell imaging methods based on the kind of sample. The goal of both methods, however, is to visualize the target RNA and extract the relevant information. In a fixed-cell imaging approach, the cells are fixed to keep the cellular biomolecules intact. In contrast, the live-cell imaging approach usually tracks the dynamic movement of target RNAs over time.In situ hybridization (ISH) is one of the oldest imaging techniques used to detect the abundance and precise localization of RNAs in cells . TraditicircHECTD1, circARHGAP10, hsa_circRNA_103809, hsa_circ_0017639, circSAMD4A, circPVT1, circTTN, EIcircEIF3J, EIcircPAIP2, circRHOT1, circFAT1, and circTADA2A [ciRS-7 in HEK293 and neuronal cells [circITCH in cardiomyocytes [circDLGAP4 in cerebral ischemia [circCCDC9 and miR-6792-3p, circRHOBTB3 and miR-654-3p in gastric cancer cells [circFAM114A2 and miR-762 in urothelial carcinoma of the bladder [cZNF532 and miR-29a-3p in the cytoplasm of pericytes [The traditional FISH technique uses multiple short fluorescently labeled DNA probes that bind to the RNA. Due to the binding of multiple probes on a single RNA, the signal to noise ratio is enhanced. The image is then analyzed using appropriate fitting algorithms that detect spots in the image. Unlike traditional FISH, smFISH can detect even a single RNA molecule . Since trcTADA2A ,66,67,68al cells ,52,69, mal cells , miR-330myocytes , miR-143ischemia , circCCDer cells ,74, circ bladder , and circircERBB2 that regulates ribosomal DNA transcription in gallbladder cancer and the association of circRHOT1 with TIP60 initiates NR2F6 expression in hepatocellular carcinoma [Additionally, ImmunoFISH is a variant of the smFISH technique where a combination of FISH and immunohistochemistry enables the detection of RNAs and proteins in the same sample. In ImmunoFISH, circRNA-associated protein is detected by immunofluorescence, while the target circRNA is detected with smFISH. ImmunoFISH confirmed the nucleolar colocalization of PA2G4 with arcinoma ,78. AlthcircSamd4 that regulates myogenesis by sponging PUR proteins [circSHKBP1 in gastric cancer tissues [circAR3 expression in prostate cancers [circPLEKHM3 in the tumor samples [circSlc45a4 in neuronal differentiation [circCACNA2D1 and circCACNA1E in rhesus macaque brains [circPANs and circK7.3s in Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and circBHLF1 in Epstein Barr virus (EBV) infection [RNAscope technology is a recently developed version of the ISH technique that detects mRNAs and ncRNAs with a length of more than 300 nucleotides . With itproteins . Similar tissues , circAR3 cancers , circPLE samples , circSlcntiation , circCACe brains , sisRNAse brains , and ciRe brains ,85. Furtnfection ,87.Fixed-cell imaging enables the observation of RNA only at a single time point in the cellular RNA pathway. It is generally useful for observing RNA localization. However, unlike this static observation, live-cell imaging adds a dynamic dimension that can track the entire RNA lifecycle. Live-cell RNA imaging captures many important events, such as RNA biosynthesis, splicing events, transport, function, and decay (reviewed in ). UnlikeRNA aptamer-based imaging systems require an aptamer sequence fused with the transcript of interest that can only be visualized when the dye is captured by the aptamer structure with no background issue during RNA tracking experiments (reviewed in ). The fiAlthough several aptamer–dye systems have been developed to visualize linear RNAs in live cells, only a few studies have used RNA aptamers for visualizing circRNAs. Given the tRNA introns are spliced to form RNA circles known as tricRNA, a recent study engineered aptamers containing circRNAs by inserting sequences corresponding to Broccoli and Spinach 2 RNA aptamers in the tRNA intron ,88. InteAlthough the CRISPR/Cas system is well known for gene silencing and genome editing, fluorescently labeled modified Cas proteins have recently been used for live-cell tracking of target RNA with high specificity (reviewed in ). SeveraThe molecular beacon (MB) was first developed in 1996 for nucleic acid amplification assays as well as RNA tracking in live cells . Unlike Multiply labeled tetravalent RNA imaging probes (MTRIPs) can be used for RNA imaging with high specificity. To increase the sensitivity of nucleic acids based on exogenous probes, Santangelo et al. developed MTRIPs by conjugating two to four fluorophores to a single nucleic acid. These nucleic acids  were combined using streptavidin such that the probe brightness was increased four fold ,102. OneCircRNAs regulate cell physiology by acting as sponges for microRNAs, a decoy for RBPs, competitors of splicing regulators, and substrates for protein translation (reviewed in ). AlthouGiven that circRNAs are generated from pre-mRNAs, the body of the circRNAs resembles the counterpart linear RNAs, limiting the specific probe to be complementary only to the junction sequence of circRNA. As discussed above, smFISH employs a fluorescent-labeled probe that is complementary to the circRNA junction sequence and useful for detecting abundant circRNAs . It mighLive-cell imaging of circRNAs using RNA aptamers has only been reported recently, utilizing the tricRNAs and racRNAs ,88,89. TFurthermore, the inactive Cas-13 system with a fluorescent tag and the sgRNA targeting the circRNA junction could help live-cell tracking of circRNAs ,97. HoweRecent advancements established that circRNAs are stable, ubiquitously expressed, abundant, and evolutionarily conserved RNA families (reviewed in ). There As discussed in this review, the spatiotemporal interaction of circRNAs with cellular machinery in live cells must be investigated in the context of (1) transcription of pre-mRNA and associated splicing machinery during circRNA biogenesis, which may change with the cellular state, stimuli, transcription speed, and disease conditions; (2) circRNA localization into different subcellular compartments ; (3) circRNA trafficking from nucleus to cytoplasm and packaging into exosomes for secretion; (4) the dynamic interaction of circRNA with RBPs and miRNAs affecting cellular processes. Given that immense research is underway to elucidate circRNA function, future studies must include these considerations to improve understanding of circRNA dynamics in human physiology and pathology.


Saccharomyces cerevisiae telomerase RNA, TLC1, has unveiled striking flexibility in terms of both structural and functional features. Results support the “flexible scaffold” hypothesis for this 1157-nt telomerase RNA. This model describes TLC1 acting as a tether for holoenzyme protein subunits, and it also may apply to a plethora of RNAs beyond telomerase, such as types of lncRNAs. In this short perspective review, I summarize findings from studying the large yeast telomerase ribonucleoprotein (RNP) complex in the hope that this hindsight will sharpen foresight as so many of us seek to mechanistically understand noncoding RNA molecules from vast transcriptomes.It is said that “hindsight is 20-20,” so, given the current year, it is an opportune time to review and learn from experiences studying long noncoding RNAs. Investigation of the  Scientists, like sailors, know that only a fraction of what actually exists in this world is currently within one’s “arc of visibility” . In terms of the physical organization of biological macromolecules and how they behave in space and time, what we can see and understand at this point is based largely on highly ordered matter. This is a consequence of methods such as X-ray crystallography and cryo-EM being our main visualization tools. These approaches are magnificently powerful, but they also only produce intelligible pictures if the purified target is structured (or can be made to be). Nevertheless, biologists ultimately aim to perceive what is beyond the proverbial horizon in terms of the molecules of life, and this requires conceiving of an expanse of physically disordered macromolecular complexes that are currently beyond our vantage. In this perspective, I briefly review results that have demonstrated pronounced organizational flexibility of the large telomerase RNA-protein complex, and then extrapolate what these findings may mean about the nature and extent of flexible RNPs possibly residing outside our current “arc.”.Life evolves based on DNA mutations that lead to fitness improvements. Genes are expressed as RNA and, in turn, frequently also as protein. If an evolutionary advantage for an organism would be obtained by building a highly flexible molecular complex based on one of these two gene-encoded polymers, one would be apt to argue that RNA provides more potential. This is because of 1) RNA’s larger size , and (2) RNA’s chemical composition, with its higher solubility in aqueous cellular environments than proteins, as evidenced by what happens in a phenol extraction of a cell lysate. Life on earth began with RNA [ RNA’s laS. cerevisiae. The telomerase RNP is required by most eukaryotes to address the inadequacy of DNA polymerases to entirely finish copying chromosome ends [7 heteroheptamer [The nature and extent of flexibility of RNA in RNP complexes has been highlighted in the last two decades by studies of the telomerase RNP enzyme in the yeast ome ends . Structuome ends ,4. Otherome ends ,6, the Kome ends ,8  [Saccharomyces, overall nucleotide sequence identity is, strikingly, less than 50% [As the genomes of budding yeast species were sequenced in the years following the completion of equence) ,10,11,12than 50% ,14. The than 50% . NeverthS. cerevisiae TLC1 gene without causing senescence, whereas central conserved segments were essential, and tended to overlap with regions required for binding to the Est2/TERT and Est1 essential subunits [The identification of budding yeast telomerase RNA sequences provided the starting point for next determining secondary structure. But the RNAs were very large, and so rapidly evolving in sequence and length that it made alignment in some sections rather difficult. The large RNAs and rapid evolution challenged computational modeling and identification of covarying nucleotides that could help solve the secondary structure. In 2002, the Cech lab reported that large truncations could be made in the subunits . At thisSaccharomyces species to identify covarying nucleotides [Mfold software prediction of a large RNA could be accurate  [Mfold predictions compared to wild type and the severity of the observed phenotypes. However, the Mfold lowest-free-energy modeling required some important constraints to accommodate covarying nucleotides; these supported the existence of some long-range base pairings and helices not present in the lowest-free-energy structure computed by Mfold. These covarying base pairs I identified with help from RNAalifold [Mfold energetics-driven modeling, adding further confidence and encouragement that the data were leading towards a compelling consensus. The model ultimately published by us [In 2004, secondary structure models for TLC1 were reported . The twoleotides ,14. It wpairings ). Neverth (2002)  while I Aalifold . Primaried by us  and anot7.  