Earth’s life may have originated as self-replicating RNA, and it has been argued that RNA viruses and viroid-like elements are remnants of such pre-cellular RNA world. RNA viruses are defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRp), whereas viroid-like elements consist of small, single-stranded, circular RNA genomes that, in some cases, encode paired self-cleaving ribozymes. Here we show that the number of candidate viroid-like elements occurring in geographically and ecologically diverse niches is much higher than previously thought. We report that, amongst these circular genomes, fungal ambiviruses are viroid-like elements that undergo rolling circle replication and encode their own viral RdRp. Thus, ambiviruses are distinct infectious RNAs showing hybrid features of viroid-like RNAs and viruses. We also detected similar circular RNAs, containing active ribozymes and encoding RdRps, related to mitochondrial-like fungal viruses, highlighting fungi as an evolutionary hub for RNA viruses and viroid-like elements. Our findings point to a deep co-evolutionary history between RNA viruses and subviral elements and offer new perspectives in the origin and evolution of primordial infectious agents, and RNA life. RNA viruses are defined by linear RNA genomes encoding an RNA-dependent RNA polymerase, while viroid-like elements consist of small, single-stranded, circular RNA genomes that, in some cases, encode self-cleaving catalytic RNAs. Here, the authors identify over 20,000 candidate viroid-like elements, and show that infectious agents of fungi display hybrid features of viroid-like RNAs and RNA viruses. Further, extant sub-viral and viral agents have been suggested to be “living fossils” that can help illuminate the molecular biology of Earth’s first life5.Serving dual functions as encoding for genetic information, and as a biological catalyst, RNA has been proposed to predate DNA and protein at the origin of life in an “RNA World”Riboviria) are infectious agents defined by a linear RNA genome encoding one of their hallmark replication polymerases (replicases), either an RNA-dependent RNA polymerase (RdRp) for RNA viruses, or a Reverse Transcriptase (RT) for retroviruses. These replicases, and thus Riboviria, are monophyletic in nature as based on alignment of the conserved palm domain of RdRp and RT6.Viruses having RNA genomes 7, hairpin (HPRz)8 and hepatitis Delta (DVRz) ribozymes10. In contrast to RNA viruses, infectious circRNAs replicate by RNA polymerases encoded in trans by the host (viroids and hepatitis Delta viruses) or helper viruses (satellite circRNAs). Additionally, while viroids are non-coding RNAs4, deltavirus-like RNAs code for the conserved Delta Antigen proteins and have been assigned the realm Ribozyviria11. Overall, the simple genomes and small ribozymes of infectious circRNAs are why these elements are regarded as molecular fossils of the prebiotic world4.Viroids and viroid-like entities , are infectious sub-viral RNA agents of plants and animals Fig. . These s13.Ambiviruses are a recently characterized and widespread group of fungal single-stranded RNA infectious agents with a bicistronic, ambisense genome of ~5 kb encoding for two conserved open reading frames (ORFs) A and B, one in each polarity strand Fig. . ORF-A hHere we report that the number of potential new viroid-like circRNAs occurring in geographically and ecologically diverse niches is much higher than previously thought. Among them, we chose to investigate the most fascinating elements, the ambiviruses. We show that they have circRNA genomes and encode paired self-cleaving ribozymes active in vivo, enabling a rolling circle mechanism of replication, the typical features of viroid-like RNAs reported so far. However, we further show that ORF-A has the typical features of a functional RdRp, like that of a classical RNA virus. Thus, ambiviruses appear to have likely arisen from the genetic recombination of a viroid-like genomic backbone with the hallmark RNA virus gene RdRp, and as such are an intermediate class of infectious RNAs. This finding bridges the RNA virospheres of viruses and viroids, posing deep questions regarding their origins and (co-)evolution.Serratus ultra-high-throughput computing architecture previously used for RNA virus discovery14 to search for ribozymes using INFERNAL across 198,194 raw metagenome/metatranscriptomes freely-available in the Sequence Read Archive16. Combining the top 5000 ribozyme-hit libraries with our previous RNA virus assemblage and the Transcriptome Shotgun Assembly (TSA), we created the “RNA Deep Virome Assemblage” (RDVA) of 58,557 libraries. From the 12.5 billion assembled contigs, 34 million contained 5’–3’ k-mer overlaps consistent with a circular molecule. Filtering the potential circular contigs further for those encoding both a plus-sense ribozyme and a negative-sense (paired-ribozymes), resulted in a discovery set of 32,393 ribozyme-bearing potentially circular elements, which clustered into 20,364 units at 90% nucleotide sequence identity. Based on their sizes, the vast majority of the clusters group into distinct populations of either small- (70% are 200–800 nt long) or medium-size genomes (25% are 800–2 kb)  unveiled sequence similarity with diverse ambivirus genomes, suggesting that their unusual features derived from 5’ RACE analyses could be due to a circular genome  revealed up to 974 unique RdRp-encoding ambiviruses, which clustered into 439 sOTUs at 90% palmprint amino acid identity19. Again, these genomes were found widespread in diverse environmental datasets  that these proteins take on the classic right-handed palm domain architecture, with highest structural similarity to negarnavirus RdRps   (two HHRzs), Tulasnella ambivirus 4 (TuAmV4) (one HHRz and one HPRz) and TuAmV1 (two HHRzs). For all tested sequences, transcripts showed self-cleavage, and termini of resultant fragments are consistent with the