would almost certainly not function. Thus, it was probable that telomerase has a different physical organization than other well-studied RNP complexes.The Ku70/80 heterodimer is well known for its key role in DNA repair, but it also binds to telomerase RNA in yeast and also in humans . In yeas7 complex, which binds to a consensus sequence near the 3′ end of TLC1 [7 subunit in these alleles, since Sm7 binding location participates in the proper processing of the 3′ terminus. Thus, since these circularly permuted Sm7-repositioned TLC1 constructs were functional, TLC1 tolerates relocation of its termini—a feature of the RNA that begets a different co-transcriptional folding pathway, due to the altered order in which the RNA polymer is synthesized, in addition to causing local structural changes at the new end sites.The Sm of TLC1 , can als of TLC1 ,29. One 7 complex components, we showed that these subunit-binding sites are functional modules, and likely all represent structured domains, in the context of the RNP as a whole. In fact, the crystal structure of yeast Ku bound to the TLC1 hairpin was recently solved, showing details of this particularly discrete module [In summary, with the above relocations of holoenzyme-specific subunits, including the essential Est1, and important Ku and Sme module . TLC1 RNWe deleted the bulk of the three rapidly evolving RNA arms to test whether they were important . These 3S. cerevisiae cells, since the organism lacks miRNA-processing components.)We stiffened the arms by converting them all (and each single- and double-arm combination) to perfect double-stranded RNA (dsRNA) rod-like struts by removing all of the bulges and loops that connect all the dsRNA segments  Figure . The triGiven that subunits’ binding sites can be repositioned on the yeast telomerase RNA with retention of their function, demonstrating that the holoenzyme subunits are modular in nature, to what extent does this show that they are orchestrated by TLC1 in a physically flexible, or pliable, manner in wild-type telomerase? In terms of what we know of TLC1 secondary structure, since the overall long-armed, Y-shaped molecule  is litteMini-T and TSA-T RNAs were functional, supporting sufficient telomerase activity in cells to maintain stable telomeres and prevent senescence. However, the fitness of a truncated-arm construct, Mini-T(500), was tested quantitatively and shown to be reduced, and all of the Mini-T constructs supported very short telomeres .Perhaps not surprising for Mini-T, due to having two-thirds of the natural telomerase RNA nucleotides removed, and therefore experiencing a simplified folding landscape (and avoiding misfolding ), these 7 function as modules that do not need to bind to each other or to the catalytic core for their function even in cis. Furthermore, these essential and important modules flexibly scaffolded by TLC1 are also unlikely to coordinate with each other in trans, since S. cerevisiae telomerase has been shown to function as a monomer [Some things that we learned collectively from Mini-T and TSA-T about the flexible arms of the wild-type TLC1 RNA in yeast include (1) the wild-type arms are required for full fitness of the cells, (2) they are not essential for basic telomerase maintenance of shorter but stable telomeres, and (3) even in the case where the arms can no longer physically bend due to excision of all of the hinges (loops and bulges), telomerase RNA still functions, even in vivo. This last result supports the mounting evidence that Est1, Ku, and Sm monomer .Approximately 40% of TLC1 RNA comprises well-conserved patches of sequence, based on alignments with closely related budding yeast species. Within these patches   or CS2a 7) of yeast telomerase RNPs, these modules have been shown to be flexibly scaffolded as reviewed in sections above, whereas, in the case of the catalytic core, there is some very interesting and impressive flexibility [Thus, the fraction of 1157-nt yeast telomerase RNA that is conserved among budding yeasts essentially represents the portion of TLC1 that is shared with the Mini-T-sized 451-nt human telomerase RNA, and this is greater than the “Micro-T”-size 150-nt ciliate  telomerase RNAs ,25. Our xibility ,30, yet xibility ,30, cons7-binding regions from coming together in cis. Second, Mini-T is functional despite its presumably pliable long arms being excised, and these truncations thus “reel in” the holoenzyme subunits in towards, and nearly abutting, the catalytic core. This would confine the amount of physical sampling of these subunits in space relative to the RNP as a whole. Thus, the most compelling and parsimonious view is that holoenzyme subunits function quite independently, with the TLC1 simply flexibly tethering these modules to the RNP complex.In theory, the structured modules that comprise the minority of the overall TLC1 RNA molecule could come together in 3D space on a large flexible-scaffolding RNA to ultimately form a holoenzyme RNP with overall precisely coordinated positioning. This is important to consider and test. The data acquired thus far do not support this possibility, however. First, as mentioned above, the triple-stiff-arm allele, TSA-T is functional in vivo despite the holoenzyme-binding modules being held apart by rigidified rod-like arms that would prevent the Est1, Ku, and SmWhat lessons can be learned from what we have discovered so far about the large yeast telomerase RNA’s flexible scaffolding of holoenzyme subunit proteins in this RNP? The repercussions of the studies summarized above should be considered in the context of the broad range of conditions where RNA may serve to scaffold a higher-order complex. One context is simply with respect to other telomerase RNPs. A second is extrapolating to other RNPs in cells, while considering that RNA can serve as a flexible scaffold for proteins in an RNP, even one that is enzymatic. We can use the case of the TLC1 flexible-scaffold RNA paradigm when considering other RNPs. How flexible are the best-characterized RNP complexes, including those with enzymatic functions? Furthermore, with respect to what is “beyond our visible horizon” in terms of flexible RNPs, is yeast telomerase a harbinger of many more that are to be found next, such as from the class of RNAs currently referred to as long noncoding RNAs?It is useful to conceive of the spectrum of physical and functional flexibility as it pertains to RNA-protein complexes. A prior review by Tom Cech and me  covered RNase P and the ribosome may be viewed as some of the most rigidly structured large RNP complexes characterized so far . It is tIn terms of the most flexible large RNPs, it is interesting to consider what would be theoretically, as well as biologically, the most extreme case. In theory, single-stranded RNA would be most flexible (this is the schematic on the far right of However, like the ribosome, yeast telomerase is an enzyme, and I have mentioned above that it has structured elements within its conserved domains. These modules bind to specific proteins and coordinate what is presumably a complex and elegant “dance” with its core-enzyme partner TERT during rounds of template re-use in telomere synthesis. Thus, additional examples of such extremely flexible RNPs are apt to be discovered, and presumably the most flexible will be classes of lncRNAs that bind to proteins for the sole purpose of scaffolding them in a flexible fashion, presumably without the constraints of also coordinating enzymatic RNP dynamics.Some of the most flexible RNAs in large RNPs are likely to be lncRNAs. Therefore, although lncRNAs are a broad classification, I have listed them to the right of yeast telomerase in Limited progress on lncRNA structure–function has been reported, although there have been a substantial number of perspectives published on the topic, given the level of interest to RNA biologists and relevance of lncRNAs to human health ,62,63,64In terms of what yeast telomerase can tell us about telomerase flexibility in other species, as pointed out in As for why telomerase in yeast is so flexible, and why it is rapidly evolving in sequence and size throughout the Tree of Life, this is a fascinating question, but the answer remains elusive. Presumably, there are facets of telomerase RNP biogenesis, assembly, catalytic mechanism, regulation, and more, which are beyond our understanding at this point, leaving such large questions too far “beyond the horizon” to be worthy of speculation just yet.In summary, investigating yeast telomerase RNA has taught us about not just this particular RNP enzyme, but also about the organization of an emerging multitude of other RNA-based complexes that are also likely to be flexible. As the anatomy of other RNA-containing complexes is characterized, it will be useful to consider the impressive possible, and likely extant, physical and functional flexibility of RNA complex organization compared to TLC1. The proposed continuum of RNP complex flexibility in One of the best ways to identify a highly flexible RNA is based on a fast rate of sequence evolution and by noting resistance to structure being captured in 3D by X-ray crystallography or ensemble-averaging of cryo-EM data. However, even presumably flexible lncRNAs can be studied biophysically, such as by AFM, EM, and other single-molecule approaches. These approaches are likely to be more frequent as more researchers consider how the term “structure” tends to presuppose rigidity of macromolecular RNP complexes. This research will seek increasingly more than structural identification of three-dimensionally organized domains, but also biophysical characterization of dynamically flexible full-length RNAs. As we have shown in studying yeast telomerase RNA, when a large RNA is rapidly evolving overall, yet contains discrete islands of conserved sequence, this affords the ability to test for functions in vivo and in vitro upon altering the relative arrangement of the conserved modules along the transcript. Few studies of RNPs have been performed in this way, but this approach will become more widespread, as it should reveal far more RNAs that function as flexible scaffolds for proteins and other modular moieties.