predicted ribozyme cleavage sites , so far only described in the mitochondrial VS plasmid of some Neurospora isolates23. The self-cleaving activity of mitovirus VSRzs was experimentally confirmed, whereas the predicted secondary structure of these agents was found to be highly branched , fungi (ambiviruses), plants (viroids), or animals (delta-like viruses), these still limited host-distributions likely reflect our ignorance of the full diversity of infectious circRNAs across all kingdoms.While computational advances will drive the exponential discovery of these elements (similar to RNA viruses)26. The phenotype associated to CpVA1 infection also hints at the possibility that these viroid-like infectious agents are new components of the holobiont and contribute to intraspecific phenotypic variability. Furthermore, these agents (ambivirus in specific) could develop into circRNA expression vectors, a further step into protein expression stabilization from RNA templates27.Our in vivo demonstration that fungi harbor infectious circRNAs expands the experimentally validated hosts for viroid-like molecules beyond plants and animals to also include the kingdom fungi. With their multiple symbiotic relationship with plant and animal hosts, fungi are indeed a hub for generating trans-kingdom horizontal transfer of infectious elements such as those described here28, the disposition of viroid-like elements to swap ribozymes (as shown here), and the limitations of phylogenetic analysis, the deep evolutionary relationships between infectious circRNAs (such as the genomic origin of ambiviruses) is likely unknowable. Further, structurally simple ribozymes, such as HHRz can arise spontaneously in evolution29, as is the case in relatively simple head-to-head ligated RNAs, supporting the notion that the simplest classes of infectious circRNAs may be evolving de novo continuously. As viroid-like circRNAs can acquire genes, such as a delta-antigen or viral RdRp, and give rise to more complex entities, it follows that host RNA polymerases may have been exapted to give rise to Earth’s primordial “viruses”. Perhaps most tantalizingly, the acquisition of an early RNA replicase ribozyme would serve as a blueprint for the simplest replicator of the RNA world, and thus a major step in determining the origins of life4.Given the rapid mutation rate of viroids14, with the INFERNAL18, an algorithm for identifying RNA elements using secondary structure and nucleotide covariation modelling. Using Serratus-INFERNAL , we performed a screen of 198,194 publicly available metagenomic/metatranscriptomic sequencing libraries in the Sequence Read Archive (SRA) for the delta, hairpin, hammerhead, twister, and Varkud satellite ribozymes . The SRA search query was randomly sub-sampled from the set of “METAGENOME” OR “METATRANSCRIPTOME” OR “metatranscriptomic”[Filter] OR “metagenomic”[Filter] NOT amplicon [All Fields] AND “platform illumina”[Properties] AND cluster_public[prop]” on 2022-01-13. We then heuristically rank ordered the libraries based on the quality and number of distinct ribozyme hits in a library as well as sequencing depth for focused sequence assembly of the SRA. Candidate viroid-like and ribozyviria sequences were identified by the following criteria, all of which must be satisfied: (1) the presence of 5’–3’ end k-mer overlaps suggestive of a circular molecule, (2) a pair of complementary hits to one ribozyme model with E-value <1e−3, and (3) <25% repetitive sequence according to the union of hits reported by dust30, Tandem Repeat Finder31 and self-hits according to ublast32. Clustering into 90% identity species-like Operational Taxonomic Units was performed using circuclust (https://github.com/rcedgar/circuclust).Observing that ribozymes are sufficiently short to be captured on a short sequence read (less than 100 nt), we reasoned it will be possible to screen large volumes of sequencing data to identify libraries potentially containing ribozyme agents. To this end we adapted the ultra-high throughput Serratus cloud computing platformhttps://github.com/rcedgar/palmscan) capable of ambivirus RdRp detection. Palmscan searches were performed in the circular contigs from the RDVA, which together with the ones obtained from GenBank resulted in 439 ambiviral RdRps at 90% sequence identity (https://github.com/ababaian/serratus/wiki/ambivirus_extended_data/). We performed multiple sequence alignment on the clustered RdRp palmprint sequences using MUSCLE33 and maximum-likelihood tree is inferred from the alignment using MEGA1134. Secondary RNA structures of minimum free energy of the ambivirus genomes were calculated with the RNAfold program from the ViennaRNA Package35. The obtained structures were visualized by circular plots obtained with the jupiter software (https://github.com/rcedgar/jupiter).RdRps (ORF-A) from either the 19 deposited ambivirus genomes at the GenBank (09-2021), or the 169 ambiviral-like sequences (either putative full circles or genomic fragments) detected though INFERNAL in public metatranscriptomic datasets (“metatranscriptome” OR “metatranscriptomic”[Filter]), were used to build a consensus sequence for the ambivirus motifs A, B and C. Based on the obtained consensus sequence for the ambiviral palm domain, we built a new version of the palmscan software (Castanea sativa) sticks of 8 cm length  autoclaved at 121 °C for 1 h. 50 mL of the mix was transferred into 100 mL conical flasks for all the samples. Then, 12.5 mL of the liquid media was collected in 50 mL conical FalconTM tubes (CorningTM Fisher Scientific) where 30 mycelia plugs were transferred for each of the replicates. The tissues were ruptured with Omni Tissue Homogenizer (Qiagen) for 1 min and 3.5 mL of the homogenized paste was inoculated in each of the conical flasks containing the liquid media. The cultures were kept constantly shaking at 120 rpm at room temperature.To obtain fungal mycelia for molecular analysis, three CpAV1-free and three CpAV1-infected isolates were inoculated in liquid media in biological triplicate (18 samples). The media contained a mix of 24 g/L potato dextrose broth (PDB) (SIGMA) with two European chestnut  connected to a vacuum pump to separate the mycelia from the liquid media; the harvested mycelia were frozen at −80 °C and lyophilized for 45 h (EDWARDS MODULYO freeze dryer). Mycelial dry weight for each replicate was determined with a top pan balance.Tulasnella spp. isolates were grown in liquid cultures and harvested and lyophilized as above.TM homogenizer from MP BiomedicalsTM for 30 seconds at setting 6. A 1 mL mixture of lysis buffer and 2-mercaptoethanol (Sigma-Aldrich) at 1:100 volume dilution was added to the samples and mycelia was extracted by another round of bead beater, as above; the sample was then centrifuged at 10,000 × g for 4 min. 700 μL of the supernatant was then transferred to filter column tubes provided in the kit and centrifuged for 30 s at the same speed. The columns were discarded and 700 μL of the binding solution was added to the flow-through. This mixture was then passed through the binding column by a further centrifugation as above. The column was collected, transferred to a fresh collection tube and washed twice with wash buffer I and II . RNA was then eluted in a 40 μL elution buffer. RNA quantification was done with Nanodrop LITE Spectrophotometer (Thermo Fisher Scientific).RNA extraction was done with Spectrum Plant Total RNA kit (SIGMA-Aldrich) following the manufacturer’s protocol with minor modifications: briefly, lyophilized mycelia (100 mg) was broken in 1.5 mL conical microcentrifuge tubes with O-rings (Biosigma) with 10, 4.5 mm diameter, ceramic beads in a FastPrep-24C. parasitica strain ACP3413 and TuAmV1 and TuAmV4 from Tulasnella spp. (respectively from strains MUT4048 and MUT4047)12 using the Hirzmann method36. Briefly, cDNA of the 5’ end of the positive and negative sense of the ambivirus genome was synthesized with the First Strand cDNA synthesis Kit #K1612 from Thermo Scientific with a specific primer  with a final elution volume of 10 µL and 1 µL of the obtained cDNAs was used as template to perform a PCR reaction with Taq polymerase (OneTaq kit from Biorad) following manufacturer’s protocol using a specific primer and anchored poly-T or poly-C primer complementary to the added tail at 51 °C in the annealing step for 30 s, 15 s at 68 °C for extension step and 98 °C for the denaturation step for 15 s. The resulting amplicons were fractionated by gel electrophoresis in 1% Agarose 1× TAE buffer and purified using a Zymo gel DNA recovery kit  and cloned in pGEM-T easy vector system  following manufacturer’s instructions. Ligation products were transformed in DH5 alpha E. coli cells prepared and transformed following exactly the protocols and materials of the Mix&Go! E. coli Transformation Kit (Zymo Research). Sanger sequencing of the obtained clones  was performed by Biofab research  using the specific primers detailed in Supplementary Data Rapid Amplification of cDNA Ends (RACE) analysis was performed on CpAV1 from 37 which is optimized for the sequencing of double-stranded RNA, but all failed. Briefly, a 5’-phosphorylated, 3’-amino-linked oligodeoxy-nucleotide primer was ligated to the 3’ termini of the total extracted RNA  with T4 RNA ligase at 4 °C overnight. The ligated RNA was cleaned in column , the cDNA was synthetized  with a primer complementary to the ligated adaptor, cleaned using Zymo clean and concentration 25 kit  with a final elution volume of 25 µL; 1 µL of the obtained cDNAs was used to perform a PCR reaction using a specific primer and the primer used for the cDNA synthesis with the same conditions described above, except the annealing temperature at 55 °C.Attempts were performed to directly sequence the 3’ end of both the positive and negative sense of the CpAV1 and TuAmV1 ambivirus genome by a method modified from Lambden et al.38 then their 5’ terminal sequence was determined by 5’RACE as described above using the primers indicated in Supplementary Data Self-cleavage activity of HHRz and HPRZ of representative ambiviruses  and VSRz of the mito-like virus detected in the SRR10849745 library was tested in vitro by transcription of recombinant plasmids containing the cDNA of the respective ribozymes. The cDNAs of viral RNA fragments containing the ribozymes were either amplified using the appropriate primers listed in Supplementary Data 12. Agarose gel electrophoresis of RNA preparations under non-denaturing conditions was carried out in 1× TAE without denaturation prior to loading.Agarose (1%) gel electrophoresis under denaturing conditions of total RNAs was performed using 6 M glyoxal and 50% v/v DMSO in HEPES EDTA buffer, keeping the RNA samples at 55 °C for 20 min prior to loading, following the protocol described previouslyAfter electrophoresis, RNA was blotted on Immobilon-Ny+ membrane  and crosslink was performed in a Biorad gs gene linker UV chamber . The membranes were hybridized with digoxigenin (DIG)-labeled riboprobes specific for (+) or (−) polarity strands of CpAV1 that were synthesized by in vitro transcription using the Dig-RNA labeling mix  and linearized plasmids containing CpAV1 cDNA inserts from positions 2798 to 3088 (located in ORFA) and from positions 1628 to 1998 (located in ORF B), respectively. Pre-hybridization and hybridization were performed in DIG easy Hyb  buffer at 65 °C. After hybridization, the membranes were washed twice in 2× SSC , 0.1% SDS at room temperature for 10 min, and twice in 0.1× SSC, 0.1% SDS at 65 °C for 15 min. The hybridization signals were revealed with the anti-DIG alkaline phosphatase conjugate AB fragments and the chemiluminescence substrate CDP-star  following the manufacturer’s instructions and visualized with a Chemidoc Touch Imaging system .TM 25 Zymo Research) and a standard qPCR was performed using