To The Editor Novel coronavirus 2019 infection is a new coronavirus disease  to calcu


RNA-binding proteins (RBPs) are involved in regulating all aspects of RNA metabolism, including processing, transport, translation, and degradation. Dysregulation of RNA metabolism is linked to a plethora of diseases, such as cancer, neurodegenerative diseases, and neuromuscular disorders. Recent years have seen a dramatic shift in the knowledge base, with RNA increasingly being recognised as an attractive target for precision medicine therapies. In this article, we are going to review current RNA-targeted therapies. Furthermore, we will scrutinise a range of drug discoveries targeting protein-RNA interactions. In particular, we will focus on the interplay between Lin28 and let-7, splicing regulatory proteins and survival motor neuron (SMN) pre-mRNA, as well as HuR, Musashi, proteins and their RNA targets. We will highlight the mechanisms RBPs utilise to modulate RNA metabolism and discuss current high-throughput screening strategies. This review provides evidence that we are entering a new era of RNA-targeted medicine. Eukaryotic post-transcriptional regulation is a critical process that controls RNA metabolism and gene expression. RNA-binding proteins (RBPs) are key regulators of post-transcriptional events such as RNA maturation, localisation, modification, silencing, translation, and turnover . This isMutations that affect post-transcriptional events often lead to serious diseases . Hence, In this review, we will explore the discovery process of FDA-approved and other pending therapies for spinal muscular atrophy (SMA) that target survival motor neuron (SMN) pre-mRNA and splicing regulatory proteins. We will also present two examples of potential RNA-targeted therapies, which could work through regulating RBPs and RNA metabolism—Lin28 and let-7; as well as HuR and Musashi proteins, and their RNA targets. We will review the drug discovery processes targeting these RNA-protein interactions in depth, which utilised high-throughput screenings (HTS). The focus will be placed on the screening strategies and the interplays between the RBP, its relevant drug, and the RNA target. Finally, we will highlight several other discoveries towards RNA-targeted therapies of human diseases. Together, this review provides insights into the pathways involved in uncovering novel, RNA-targeted therapies.Homo Sapiens [MicroRNAs  are a class of short non-coding RNAs that silence gene expression post-transcriptionally. To date, more than 2000 miRNAs have been identified in  Sapiens . In anim Sapiens . Dicer c Sapiens . The mRI Sapiens .C. elegans. There are nine mature let-7 miRNAs in the human let-7 family, encoded by 12 different genomic loci [Let-7 was first discovered as a regulator of stem cell differentiation in mic loci . The biomic loci ,30,31,32mic loci . Lin28 imic loci . Lin28A mic loci  -based screenings are widely used in the study of protein–protein interactions and have recently been applied in RNA-protein targeted screenings. Normally, the two components are tagged with a donor or acceptor fluorophore, respectively. Quenching of the donor fluorescence or enhancement of the acceptor fluorescence can be detected, allowing interpretation of the interactions. The first small molecule disruptor of Lin28/let-7 was identified through FRET screening of 16,000 drug-like small molecules from the Maybridge Hitfinder library . InsteadAnother FRET-based screening assay labelled the terminal loop of pre-let-7a-1 at the 3’-end with the quencher molecule BHQ-2 . The fluAnother common screening strategy is fluorescence polarisation (FP). Excited by the corresponding polarised light, a fluorophore linked to an unbound ligand depolarises the light emitted. While the ligand is associated with a protein, the polarisation is retained due to a slower rotation of the bound ligand. The affinity of ligand binding can be measured according to the difference of the polarised light emitted by the fluorophore . A previA more recent FP-based screening was conducted using an extensive 101,017-compound pool, combining six commercially available libraries . The FP A novel fluorescence intensity-based binding assay (FL assay) was developed recently to screen modulators of RNA-protein interaction, as protein-binding induced fluorescence enhancement can be detected when nucleic acid fragments are conjugated with environment-sensitive organic fluorophores . Based oClick chemistry is often applied in HTS by joining two biomolecules efficiently. The Garner group developed a catalytic enzyme-linked click chemistry assay (cat-ELCCA) to monitor protein–protein interaction and miRNA biogenesis in a high-throughput format ,57,58. BLin28/let-7 is the best characterised RBP-miRNA interaction. These HTS are important examples for drug discovery of RBP-miRNA modulators. Our group have identified Lin28A as a negative regulator of the biogenesis of miR-9, through a distinct mechanism . AdditioPre-mRNA splicing is a critical process in the production of functional mature mRNA in eukaryotic cells. During the process, introns are removed, and exons are joined. Pre-mRNA splicing is catalysed by a macromolecular ribonucleoprotein complex known as the spliceosome. The major spliceosome is composed of five small nuclear RNAs , which combine with proteins to form small nuclear ribonucleoproteins (snRNP). These snRNPs recognise conserved sequence elements at 5’ splice sites (5’ss), 3’ss and branch points . AlternaSMA is an autosomal recessive disorder characterised by motor neuron degeneration, leading to progressive muscle weakness, atrophy and paralysis. SMA occurs in 1 in 10,000 new-borns and is dominated by SMA type I (Werdnig–Hoffmann disease), a fatal, infantile-onset subtype ,71. The th–24th position in intron 7) [Spinraza (nusinersen) is the first FDA approved drug for the treatment of SMA. The drug is a 2′-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASO. The modification endows the ASO drug with an improved in vivo stability . The disntron 7) . Thirty-ntron 7) . After entron 7) . The leantron 7) ,79,80,81Spinraza has been shown to improve various clinically important outcomes, including increased survival and enhancement of motor function. This is true for both early-onset type I disease and later-onset type II and III diseases ,83. As tIt is clear that Spinraza functions by sequestering ISS-N1, but the mechanism of how ISS-N1 works as a negative SMN2 splicing regulator remains contentious. It was previously assumed that the two putative hnRNP A1/A2 binding motifs in ISS-N1 were responsible for the inhibitory effects . HoweverRecently, a gene replacement therapy named Zolgensma  was approved by the FDA for paediatric SMA patients with bi-allelic mutations in the SMN1 gene . ZolgensCurrently, two orally bioavailable small molecules targeting SMN2 splicing correction, risdiplam and branaplam, are under evaluation in clinical trials. If approved, these drugs would advance SMA treatment and improve patient accessibility beyond Spinraza and Zolgensma.in vitro assays [Risdiplam (RG7916) is structurally optimised from RG7800, the first small-molecule SMN2 splicing modifier to enter human clinical trials . Unfortuo assays . Additioo assays ,101. Howo assays . Compareo assays . The druo assays . RisdiplBranaplam (LMI070) was discovered from a 1.4 million-compound library (Novartis) using a pair of opposite SMN2 minigene luciferase reporters expressed in an NSC34 motor neuron cell line . The scrSecondary RNA structures are potential targets for small molecule splicing modifiers. Terminal stem-loop 2 (TSL2) is a 19-nt RNA hairpin formed at the exon 7/intron 7 junction of SMN2, with its 3’ end partially overlapping with 5’ss of exon 7 . Prior tLuciferase SMN2 minigene reporters were extensively applied in the previous HTS of SMN2 splicing modulators ,108. RigAfter the development and approval of Spinraza, the field of RNA-targeted drug development for the therapy of SMA has expanded exponentially. The development of ASO drugs raised hope for RNA-targeted therapies aimed at RBPs, previously considered undruggable. Additionally, the identified splicing modifiers are useful tools to study the mechanism of how RBPs regulate alternative splicing. A deeper understanding of the mechanism will, in turn, facilitate the development of more specific therapies. Finally, minigene reporters are good models for screening splicing modulators, if the hits are validated using more physiologically relevant models.MicroRNA-7 (miR-7) is a brain-enriched miRNA. Dysregulation of miR-7 could contribute to severe diseases, especially neurological disorders . In glioOur group has identified that an RBP HuR (ELAVL1) binds to the terminal loop of pri-miR-7 and recruits another RBP MSI2 (Musashi RNA-binding protein 2) to inhibit the biogenesis of functional miR-7 . We alsoHuR is expressed ubiquitously across human tissues. The protein often stabilises mRNAs bearing AU-rich elements (AREs) in their 3’-UTRs. Most of the targeted ARE-RNAs encode oncogenic proteins, highlighting a crucial role of HuR in regulating the development and progression of multiple human cancers . HuR inh12 (containing only RRM1 and RRM2), and tetramethylrhodamine (TMR) labelled ARE-RNA (unspecified) using a 50,000-compound library composed of microbial, mycological, and plant extracts (Novartis) [A confocal fluctuation spectroscopic screening assay was performed with recombinant HuRovartis) . In compovartis) . Three oovartis) . The throvartis) . MS-444 ovartis) ,127,128.ovartis) ,129, colovartis) ,131, melovartis) , and gliovartis)  in vitro12) with the highest intensities were found in sub-libraries of pyrrole and dicarboxylic acid scaffolds [Confocal nanoscanning (CONA) screening from one-bead-one-compound libraries was used to look for HuR ligands, specifically targeting RRM3 and elucidate the controversial functions of the orphan RRM3 pocket . Eighty-caffolds . Forty-scaffolds . With thcaffolds . Notablycaffolds . The on-A small-scale screen targeting HuR and TNF-α ARE-RNA was completed using EMSA. One hundred seventy-nine compounds, obtained from the Korea Research Institute of Chemical Technology, were tested . Three cAn AlphaScreen (PerkinElmer)-based HTS was developed to identify inhibitors of HuR binding to TNF-α mRNA . The intAn FP-based HTS has been reported to screen a library of around 6,000 compounds (an in-house library and a library of FDA approved drugs) for HuR/MSI1 ARE-RNA disruptors . The scrThe above FP assay was applied to screen about 2,000 compounds from the National Cancer Institute library  and an in-house library . The hitAnother FP-based HTS was carried out from the NCI library  to identify small HuR inhibitors . The scrUsing differential scanning fluorimetry (DSF), suramin was recognised as an HuR ligand from an FDA-approved drug library  . Althoug1–3AGU sequences, whereas MSI2 prefers ACCUUUUUAGAA and UAG motifs [Human Musashi proteins have two paralogues MSI1 and MSI2, sharing approximately 80% amino acid similarity. Both proteins have two highly conserved N-terminal RRMs, with MSI1 mainly binding (G/A)UG motifs ,150,151.G motifs . MusashiG motifs . AberranG motifs . OverexpG motifs ,154,155.G motifs .A 1536-well format FP-based HTS has been developed to identify inhibitors of MSI RNA binding activities . The scrAnother FP-based HTS uses a previously identified RNA probe (AGCGUUAGUUAUUUAGUUCG) containing two MSI1 consensus recognition motifs . The int(−)-gossypol is a natural product extracted from cottonseed. The molecule targets anti-apoptotic activities of Bcl-2 family proteins and has been studied in clinical trials for the treatment of a variety of cancers . (−)-gosAn MSI1 inhibitor, luteolin, was discovered from an FP screening using a total of 25,588 compounds from several libraries, including Prestwick, LOPAC, Cambridge NovaCORE, and the Life Chemical FSP3-enriched libraries . The assSo far, structural knowledge of HuR and Musashi proteins is very limited. The structures of HuR RRM1-2 and RRM3 were solved separately ,124,164.Aside from the above three stories, there are more potential RNA-targeted therapies being developed. The following section will briefly outline some notable examples.Myotonic dystrophy type 1 (DM1) is caused by the expansion of a (CTG)n repeat sequence in the dystrophia myotonica protein kinase (DMPK) gene 3’UTR . From an4C2 repeats in the c9orf72 gene is the most causative factor of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The transcribed G4C2 repeats are responsible for the accumulation of toxic RNA foci, sequestration of proteins, as well as the production of dipeptide repeat proteins (DPRs), which are a result of non-ATG (RAN) translation [4C2 repeat RNA G-quadruplexes were identified. These RNA binders could reduce the G4C2 foci and DPRs in c9orf72 patient-derived neurons [4C2 repeat RNA was discovered from a fluorescence displacement assay. According to in vitro and cell-based assays, this molecule inhibits RAN translation and hnRNP-H sequestration of the target, blocking these two potential mechanisms driving the c9ALS/FTD pathogenesis [A different expansion of Gnslation . From a  neurons . Anotherogenesis .In another study, a small molecule microarray (SMM) containing 20,000 drug-like molecules was previously amassed. SMM screening was performed against a fluorescently labelled RNA element, the HIV-1 transactivation response (TAR). This resulted in the discovery of TAR hairpin stabilisers, which provide anti-HIV activity in T-lymphocytes . FurtherThe discovery of small-molecule miRNA regulators has been previously reviewed, summarising three major strategies, including HTS from large libraries, focused screening using known RNA binders, such as aminoglycosides, as well as drug design targeting RNA secondary structures or relevant RBPs . The useStreptomycin is a well-known aminoglycoside antibiotic. It was previously identified as a miR-21 inhibitor, from a focused screen using a cell-based luciferase reporter assay . StreptoFinally, Disney et al. developed a novel focused screen platform named dubbed two-dimensional combinatorial screening (2DCS). This is a library-vs-library screen where a small molecule microarray is hybridised with an RNA element library, allowing the definition of interactions between the compounds and steric RNA structures ,179. UsiThese RNA binders have shown great potential as future therapeutics, targeting viral RNA, oncogenic miRNA, as well as abnormal mRNA repeats. To find out whether and how the relevant RBPs become involved in the field will be a topic of great interest.Throughout this review, we have summarised commercialised RNA-targeted therapies and those under evaluation in clinical and preclinical studies. These drugs are based on different formats including ASOs, siRNAs, miRNA mimics and small molecules. The therapeutic effects are achieved through the regulation of RNA maturation, splicing, interference, translation and stabilisation. More possibilities of drug formats and therapeutic mechanisms have been discussed, including peptides , aptamerBy reviewing a number of HTS targeting RBPs and their regulatory RNAs, we have reached several important conclusions, which may benefit strategy making for future RNA-targeted drug discovery in the preclinical stage. First of all, we have shown a collection of different screening assays. FRET and FP are the most common assays applied in the identification of disruptors between two-component RBP-RNA interactions. Luciferase minigene reporter assays have been frequently used to identify splicing modifies. This is a paradigm of phenotypic screens. Alternatively, some novel screening strategies are being developed, such as CONA and FD assay ,122. MorSeveral aforementioned hit compounds have shown promising therapeutic effects in the preclinical and clinical tests. Risdiplam is the most advanced investigational medicine, awaiting the FDA’s decision under priority review. It is being studied in all types of SMA patients aged from 0-60 years old. If approved, risdiplam will become the first self-administrated SMA medicine. Some of the Lin28/let-7 disruptors (1632 and KCB3602), as well as HuR  or MSI inhibitors -gossypol and Gn), have exhibited anti-cancer properties in vitro or in vivo. However, further research needs to be undertaken to evaluate their efficacy, bioavailability and safety before entering the clinical studies. Beyond these three examples, therapies targeting viral RNAs ,188, RNA


As of 2 April 2020, over one million people have been infected with a novel viral pneumonia (COVID-19) that emerged from Wuhan, China late December of 2019  of the variance in dysfunctional coronavirus anxiety and correctly classified 75% of cases.This sample consisted of 675 males, 558 females, 4 other gender, with a median age of 35 years (range 18−65). The rates of coronavirus infection, dysfunctional coronavirus anxiety (CAS ⩾ 9; Lee, These results suggest that people with dysfunctional coronavirus anxiety suffer from a wide range of psychological difficulties and that having the coronavirus infection poses a major risk factor for this form of psychopathology. These findings are part of a disturbing trend of coronavirus anxiety found among people in the U.S. Lee, , China , and mor