the same primers: briefly, qPCR reaction was performed in 10 μL final volume using the SYBR® Green Master Mix (Biorad). Each 10 μL reaction mix contained 5 μL mix, 4 μL of sterile water and 0.2 μL of forward and reverse primers  to each samples and incubating at 65 °C for 20 min. Denatured samples were then analyzed through denaturing agarose gel electrophoresis and northern blotting as reported above.RNAse R (Lucigen) was used to test the circular nature of the ambivirus genomes on CpAV1. Samples containing 2 µg of total RNA from infected Castanea sativa) with evident cankers in Azerbaijan13. The C. parasitica isolates were maintained on PDA media at 4 °C.Isolate -ACP34- was originally collected from a diseased tree (−1 to 1:10−10). Single spore isolates were transferred after 5 days, and single fungal colonies were tested for ambivirus presence/absence by qRT-PCR. cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit  following the manufacturer’s protocol.Isolate ACP34 was inoculated in 90 mm Petri plates with PDA for two months and sixteen days with light/dark cycles of 12 h. Conidia were harvested from the culture by pipetting 10 ml of sterile water in the Petri plate for two minutes and scrubbing the mycelia twice at 1-min intervals. The spore suspension aliquot was collected and filtered with cotton placed in a 5 mL tip. Isogenic monoconidial isolates were obtained by serially diluting the harvested spore suspension and plating 300 mL of the diluted conidia in 150 mm Petri plates with serial dilutions . The PCR reaction was performed in 10 μL using the I-Taq supermix (Biorad) and Taqman probe. Each 10 μL reaction mix contained 5 μL supermix, 0.15 μL probe, 4 μL of sterile water and 0.2 μL of forward and reverse primers . The isolates were inoculated in 150 mm Petri plates and incubated in potato dextrose agar (PDA) (Sigma-Aldrich) at 26 °C for 14 days. For each isolate we included three biological replicates and mycelia diameters were measured for all isolates at three days intervals post-inoculation until the full diameter of the Petri plate is covered.Castanea sativa in Salò-BS, Italy). Each stem cutting was disinfected with 70% ethanol then the cuttings were coinfected with virus-positive and negative mycelia plugs at a 17 cm distance from each other. The experiment was performed with three biological replicates randomly distributed to have CpAV1-infected and CpAV1-free mycelia plug at the upper and lower side of the stems. A cork borer of 5 mm diameter was used to artificially displace a section of the bark. Mycelia plugs were then placed under the removed barks and tightly taped for wound closure. Also, negative controls without mycelia plugs but with similar wounds were made. The cuttings were stored in 1.5 L of vermiculite soaked with water for 21 days.Live chestnut stems of 30 cm were prepared for inoculating the isogenic CpAV1-free and infected isolates . Cuttings to be used in the experiment were obtained from chestnut suckers from a chestnut orchard  were also used to verify virulence. Each apple was inoculated in 4 points with a CpAV1 infected and a CpAV1 free strain . Inoculated apples were kept at room temperature for 14 days.39. Significant differences were assessed with Tukey’s post hoc test (p < 0.05).Analyses of the data referred to the measures of lesion or canker size in the virulence assays (apples and chestnut cuttings) were performed with one-way analysis of variance (ANOVA) using the R-statistical programRhizoctonia solani isolates40 and PRJNA629308 corresponding to an orchids and ericoid mycorrhizal fungi collection12 using the same pipeline described above. All the selected contigs were checked in each of the biological samples to track contig-positive samples using the primers in Supplementary Data We screened HTS read libraries present in two bioprojects: PRJNA524447 corresponding to a collection of Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The RNA polymerase III (Pol III) transcriptome is universally comprised of short, highly structured noncoding RNA (ncRNA). Through RNA–protein interactions, the Pol III transcriptome actuates functional activities ranging from nuclear gene regulation (7SK), splicing , and RNA maturation and stability , to cytoplasmic protein targeting (7SL) and translation . In higher eukaryotes, the Pol III transcriptome has expanded to include additional, recently evolved ncRNA species that effectively broaden the footprint of Pol III transcription to additional cellular activities. Newly evolved ncRNAs function as riboregulators of autophagy (vault), immune signaling cascades (nc886), and translation . Notably, upregulation of Pol III transcription is frequently observed in cancer, and multiple ncRNA species are linked to both cancer progression and poor survival outcomes among cancer patients. In this review, we outline the basic features and functions of the Pol III transcriptome, and the evidence for dysregulation and dysfunction for each ncRNA in cancer. When taken together, recurrent patterns emerge, ranging from shared functional motifs that include molecular scaffolding and protein sequestration, overlapping protein interactions, and immunostimulatory activities, to the biogenesis of analogous small RNA fragments and noncanonical miRNAs, augmenting the function of the Pol III transcriptome and further broadening its role in cancer.This article is categorized under:RNA in Disease and Development > RNA in DiseaseRNA Processing > Processing of Small RNAsRNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications In humaBeyond its central role in translation, tRNAs engage in multiple noncanonical functions, many of which are actuated by tRNA-derived small RNAs (tsRNAs), small regulatory RNAs that go on to drive changes in gene expression, translation, and other biological processes. tsRNAs include tRNA halves generated by Angiogenin-mediated cleavage of tRNA anticodons, and multiple subgroups of tRNA-derived fragments (tRFs), generated by Dicer, RNase Z, and other RNase enzymes, which are classified based on biogenesis . As an RARGCCG and tRNAGluUUC, are upregulated in highly metastatic breast cancer cells and increase the translation of oncogenic transcripts bearing cognate codons, driving a proteomic shift that promotes metastatic progression (iMet), which alters tRNA expression profiles and increases cell proliferation . 