In recent years, there have been major advances in the application of non-invasive techniques to predict pregnancy-related complications, for example by measuring cell-free RNA (cfRNA) in maternal blood. In contrast to cell-free DNA (cfDNA), which is already in clinical use to diagnose fetal aneuploidy, circulating RNA levels can correspond with tissue-specific gene expression and provide a snapshot of prenatal health across gestation. Here, we review the physiologic origins of cfRNA and its novel applications and corresponding challenges to monitor fetal and maternal health and predict pregnancy-related complications. For years, ultrasound has been the primary tool used to monitor fetal health allowing for the direct measurement of fetal shape, size, and some aspects of fetal physiology, all of which are important for development. It would be very useful to have a companion tool, which enables measurement of the molecular details of development, like the gene expression profile of the fetus over time. Liquid biopsies that rely on a simple maternal blood sample offer a possible avenue to realize this goal. Indeed, the measurement of circulating cell-free DNA (cfDNA) to screen for fetal aneuploidy as first demonstrated 12 years ago , preeclaCirculating RNA in pregnant women was first identified by Mandel and Metais in 1948 —long befSeveral groups have now confirmed through orthogonal methods that both the fetus and mother contribute to measured cfRNA levels , 11, 12.Furthermore, cfRNA from many types of RNA, including circular and non-coding, can be measured in the cf-transcriptome throughout pregnancy . As Koh To address where in the body prenatal cfRNA originates from, researchers used available body atlases  to identWhat specific cellular process—death, active signaling, or both—gives rise to cfRNA in circulation remains an open question , 23–25. Finally, there remains the question of cfRNA clearance or how long do RNA molecules remain in circulation following their release. In general, for healthy pregnancies, we expect all cell-free nucleic acids to clear within hours of delivery , 8, 31. Intriguingly, several groups have shown that cfRNA levels for specific pregnancy-related genes and circular RNAs change over gestation in predictable patterns , 6. LateLongitudinal changes in gene expression follow three main trends: those that change continuously over pregnancy, those that change early in pregnancy and reach a plateau late in the second trimester, or, conversely, those that remain constant early in pregnancy only to change dramatically later in gestation , 4, 6, 8Until the last 2 years, research with the goal of predicting pregnancy-related complications has focused on measuring very few genes using RT-qPCR in small, single-hospital cohorts without external validation. These studies have been thoroughly reviewed elsewhere  and willPrediction of spontaneous PTB has been reported in Black women at two sites in the USA, where women either presented with symptoms of early labor or experienced a prior spontaneous PTB in the discovery and validation cohorts, respectively. In validation, cfRNA levels for seven genes successfully identified women who delivered preterm up to 2 months in advance of delivery; however, both discovery and validation cohorts were small . Larger Another study used cfRNA to characterize preeclampsia. They focused on women with early onset, defined as a diagnosis prior to 34 weeks of gestation and severe features. Blood was sampled at the time of diagnosis between 24 and 34 weeks of gestation. The cfRNA levels of 49 genes, of which 19 were specific to fetal or placental tissue, successfully identified women who experienced early-onset preeclampsia with an accuracy of 85–89% in a small, independent validation cohort  and, more specifically, for prediction of early-onset pre-eclampsia across paired samples for which plasma was isolated 0 to 5 days post-collection of blood in Streck cfDNA tubes.A major concern when using plasma has been the recommendation to isolate plasma immediately after whole blood collection to ensure sample quality. Recent work by Munchel et al.  has showcfRNA can be successfully extracted and amplified from a minimum of 0.5 ml of plasma with strong agreement between technical replicates . In a diFinally, extraction and amplification of sufficient cfRNA have persisted as challenges. Recent work has converged on several best practices. Like most RNA-based discovery work, cfRNA analysis can be separated into three parts—extraction, amplification, and downstream analyses.Published work today relies on commercially available RNA extraction kits from Norgen Biotek Corp., Qiagen, or Thermo Fisher Scientific for example. Importantly, RNA extraction must be followed by DNA digestion as recently emphasized in a re-analysis , 42 of dFollowing RNA extraction, samples may be prepared for sequencing or RT-qPCR. Library preparation for sequencing has converged on two key methods—total RNA sequencing with ribosomal RNA (rRNA) depletion  and exonEven with best practices, sample quality can still vary. Several quality metrics can be used to identify samples with unusually high levels of RNA degradation, DNA contamination, and/or ribosomal RNA . Any samFor degraded input material like cfRNA, the RNA integrity number (RIN) may not be informative. Instead, to assess RNA degradation, researchers can leverage that ribonucleases present in human plasma exhibit 5′-3′ catalytic activity owing to poor 3′ poly(A) cleavage . SpecifiTo assess DNA contamination, one can estimate the intron-to-exon read ratio by counting the number of reads that map exclusively to intronic as compared with exonic sequences . Here, wFinally, to estimate the rRNA fraction, researchers can count reads that map to known ribosomal sequences as compared with the total number of reads for a given sample. Here, we expect values of 10–20% for total RNAseq as previously noted and <10% for whole-exome enriched RNAseq , 8, 44.Most computational methods for cfRNA data analyses mirror those used for any RNA-based application. RNAseq data pre-processing relies on tools to remove adapter sequences from reads , map reads , remove PCR duplicates , and finally count the number of reads that map to any given gene . RT-qPCR analysis requires far fewer pre-processing steps, namely, just normalization as described in more detail below. Subsequently, to identify genes associated with pregnancy-related complications, researchers frequently use methods like differential expression  or machine learning . Despite the numerous pre-processing steps in common between cfRNA and general RNA analyses, there remain several important differences as well.Unlike RNA measurements from specific tissues, cfRNA levels represent a mixture of RNA from many tissues and both mother and fetus for any given gene . InterprFurthermore, standard practice when analyzing cellular RNA may not apply as expected to cfRNA since it represents a mixture of RNA levels from across the body. One such example is the use of reference genes to normalize RNA levels across samples. When planning RT-qPCR experiments and choosing an appropriate number of reference genes, we point the reader to the MIQE guidelines . In the Finally, cfRNA discovery work is also plagued by the same issues as any method that seeks to identify predictive signatures , 49. BuiRecent years have seen several proof-of-concept studies, which suggest that cfRNA can be a powerful diagnostic tool. However, owing to the study scale required to prospectively collect enough samples from women with pregnancy-related complications, all of the highlighted studies used at least some samples collected at onset of symptoms or at diagnosis. While encouraging, this present emphasis on prediction at diagnosis cannot replace the required work to show that cfRNA diagnostics can also anticipate complication onset and consequently allow for clinical intervention. To address this, large-scale, prospective studies hold promise as they can both confirm prediction at and prior to diagnosis.In contrast to several studies so far that have focused on cf-mRNA, future applications may explore the predictive power of different types of RNA. Data from total RNA-sequencing like that from Koh et al.  have revFuture applications may also benefit from incorporating measurements from other nucleic acids entirely like cfDNA or other molecules like proteins and metabolites as has recently proven informative for prostate cancer  and PTB Separate from the prediction of pregnancy-related complications, another goal is to monitor fetal health in real-time. Despite encouraging results, researchers have thus far only identified a small number of fetal genes in circulation and noted the variabilities of such detection early in gestation when the fetus contributes <4% of the total cfRNA measured. Creating signatures, which combine information from groups of co-regulated genes may prove key to developing fetal tissue-specific signatures that can be reliably measured across gestation.Furthermore, extension of such work from correlative observations to causative inference remains a major challenge not only for cfRNA analyses but also for RNA discovery work broadly. Here, we can imagine applying both cutting-edge computational and experimental methods like the construction of generative models or the application of correlative cfRNA insights to refine experimental hypotheses. Nonetheless, such experimental or computational work must be informed by our knowledge about the physiology of cfRNA. Here, one important question remains unanswered: What biological process—active secretion, cell death, or both—produces prenatal cfRNA?Step by step, researchers have answered many important questions in a field that is only two decades old. Excitingly, groups over the past few years have clearly shown that cfRNA is not only a rich data source but also one with diagnostic potential. Yet still, key questions must be addressed for cfRNA diagnostics to be clinically used and impact prenatal care.MNM, RJW, GMS, DKS, and SRQ conceptualized this review. MNM wrote the original manuscript draft and created the overview figure. All authors revised the manuscript and approved it for publication.SQ is a founder and shareholder of Mirvie, and Stanford University and the Chan Zuckerberg Biohub have filed patents based on the work of SQ and MM on the use of cfRNA in maternal and fetal health. The remaining 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.


Increasing evidence has demonstrated that circular RNA (circRNA) exerts important function in the pathogenesis of some diseases. While, the contributions of circRNAs to aseptic loosening after total hip arthroplasty (THA) remain largely unknown. Our research is to explore the differentially expressed circRNAs (DEcircRNAs) and elucidate complex regulated mechanism of circRNAs in aseptic loosening. The DEcircRNAs were identified by RNA sequencing (RNA-seq) analysis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was adopted to corroborate these DEcircRNAs. The potential function of circRNAs in aseptic loosening tissue was identified by competing endogenous RNA (ceRNA) analysis. Enrichment analysis was performed for target mRNAs and host genes of the DEcircRNAs by Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 257 DEcircRNAs were obtained from RNA-seq results. Following the RT-qPCR corroboration, 6 circRNAs  were selected for further analysis. By circRNA-miRNA and miRNA-mRNA prediction, 6 circRNAs, 138 miRNAs and 1667 mRNAs were identified. Then, circRNA-miRNA-mRNA network was established. The result of GO and KEGG enrichment analysis suggested that the circRNAs were related with some biological functions and pathways of aseptic loosening. A novel pathogenesis and treatment strategy about aseptic loosening after THA was revealed from our study of circRNA-miRNA-mRNA network. Total hip arthroplasty (THA) is one kind of successful treatment for the management of pain and function 7 There is growing evidence that circRNAs have multiple biological functions, including microRNA sponges, regulating target gene transcription, and forming RNA-protein complexes Circular RNA (circRNA) is a special type of endogenous noncoding RNA and formed by intron or exon circularization The underlying mechanism of circRNAs in aseptic loosening after THA was investigated in our reseach. First, the RNA-seq analysis of circRNAs was used to detect the differentially expressed circRNAs (DEcircRNAs) profiles in aseptic loosening and non-aseptic loosening synovial tissue of the hip joint. Then, the DEcircRNAs were corroborated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We predicted their target miRNAs and target mRNAs, then constructed a circRNA-miRNA-mRNA network to reveal whether DEcircRNAs acting as ceRNAs in aseptic loosening. We conducted Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the downstream mRNAs and host genes of the DEcircRNAs to reveal the possible pathogenesis of aseptic loosening. Therefore, our finding might further elucidate the complex RNA regulatory mechanism by constructing ceRNA network and provide a novel perspective for the pathogenesis and latent diagnosis of aseptic loosening.The experimental specimens were obtained from the hip joint synovial tissues of patients, who received revision operations for aseptic loosening after THA. The hip joint synovial tissues from patients with femoral neck fracture during their primary THA were acting as control specimens. 6 aseptic loosening tissues and 6 non-aseptic loosening tissues were obtained between January 2018 and November 2019 from the First Affiliated Hospital of China Medical University. 3 pairs of specimens were selected for RNA-seq, and the 6 paired specimens were used to identify the accuracy of the RNA-seq results by RT-qPCR. All specimens were quick-freezing and preserved at - 80 °C before use. Our study meet the specification of the Declaration of Helsinki, and approved by the Research Ethics Committee of the First Affiliated Hospital of China Medical University.Total RNA from specimens for RNA sequencing was isolated by a TRIzol™ reagent  as per the manufacturer's instructions. NanoDrop ND-1000  was used for determinating the RNA quality and quantity. Denaturing agarose gel electrophoresis was used for testing the RNA integrity and gDNA contamination. The remnant RNA was preserved at - 80 °C for later use.Approximately 1-2 μg total RNA from each specimens was used for library construction. Extracted mRNA was enriched with NEB Next® Poly (A) mRNA Magnetic Isolation Module (New England Biolabs). KAPA Stranded RNA-Seq Library Prep Kit (Illumina) was used for the library construction. RNA-seq libraries were subjected to quality control analysis by Agilent 2100 Bioanalyzer. Libraries were quantified by qRT-PCR. Samples were sequenced using Illumina X-ten/NovaSeq. The RNA-seq data were deposited in GEO (Accession code: GSE149315).Illumina X-ten/NovaSeq produced raw sequencing data in FASTQ format. The raw sequencing data were submitted to sequencing quality control by FastQC to assess whether to be used for subsequent data analysis. The trimmed data generated by the pre-processing and filtering steps were mapped to the reference genome. First, StringTie software was used to compare the results to the known transcriptome and calculate the transcriptional abundance. Then, Ballgown was used for quantification of gene expression levels, as well as analysis of intergroup differential expression of genes. The differentially expressed genes were screened with a width of the difference ≥1.5 times, a P-value ≤ 0.05 and an internal FPKM (Fragments Per Kilobaseof gene/transcript model per Million mapped fragments) mean value ≥0.5. Backsplice junction reads count of circRNA was quantified by STAR software through mapping to the reference genome, and its backsplice junction reads detection and reads count statistics were done by the CIRCexplorer2 software. EdgeR package was used for determining the significantly DEcircRNAs with the filter criteria of P-values <0.05 and |log2FC| ≥ 0.585.-ΔCT method. GAPDH was used as internal control. Primer 5.0 software was used for the design of primer sequences in our study. Paired T-test was adopted for analysis of significance between experimental and control group by SPSS 22.0. And P-value < 0.01 indicates a statistical significance.We selected six aseptic loosening synovial tissues, matched with 6 control samples. Total RNA from specimens was isolated by a TRIzol™ reagent  following the manufacturer`s instruction. To quantify the amount of each circRNA, cDNA was synthesized by reverse transcription using SuperScript™ III Reverse Transcriptase (Invitrogen) on Gene Amp PCR System 9700 (Applied Biosystems) from 1 μg of total RNA. Subsequently, ViiA 7 Real-time PCR System (Applied Biosystems) was used for qRT-PCR by 2X PCR master mix (Arraystar) following the manufacturer`s instruction. The relative expression of each circRNA was determined by 2http://www.targetscan.org/vert_72/) http://www.microrna.org/microrna/) https://cytoscape.org/index.html) CeRNA hypothesis that the circRNA shared the same miRNA with mRNA was acted as the theoretical basis for network construction. MREs were the miRNA binding sites which were foundation of ceRNA hypothesis, and crosstalk of RNA transcripts can be caused by competing for common miRNAs with same MREs. Home-made miRNA target prediction software based on TargetScan (http://david.abcc.ncifcrf.gov/) Database for Annotation, Visualization and Integrated Discovery (DAVID)  included in circBase; (2) |log2FC| >2; 3) p-value <0.05; (4) exonic-related circRNAs; and (5) CPM value was not zero. RT-qPCR was performed in 6 pairs aseptic loosening and non- aseptic loosening tissues to validate the expression of the 10 DEcircRNAs. Nine of ten selected DEcircRNAs were the same change tendency though the data from RNA-seq and RT-qPCR. Among them, 1 upregulated circRNA (hsa_circ_0007482) and 5 downregulated circRNAs  were statistically significant and these 6 circRNAs were candidates for further study  were enrichment in cGMP-PKG signaling pathway, and the target mRNAs of downregulated circRNAs  were enrichment in chemokine signaling pathway and Toll-like receptor signaling pathway. According to current study, activation of cGMP-PKG pathway could promote rat osteoblast differentiation and maturation In general, 171 circRNAs were upregulated and 86 circRNAs were downregulated in aseptic loosening synovial tissues compared to non-aseptic loosening synovial tissues. GO and KEGG analysis of the DEcircRNAs host genes shows that PI3K-Akt signaling pathway, Rap1 signaling pathway, and autophagy are the most common terms. CeRNA network was constructed based on the 6 validated DEcircRNAs. Enrichment analysis of the target genes of these 6 validated DEcircRNAs shows that the target genes mainly took part in the Toll-like receptor signaling pathway and chemokine signaling pathway. The signaling pathways mentioned above are closely related to osteolysis. Therefore, the ceRNA network may reveal novel mechanisms for aseptic loosening, and circRNAs may serve as candidates in aseptic loosening. The functions of DEcircRNAs from our RNA-seq data warrant further investigations.


Dear Editor,novel SARS coronavirus which was first isolated from three individuals having pneumonia with connection to the Wuhan epidemic of the severe respiratory illness [zoonotic property. Genomic analysis of the virus has shown that it clusters genetically with the Beta coronavirus genus, alongside two other strains derived from bat. It shares a 96% identity with other bat coronavirus samples (Bat Cov RaTG 13) at the whole genome level. Chinese researchers in February 2020 discovered the amino acid difference in specific parts of the human and pangolin virus genome sequences, however, whole-genome comparison between the pangolin coronavirus, and the 2019-nCoV found a maximum of 92% identical genetic materials, which has so far not been sufficient enough to confirm pangolins to be the viral intermediate host [The 2019-nCoV is a canine coronavirus, the infectious bronchitis virus of birds, and feline coronavirus. Previous efforts aimed at the development of antiviral vaccines for the Coronaviridae family that majorly affects humans that have been targeted at the Middle East respiratory syndrome and severe acute respiratory syndrome coronavirus. The MERS and SARS vaccines have been tried in animal models and up till February 2020, there has been no cure or protective vaccine that has exhibited safety and efficacy in humans [Vaccines have been produced to target several animal coronavirus diseases, which includes the n humans .The historical immunotherapy consensus has been about the targeting of easily accessible antibody-binding extracellular antigens only. The reason for this is because the antibodies which are of higher molecular weight stop the antigens from gaining access to their intracellular targets through the crossing of the cell membrane. In consistence with this thought train, approved therapeutic antibody targets are mostly extracellular antigens . Three bhttps://www.ncbi.nlm.nih.gov/nuccore/MN908947.3?report=fasta) with the accession number “MN908947.3” . The sequence which ranges from the 10055 to 10972 nucleotides of the viral genome was translated, and the amino acid sequence was used in the 3D structural homology protein model prediction. A total of 120 templates were found, and an initial HHblits profile was designed by making use of the outlined procedure in Remmert et al. [With reference to previous virus related in-silico vaccine design studies , 7, we dWe went further to confirm the potential of the predicted B-cell epitope in generating high affinity antibodies through T-cell epitope prediction. This was achieved using the SYFPEITHI prediction server (database for MHC ligands and peptide motifs) . This toN-glycosylation sites on the 2019-nCoV main proteinase were therefore predicted using the NetNGlyc 1.0 prediction tool  . The conpI of 10 indicating an alkaline protein. The half-life assessment was predicted to be 30 h in vitro in mammalian reticulocytes, > 20 h in yeast, and > 10 h in vivo in E. coli. The aliphatic index estimation predicted a score of 52.00, which indicates thermostability. The predicted GRAVY score was − 0.407, indicating a hydrophilic protein which is consistent with the 3D structural view of the protein (Fig. The physiochemical properties of the final peptide as predicted by the Expasy ProtParam server  predicteThe secondary structures of the protein antigenic region as viewed in the Pymol molecular visualizer showed a loop dominated peptide with no helices Fig. . We hereAdditional file 1.http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction?Motif=HLA-A*01&amers=9&SEQU=DTANPKTPKYKFVRI&DoIT=++Run++, https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.