5S rRNA is fundamentally important for both ribosomal assembly and function and, in humans, is encoded by repetitive arrays of canonical genes largely organized within clusters of tandem repeats on chromosome 1 . 5S rRNAIn addition to chaperone protein La, 5S rRNA interacts with ribosomal proteins RPL5 and RPL11 and together assembles a pre-ribosomal 5S RNP that is subsequently incorporated into nascent 60S ribosomes in the nucleolus. 5S rRNA also directly interacts with PICT1, RRS1, and BXDC1, factors involved in 5S RNP localization and ribosome incorporation , and witWhether upregulation of Pol III transcription, a hallmark of cancer, contributes to cell proliferation status through a non-ribosomal 5S rRNA-HDM2/4 regulatory axis requires further investigation. However, mutation and/or misregulation of p53, HDM2, and HDM4 occur in most human cancers , and rec3 |Most small nuclear RNA (snRNA) species are generated by Pol II transcription, however, Pol III transcribes genes encoding 7SK snRNA, the molecular scaffold of the 7SK snRNP involved in Pol II transcription regulation, and U6 and U6atac snRNA, components of splicing machinery retained in the nucleus. 7SK snRNA was first described as a predominantly cytoplasmic small RNA in HeLa cells , though 3.1 |7SK is derived from a single constitutively active Pol III-transcribed gene possessing a “type 3” promoter architecture that relies on proximal and distal upstream regulatory elements instead of gene internal elements . NascentThe highly abundant Pol III-derived 7SK snRNA engages in a sophisticated, dynamic network of protein interactions central to its role in sequestering P-TEFb. The core 7SK snRNP complex is comprised of LARP7, a La-related protein that similarly interacts with 3’-uridines in 7SK snRNA , and MePThe P-TEFb-bound 7SK snRNP itself is reversibly regulated through stress and growth signaling pathways, which can facilitate the release and re-activation of P-TEFb . ProteolAs a generally repressive regulator of transcription, both 7SK and the 7SK-snRNP have been described as putative tumor suppressors: loss of LARP7 and the 7SK snRNP leads to upregulation of epithelial-mesenchymal transition (EMT) associated genes and increased invasion and metastasis in breast cancer , and exo3.2 |6A and m2G methylation at specific residues  ncRNA is the catalytic RNA component of RNase P, the site-specific endonuclease responsible for the cleavage of pre-tRNA 5’-leader sequences previously discussed . The clo4.1 |RPPH1 gene is also occupied by RNA polymerase II (Pol II) and sensitive to Pol II inhibition, suggesting both Pol II and Pol III contribute to the biogenesis of RPHH1 ncRNA  is 341 nucleotides in length and generated from a single gene with type 3 upstream promoter elements . The RPPH1 ncRNA . While cH1 ncRNA . RPPH1 sH1 ncRNA .RPPH1 ncRNA is bound by an ensemble of protein subunits, including RPP14, RPP20 RPP21, RPP25, RPP29, RPP3’, RPP38, RPP40, hPOP1, and hPOP5  which fuIndependently of RNase P, RPPH1 ncRNA interacts with additional proteins as well as specific miRNAs to functionally promote cell growth and proliferation, and growing evidence suggests that these mechanisms drive tumorigenesis and metastasis in a variety of cancers. RPPH1 is upregulated and associated with poor survival outcomes in patients with gastric cancer , colorec4.2 |6A methylation enhances RMRP stability . Mitochondrially imported RMRP is retained through interactions with an additional RNA-binding protein, GRSF1, ostensibly important for RMRP-mediated mitochondrial DNA replication priming . Non-nucLike RPPH1, RMRP ncRNA is similarly overexpressed and associated with unfavorable prognoses across a multitude of cancer contexts, and evidence suggests RMRP also contributes to proliferation through novel protein interactions and miRNA sequestration. RMRP is upregulated in non-small cell lung cancer (NSCLC) and promotes proliferation, invasion, and migration by interacting and recruiting YBX1 to the TGF-β receptor 1 (TGFBR1) promoter, upregulating TGFBR1 and enhancing the TGFBR1/SMAD2/SMAD3 growth signaling pathway . RMRP is5 |BCYRN1, for example, is evolutionarily derived from an Alu monomer sequence that underwent a series of substitution and insertion events (BCYRN1 (BC200) shares strong sequence similarity with 7SL and Alu but includes more recently evolved, divergent sequence and structure features in its 3’-end  RNA, 7SL, functions as a core component of the SRP RNP, which recognizes and directs presecretory proteins to translocation machinery at the ER membrane . In human events . As suchs 3’-end . Like BCs 3’-end , evolveds 3’-end . Like 7S5.1 |First recognized as a low molecular weight RNA component in oncornaviruses in the 1970s, 7SL ncRNA was subsequently identified as an endogenous human cytoplasmic small RNA in 1976 . 7SL RNAAs described for RMRP, 7SL is bound by DIS3L2, and accumulation of 3’-uridylated 7SL in cells lacking DIS3L2 suggests that modified and potentially misfolded 7SL RNA is ordinarily recognized and turned over by cytoplasmic DIS3L2-mediated decay . 7SL strUpregulation of 7SL RNA is observed in multiple cancer contexts and, like Pol III-transcribed 5S rRNA and RMRP, 7SL also promotes cell proliferation through modulation of p53 homeostasis . Specifi5.2 |AluI (Alu repeats represent the most abundant subtype of short interspersed nuclear elements (SINEs), a non-autonomous class of retrotransposons constituting over 10% of the human genome . First dAluI , Alu eleAluI . Alu eleAluI . Alu eleAluI .While most Alu elements are epigenetically repressed by histone H3K9 trimethylation , a limitIn contrast to the inhibitory activities described for Alu RNA and the Alu RNP, overexpression of Alu ncRNA in cancer and other disease states leads to changes in transcription, translation, and cell proliferation in varying ways. Ectopic expression of specific Alu sequences in IMR90 cells reprograms the expression of cell cycle genes and promotes transition from G1 to S phase . This co5.3 |First discovered as a 200-nucleotide cytoplasmic RNA in monkey and human brain tissues , BC200 RBC200 is upregulated in cancer and shown to promote cell proliferation, invasion, and migration through multiple mechanisms. In MCF-7 cells, overexpression of BC200 leads to increased global translation and proliferation, whereas targeted downregulation decreases translation and cell growth, contradicting a generalized role for BC200 in translation repression . Knockou5.4 |Small NF90-associated RNA (snaR) was first identified as a novel ncRNA upregulated in Epstein–Barr virus (EBV)-infected human B cells , and shoDespite the comparatively limited understanding of snaR RNA interactions and function, studies examining the expression of snaR-A and its effects on cancer proliferation uncover a common pattern. In patients with ovarian cancer, snaR RNA is significantly upregulated and positively associated with the expression of GAB2, a signaling protein involved in transducing growth factors and cytokine responses . snaR-A 6 |Y RNA was first discovered as an RNA component of Ro60 RNPs immunopurified by autoantibodies derived from patients with a form of systemic lupus . RNA-binThe protein interactome of Y RNA includes numerous factors that altogether implicate Y RNA in a wide range of cellular processes beyond Ro60 RNP function. In the nucleus, Y RNA interacts with replication initiation proteins, likely in complex , and mayAs described for other Pol III-derived ncRNA, Y RNA is significantly overexpressed in multiple cancers, including solid tumors derived from bladder, cervix, colon, kidney, lung, and prostate cancers , and alt7 |Vault RNAs (vtRNAs) were discovered as stably associated small RNA components of vault RNPs , massive7.1 |VTRNA1-1, VTRNA1-2, and VTRNA1-3, located on chromosome 5. Vault RNA genes possess type 2-hybrid promoter architectures that contain gene internal as well as additional upstream regulatory and response elements  element located downstream  and additional proteins, TEP1 and VPARP. The vault RNP features a massive barrel-like structure that is capable of rapid redistribution in response to external stimuli and is hypothesized to facilitate protein exchange between the cyto-plasm and nucleus . TEP1 biIn addition to the pro-survival role of vtRNA1–1, overexpression and knockout experiments demonstrate that vtRNA1–1 promotes cancer cell proliferation as described for most of the Pol III transcriptome . A uniqu7.2 |VTRNA1–1, VTRNA1–2, and VTRNA1–3  binding by chaperone La are likely events. nc886 RNA is also enriched in NSUN2 IP experiments  based on the capture of small, putative miRNA species, miR-886–5p and miR-886–3p, in sequencing experiments. Following identification instead as a novel vtRNA-like ncRNA , a revisVTRNA1–3 . Subsequeriments , suggestProtein Kinase R (PKR), an interferon-induced serine/threonine kinase and dsRNA-binding protein, binds nc886 with high affinity . nc886 uIn contradiction to the pattern that emerges for most of the Pol III transcriptome, downregulation rather than overexpression of nc886 is observed in multiple cancer contexts and associated with poor survival outcomes. Promoter methylation, which drives epigenetic silencing of nc886, increases in AML , esophag8 |The Pol III transcriptome extends beyond the genes and small noncoding RNA species highlighted in this review. In addition to Alu elements, mammalian-wide interspersed repeat (MIR) elements represent a highly abundant and ancient SINE subclass in humans . MIR repThe boundaries of Pol III activity extend also into the domain of viral-host interactions. It is well established that Pol III transcription, which is upregulated by Epstein–Barr Virus (EBV)-encoded nuclear antigen 1 (EBNA1), is responsible for the expression of EBV-encoded RNAs EBERs; . Pol III9 |The small noncoding RNAs derived from Pol III transcription are understood to play critical roles in basic and diverse cellular processes. In many cases, ncRNA species were historically identified as core components of the ribonucleoprotein complexes central to these processes . This rePerhaps most apparent among the shared motifs is the molecular sequestration activities described for most ncRNAs. Though sequestration of PTEF-b is well established for 7SK, recent work has uncovered analogous functions for vault RNA (p62 and autophagy), 5S rRNA (HDM2 and p53 homeostasis), and tRNA (YBX1 and RNA stability). Like 7SK, nearly all Pol III-derived ncRNAs also modulate gene expression in some form, including examples of direct transcription regulation (RPPH1/RNaseP), altered mRNA stability (RMRP and YBX1), and the biogenesis of secondary small RNA fragments with reported miRNA function . In parallel to protein sequestration, many reports identify similar roles for the Pol III transcriptome in sequestering miRNAs , counteracting miRNA-mediated downregulation of genes typically involved in cell growth.In the case of sequestration, questions remain as to what degree such interactions modulate the availability and/or functionality of the molecular sponge itself. In some cases, noncanonical ncRNA interactions may conceivably help to shield cytoplasmic and immunostimulatory signatures, such as detection of 5’-PPP and ensuing activation of RIG-I interferon responses . Shared Collectively, the Pol III transcriptome is overwhelmingly systematized for supporting and promoting cell growth . While