Caulobacter crescentus is a model alphaproteobacterium with a well-studied genetic network controlling its cell cycle. Essential for such studies is an accurate map of the expressed features of its genome. Here, we provide an updated map of the expressed RNAs by integrative analysis of 5′ global rapid amplification of cDNA ends, transcriptome sequencing, rifampicin treatment RNA sequencing, and RNA end-enriched sequencing data sets. Caulobacter crescentus is a model alphaproteobacterium with a well-studied genetic network controlling its cell cycle. Essential for such studies is an accurate map of the expressed features of its genome. Here, we provide an updated map of the expressed RNAs by integrative analysis of 5′ global rapid amplification of cDNA ends, transcriptome sequencing, rifampicin treatment RNA sequencing, and RNA end-enriched sequencing data sets. Caulobacter crescentus GenBank entry (GenBank accession number NC_011916.1) (C. crescentus genome (The operon map presented here is based on the latest 11916.1) , with th11916.1) . The oris genome . For eacGSE157432) to map the coordinates of stable processed RNAs. We used RIF-seq data from 15 min after rifampicin treatment, scanned along the length of the genome, and annotated stable RNAs that were longer than 20 nt, for which each nucleotide was above 30 reads, and as a whole averaged above 43 reads/nucleotide. The average read cutoff value was determined empirically using tRNAs since their biogenesis requires both 5′ and 3′ processing. Rend-seq-derived 5′ ends were not utilized for this analysis since they could arise from RNA decay intermediates. Since RNA-seq coverage can be noisy, if multiple stable RNAs were identified within 20 nt and the RNA-seq reads remained above zero between them, then the regions were manually combined into a single stable RNA feature.Because RNA processing can lead to the generation of stable functional RNAs , 6, we aFinally, we classified operons as simple if the RNA was made from a single TU defined by a single TSS and lacked any observable RNA processing sites . OperonsC. crescentus operons, such as the ctrA operon, have multiple regulatory inputs that help to coordinately regulate the expression of this essential cell cycle master regulator , GSE95211 (Rend-seq data), GSE54883 (RNA-seq data), and GSE157432 (RIF-seq data).The TU map described is accessible at


Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes. In virus-infected cells, captured sequences derived from the host or virus represent a powerful tool to study the mechanisms underlying host-pathogen interactions. We have previously deployed these methods to gain mechanistic insights into the pathophysiology of oncogenic viruses, such as Epstein-Barr virus (EBV), hepatitis B virus (HBV) and human papilloma virus (HPV)5.Advances in, and availability of, high throughput sequencing technologies have enabled the accumulation of detailed molecular level information from cells, including genome variations, gene transcription, and gene regulation. These technologies are extremely sensitive at capturing nucleic acid sequences regardless of their origin. As such, the data from these techniques contain not only sequences encoded by the cell itself, but also sequences encoded by infecting pathogens, and/or common contaminating agents 8. Similarly, in virus-induced cancer cells, chimeric reads that are partly mapped to one gene and partly mapped to another gene are the markers of genomic rearrangement and/or gene fusion10. Thus, chimeric reads can represent real biological events.RNA-sequencing data from virally infected cells contain reads that map perfectly to either the host genome or the viral genome. However, a significant portion of host sequencing reads can also be aligned to discontiguous sections of the genome and often represent canonical forward splicing or back splicing events generated from mRNAs and circular RNAs, respectively. In cells infected with DNA viruses that integrate into the host genome (e.g. HPV or HBV), a chimeric read that is partly mapped to the host genome and partly mapped to the virus genome, is a signature of transcribed segments of the host genome containing integrated viral DNA14 but still not fully understood. In particular, relatively little is known about the processes following viral infection and why some individuals develop little to no symptoms, while others develop life-threatening or persistent (“long”) COVID-19. Recent studies have identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV2 infected cells and samples from COVID-19 patients16. Both studies have suggested that HVC events support potential “human genome invasion” and “integration” by SARS-CoV2. This suggestion has fueled concerns about the long-term effects of current vaccines that incorporate elements of the viral genome17. SARS-CoV2 is a positive-sense single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events.The pathogenic mechanisms underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus responsible for pandemic coronavirus disease of 2019 (COVID-19), are under investigationHere we investigated the presence of HVC events in a large number of currently available RNA-sequencing samples from SARS-CoV2 infected cells and patients with COVID-19. Consistent with previous studies, we found that 0.01–1% of viral mapped reads could be characterized as HVC reads. In contrast to reads originating from known or novel splicing junctions, HVC events were not reproduced between different libraries, suggesting that they are either stochastic or artifactual. By counting chimeric events observed in unrelated human RNA-seq libraries prepared in the same manner but containing spike-in of fruit-fly RNA, we estimated that errors in reverse transcription result in ~1% of RNA-seq reads being artifactually chimeric, approximately the same frequency as observed HVCs from SARS-CoV2 infected cells. Finally, we developed a novel experimental approach to enrich for viral sequences from infected cells during RNA-seq library preparation. Despite achieving >30-fold enrichment of viral sequences using our method, HVC events were not enriched, indicating that the low frequency of HVCs observed are likely introduced during reverse transcription steps of RNA-seq library preparation. In summary, we conclude that current data do not support the authenticity of HVC events in SARS-CoV2 infected samples.16 suggesting the presence of HVC events in SARS-CoV2-infected cells have been interpreted as supporting viral integration into the human genome as a mechanism of viral persistence. To gain insights into the authenticity of these events, we re-analyzed the 3 available RNA-seq datasets from patients with COVID-19 (n=57 samples) and in vitro SARS-CoV2 infected cells (n=64 samples). We categorized sequencing reads to those that perfectly aligned to the human genome (build hg38) in a contiguous or discontiguous manner (i.e. reads originating from one exon or reads spanning exon-exon junctions), those that perfectly aligned to the SARS-CoV2 viral genome, and those that partly aligned to both host and viral genomes  , middle east respiratory syndrome (MERS) and respiratory syncytial viruses (RSV), were similar  . Viral m events) . SARS-Co events) . The fre similar .16  of chimeric events created as artifacts of RT and/or alignment errors. We observed ~1% of all Drosophila-mapped reads to participate in chimeric events  are error-prone and susceptible to a process called random template switchingnome see . Nearly c events . InteresDrosophila chimeras. Similar to what we had observed in SARS-CoV2 infected cells . For comparison, we also prepared cDNAs from RNAs of infected cells without any enrichment  of viral N gene mRNA in enriched libraries compared to the control . Thus, appropriate positive and negative controls during analysis are essential to distinguish real from artifactual events.Sequencing reads from high throughput assays, if appropriately analyzed, are invaluable resources for identifying novel biological events and provide exceptionally detailed information about host-virus interactions occurring SARS-CoV2 is a good example of a novel pandemic coronavirus of humans that is as yet relatively understudied. In particular, little is known about host-pathogen interactions that determine clinical outcomes, including why some people develop little to no symptoms, while others succumb to life-threatening disease and others progress to chronic disease (“persistent” COVID-19). In this setting, sequencing assays are key methods for uncovering as yet undiscovered mechanisms of pathogenesis. Particular attention has focused recently on reports of HVCs forming between host and viral mRNAs, which have been interpreted as evidence of viral incorporation into the host genome and raised questions about long-term safety of vaccination strategies using viral RNA. Here we found several lines of evidence that indicate that the reported HVC events observed in sequencing libraries are most likely artifactual.20. Likewise, we also observed that chimeric events incorporated the more highly expressed host genes. A model consistent with these observations is that HVCs are likely the result of stochastic events occurring at the RNA level that incorporate components of more highly expressed transcripts (templates) from both the host and virus.We found that the precise location and the nucleic acid sequence of HVC events are not reproducible across different libraries prepared by different labs. Consistent with previous reports, we concur the viral part of HVC events to be enriched in sequences from the 3’ end of SARS-CoV2 virus. This is the portion of the virus that contains the most highly expressed gene encoding N-protein18. Not surprisingly, we found that the host part of HVC events was enriched in RNAs with highly repetitive structures, including rRNAs and tRNAs . We found that the frequency of HVC events from all SARS-CoV2 infected samples was below 1%, indicating that these events are likely to be artifacts of RTs. Moreover, repeat sequences of RNA are known substrates for template switchingnd tRNAs . This fuin vitro RTs. A benefit of our technique is that it is a general method that can easily be used for enrichment of any RNA and its chimeric partners, as long as sequences for oligonucleotide design are known . This is particularly useful because cellular RNAs in infected cells typically dominate over RNA derived from infecting pathogens, especially when infection rates and/or viral titers are low. One example for the utility of this method is to help identify “cap-snatching” and “start-snatching” events. In IAV-infected cells, for example, viral transcripts form chimeras with the 5’ portion of host transcripts containing 5’ caps in order to stabilize viral transcripts and create bona fide fusion proteins22. Although there are computational challenges in aligning sequencing reads if very short fragments (<18 bp) are “snatched” from the host, one would anticipate seeing enrichment of host 5’ UTR elements in HVC events if similar cap-snatching mechanisms were utilized by SARS-CoV2. However, we observed quite the opposite, if any  cells  were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% Fetal Bovine Serum , HEPES, non-essential amino-acids, L-glutamine and 1X Antibiotic-Antimycotic solution (Gibco). All cells were maintained at 37°C and 5% CO2. SARS coronavirus 2 (SARS-CoV-2), isolate USA-WA1/2020 (NR-52281) was obtained from Bei resources and was propagated in Vero E6 cells in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM Non-Essential Amino Acids, 1 mM Sodium Pyruvate and 10 mM HEPES. Infectious titers of SARS-CoV-2 were determined using TCID50 method (Reed and Muench). Fifty thousand Calu-3 were seeded in 48 well plate and allowed to form 80% confluent monolayer. SARS-CoV-2 virus was pre-treated with porcine trypsin (10ug/ml) for 15 minutes at 37 degrees. Cells were then infected with the pre-treated virus prep at a multiplicity of infection (MOI) of 10 for 1h in culture media . After absorption, virus inoculum was removed and replaced with fresh culture media. 48hrs post infection, cells were harvested and RNA was isolated. Briefly, infected cells were lysed in TRIzol (Invitrogen) and RNA was extracted using Direct-zol RNA Miniprep kit (Zymo Research) as per manufacturer’s instructions. Experiments using SARS-CoV-2 were performed at the University of Michigan under Biosafety Level 3 (BSL3) protocols in compliance with containment procedures in laboratories approved for use by the University of Michigan Institutional Biosafety Committee (IBC) and Environment, Health & Safety (EHS).in vitro amplification steps using SARS-CoV2 virus specific primers (VSP) as following. The VSP pool contained ~30 oligonucleotides that span all SARS-CoV2 genes (at least one oligo per gene and nearly 1 oligo per 1kb of the genome). Given our goal to additionally enrich for potential HVC events, we used two approaches, enrichment methods 1 (5’->3’ host-virus chimeras) and enrichment method 2 (5’->3’ virus-host chimeras)  .™ RNase A (20 mg/mL)  and 1 μl (5 units) of RNase H  and incubated at 37°C for 1 hour. We then purified cDNA using 1X Mag-Bind TotalPure NGS  as per manufacturer’s instructions and eluted the cDNA in 15 μl of sterile water. To further remove the excess single-stranded short oligos, we treated the purified RT reaction with 1 μl of Exonuclease I  at 37°C for 30 minutes. Next, we added excess sterile water to sample to get a total volume of 40 μl. The reaction was them purified with 1X Mag-Bind TotalPure NGS beads and eluted in 15 ul of sterile water.To capture and enrich for viral, virus-host or host-virus transcripts, we first set up a 20 μl reverse transcription (RT) reaction using 100 ng of mRNA isolated from SARS-CoV2 infected using Superscript III reverse transcriptase. We used 2 pmol T7-P5-VSP oligo pool (for enrichment 1) or 50 pmol of T7-P5-Oligo dT (for enrichment 2) as “gene-specific primer” for RT reaction see . We alsoFollowing this, we performed second strand synthesis using the NEBNext Ultra II Non-Directional RNA second strand synthesis module as per the suggested protocol . The synthesized DNA was purified via 1X Mag-Bind TotalPure NGS beads and eluted in ~12 ul of sterile water. 10 ul of this was then used as an input for T7 polymerase mediated In vitro Transcription (IVT) using the NEB HiScribe T7 High Yield RNA Synthesis Kit . Briefly, all the components were mixed as mentioned in the kit protocol and incubated at 37°C (lid at 50°C) for 16 hours. The reaction was eluted in 20 μl of sterile water after a round of 1X Mag-Bind TotalPure NGS bead cleanup. This newly transcribed RNA was quantified using a Nanodrop and to improve the hybridization kinetics and enhance signal, 500ng of the amplified RNA was fragmented by RNA Fragmentation reagent in a total reaction volume of 10 μl as per specifications .Next, to generate final enriched libraries, we performed reverse transcription of the fragmented RNA with 50 pmol of P7–N6 for enrichment (1) and 2 pmol of the P7-VSP primer pool for enrichment  respectively see  using SS−dCt method and normalized to that of indicated housekeeping gene in the same sample.The enrichment of viral genes was determined by performing a qRT-PCR assay on the libraries generated. Briefly, the cDNA generated by RT, prior to library amplification by Q5-PCR was diluted 10 −20 fold and used to amplify target gene ‘N’ of SARS-Cov2 using CDC recommended primers for 2019-nCoV_N1-F: 5’-GAC CCC AAA ATC AGC GAA AT-3’, 2019-nCoV_N1-R: 5’ TCT GGT TAC TGC CAG TTG AAT CTG-3’, 2019-nCoV_N2-F: 5’ TTA CAA ACA TTG GCC GCA AA-3’, 2019-nCoV_N2-R: 5’ GCG CGA CAT TCC GAA GAA3’ and UBC as the housekeeping gene, UBC-F: 5’-CCT GGA GGA GAA GAG GAA AGA GA-3’ and UBC-R: 5’-TTG AGG ACC TCT GTG TAT TTG TCA A- 3’. The qRT-PCR was performed on Bio-Rad CFX connect. All experiments were performed in independent triplicates in total reaction volumes of 15 μl using PowerUp SYBR Green Master Mix . The expression was calculated by the 2The raw sequencing files were download from the Sequence Read Archive (SRA) as shown in the To estimate the background level of chimeric reads in RNA-seq libraries, a fruit-fly RNA spike-in control libraries (PRJNA311567) were used. Briefly, a chimeric genome between human (hg38) and fruit-fly chr4 (dm6) was constructed and the sequencing reads were aligned by the STAR aligner using the same parameters as --outFilterMultimapNmax 1 --outFilterMismatchNmax 3 --chimSegmentMin 30 --chimOutType Junctions SeparateSAMold WithinBAM SoftClip --chimJunctionOverhangMin 30 --chimScoreMin 1 --chimScoreDropMax 30 --chimScoreJunctionNonGTAG 0 --chimScoreSeparation 1 --alignSJstitchMismatchNmax −1 −1 −1 −1 --chimSegmentReadGapMax 3.The known annotated and novel unannotated splicing junctions were extracted from the STAR output as positive controls. The chimeric junctions for human-virus and human-fly were extracted from the STAR chimeric output. The unique chimeric junctions were considered as our chimeric events. To estimate the reproducibility, for each independent study and each cell type, the number of unique junctions were extracted. For every cell type, the mean value of overlapping junctions from two independent studies to the number of junctions in each study was recorded as the reproducibility.To examine the genomic features of the HVC reads, HOMER (v4.11) annotatePeaks.pl was used to annotate the HVC junctions and the corresponding RNA-seq library. In brief, reads in each RNA-seq library were converted to genomic regions by bamTobed  and the unique regions were kept using the following command“sort -k1,1 -k2,2n | uniq”. The reported “Log2 Ratio (obs/exp)” for each annotation  were compared between HVC junctions and the corresponding RNA-seq library. Mann-Whitney’s U test was used for statistical analysis.Supplement 1Supplement 21