iNevertheless, our generalized model of the Pol III transcriptome, which emphasizes recurrent patterns and roles of small ncRNA in cancer, is inherently oversimplified. Patterns of enhanced proliferation and cancer progression are undeniably complicated by counterexamples in which Pol III-derived ncRNAs play prominent roles in either suppressing transcription and translation or otherwise promoting cellular differentiation. Perhaps most notably, 7SK snRNA is described as a putative tumor suppressor and is primarily recognized for mediating transcriptional repression. Similarly, 7SL and 7SL-derived ncRNAs mediate translational repression though, in the case of 7SL, this intermediate step contributes to further protein synthesis. We highlight evidence that full-length vtRNA promotes cell survival and proliferation, yet dynamic regulation of vault-associated small RNA (vsRNA) contributes to efficient cellular differentiation , indicat


Recent studies have confirmed that circRNAs are present in viruses, including DNA and RNA viruses, and play various important functions such as evading host immune response, disease pathogenesis, protein translation, miRNA sponges, regulating cell proliferation, and virus replication. Studies have confirmed that circRNAs can be biological signatures or pathological markers for autoimmune diseases, neurological diseases, and cancers. However, our understanding of circRNAs in DNA and RNA viruses is still limited, and functional evaluation of viral and host circRNAs is essential to completely understand their biological functions. In the present review, we describe the metabolism and cellular roles of circRNA, including its roles in various diseases and viral and cellular circRNA functions. Circular RNAs are found to interact with RNA, proteins, and DNA, and thus can modulate cellular processes, including translation, transcription, splicing, and other functions. Circular RNAs interfere with various signaling pathways and take part in vital functions in various biological, physiological, cellular, and pathophysiological processes. We also summarize recent evidence demonstrating cellular and viral circRNA’s roles in DNA and RNA viruses in this growing field of research.Circular RNAs (circRNAs) represent single-stranded RNA species that contain covalently closed 3′ and 5′ ends that provide them more stability than linear RNA, which has free ends. Emerging evidence indicates that circRNAs perform essential functions in many DNA viruses, including coronaviruses, Epstein Circular RNA creates a continuous loop that is covalently closed and is a form of single-stranded RNA . BacksplCircRNAs have a unique structure that gives them more stability, a better half-life, and increased RNase R resistance as compared to linear RNAs  11]. Ma. Ma11]. –Barr virus, and human papillomaviruses (HPVs), viral-encoded circRNAs were found to be present. Further findings demonstrated that circRNA is a critical part of the coronavirus transcriptome [Functions of circRNA include but are not limited to miRNA inhibition, epithelial-mesenchymal transition, and tumorigenesis ,17,18. ESeveral studies have been published in recent years about circRNA’s roles in DNA and RNA viruses. Considering the growing field of circRNA research, this review will be examining the function of circRNAs in DNA and RNA viruses. However, to fully understand the function of circRNAs in these viruses, we must first discuss how circRNAs metabolize, their functions, and how circRNAs interact with proteins first.circRNA and lncRNA represent two different types of noncoding RNA species that usually do not code for any proteins . CircRNAA major difference between lncRNA and circRNA is their structure and origin. LncRNAs are spliced, capped, and polyadenylated with no ability to code protein. The length of lncRNAs is more than 200 nt and is considered to be part of a group of ncRNAs . LncRNAsRNA polymerase II synthesizes pre-mRNA that consists of exons and introns. This pre-mRNA molecule has a 7-methylguanosine at the 5′ end and a poly-adenosine tail at the 3′ end. The genes are interrupted by the presence of introns in the eukaryotic cells; therefore, the pre-mRNA molecules must undergo splicing to remove these noninformational intronic sequences. Additionally, pre-mRNA produces a translatable mature mRNA molecule through splicing at canonical splicing sites and spliceosomes. As previously mentioned, the biogenesis of circRNA has been found to be dependent on spliceosomes, canonical splicing machinery, and splice signal sites . RNA binDrosophila melanogaster cells. This demonstrates that growing RNA can be processed via other alternative cellular pathways that expedite backsplicing events when the processing of pre-mRNA is reduced [CircRNAs are produced by backsplicing, meaning that the 3′ and 5′ ends of an exon are ligated to create a sealed circular molecule that consists of a site of backsplicing junction region. It also means that the circular RNA structure is formed through an exon that has upstream 3′, 5′-phosphodiester bonds . This sh reduced . Studies reduced ,44,45.Studies have shown that mammalian-wide interspersed repeat (MIR)-mediated RNA circularization is required to create mammalian circRNAs . MoreoveCircRNAs are abundant and stable notably in various cell types . Recent A recent study found that one way cells could clear circRNAs is to secrete them using extracellular vesicles (EVs). CircRNAs are more enhanced compared to linear forms of identical genes and readily identified in EV preparation, thus supporting the idea that clearing circRNAs via EVs is a mechanism that cells can use . Studies6A). Concurrently, YTHDF3 identifies an m6A-modified start codon and interferes with the translation initiation [6As control the buildup of circ-ZNF609. Circ-ZNF609 backsplicing reaction must be directed by YTHDC1 and METTL3. There were substantial correlations found amid the capability of m6A exons tolerating backsplicing, needing METTL3, and the binding of YTHDC1 [Several researchers have found that circRNAs increase in number during murine spermatogenesis. Backsplicing in a subgroup of circRNAs takes place in areas that are enhanced with N6-methyladenoise  caused alternative splicing genes to be regulated, leading to the inhibition of gastric cancer metastasis . AnotherResearchers have found that circRNAs can be used as therapeutic targets or pathological biomarkers in various neurological diseases and manifestations . A deterDendrites and neuropils in the brain consist of enriched circRNAs that aid in controlling neural plasticity and synaptic function. This suggests that circRNAs could have a crucial role in diseases such as Alzheimer’s diseases (AD), Parkinson’s diseases (PD), and epilepsy that affect the nervous system . In PD, The lack of immune tolerance to self-antigens and an impaired immune system are defining characteristics of autoimmune diseases. CircRNAs working as non-invasive biomarkers in diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and multiple sclerosis (MS) have been demonstrated in recent studies . It was + T cells in patients with SLE consisted of several downregulated and upregulated cricRNAs. DNA methyltransferase 1 expression improved, while CD70 and CD11 expression on CD4+ T cells in active and inactive SLE patients was reduced due to the downregulation of circRNA. Autoantibody production in SLE was stimulated because of CD70 and CD11a overexpression. [Approximately, 127 differentially expressed circRNAs were identified in patients with SLE . It has ression. . In PBMCression. . Much reression. . With a ression. .circRNAs can be potential biomarkers in diagnosing different illnesses, but the expression of some host circRNAs is different after viral infections. In recent years, there has been tremendous development in approaches of bioinformatics and transcriptomic sequencing technology. This led to the advancements in recognition of various individual host circRNAs expressions in viral infections and viral circRNAs . Methods–Barr virus (EBV) infection, and early detection of NCP could be lifesaving for patients affected by this type of cancer. CircRNAs such as hsa_circRNA_001387 (host) and circRPMS1 (EBV-encoded) can be specifically diagnosed in NPC tissues that have EBV infection compared to NPC tissues that are EBV-negative. This makes both hsa_circRNA_001387 (host) and circRPMS1 (EBV-encoded) diagnostic biomarkers of NPC [Nasopharyngeal carcinoma (NPC) is challenging to diagnose because it is induced by Epsteins of NPC . Moreoves of NPC . Furthers of NPC . Anothers of NPC . The RNAs of NPC .Studies have shown that circRNAs can be used as therapeutic agents. Certain features of circRNAs make them potentially useful in the development of vaccines. Vaccines are mostly mRNA, DNA, and cells based on different specific characteristics . There aAs previously mentioned, some of the many roles of circRNAs include its behavior as a sponge miRNA and its effects on protein translations ,174. In S. aureus infection substantially changed the profiles of circRNAs in EVs. It also showed that these circRNAs were selectively packaged into EVs. Furthermore, differentially expressed circRNAs involvement in immune functions through bioinformatic analyses was proposed [Mycobacterium tuberculosis (Mtb) infection causes tuberculosis (TB) and continues to be a prevalent, deadly, and communicable disease [CircRNAs have displayed many roles in bacterial infection. It is speculated that one method of battling against bacterial infections is using EVs because bacterial infections continue to be one of many primary global health issues . Severalproposed . Therefo disease . PBMCs f disease . Through disease . Additio disease . Another disease . In concAlthough induced modifications in cellular circRNA transcriptomes and viral circRNA expression have been found in DNA virus infections , those iAlphaherpesvirinae and known for causing cold sores [vhs) protein is essential in HSV-1 lytic infections. It can quickly split viral and cellular circRNA when delivered to infected cells via incoming virus particles [vhs prompts an enhancement of circRNAs compared to linear mRNAs during HSV-1 infection. Most circRNA enrichment is due to a decrease in linear RNA rather than an increase of circRNA synthesis. On the contrary, IAV and HSV-1 infection both demonstrated synthesis of circRNA arising from NEAT1_2, which is the long isoform of the nuclear paraspeckle assembly transcript 1 (NEAT1). This was linked with circRNA downstream and new linear splicing of NEAT1_2 induction within [A human pathogen known as herpes simplex virus type 1 (HSV-1) is part of the subfamily ld sores ,187. Virarticles  and cleaarticles . Recent n within . NEAT1_2n within ,191,192.n within .Picornaviridae family that causes an array of diseases such as encephalitis, pneumonia, hepatitis, myocarditis, aseptic meningitis, and gastrointestinal illness [Coxsackievirus group B (CVB) is part of the  illness . Interac illness . Past re illness ,196. Up  illness .Recent emerging evidence has shown that the pyroptosis process specifically seen in diabetic cardiomyopathy is regulated by circ_0076631 . Qin et EBV infection occurs in over 90% of the adult population globally. Currently, no vaccine is available to treat EBV infection. EBV infections have been associated with infectious mononucleosis, Hodgkin’s lymphoma, Burkitt’s lymphoma, and nasopharyngeal carcinoma. Studies have shown that miRNA, lncRNAs, and EBV-encoded small RNAs are non-coding RNAs (ncRNAs) that are produced in EBV during infection . EBV cirKSHV can cause many diseases, including a plasmablastic form of Castleman’s disease (MCD), primary effusion lymphoma (PEL), and Kaposi’s sarcoma. Like many other viruses, KSHV encodes miRNAs and aims for human or viral transcripts . SeveralHPVs are circDNA viruses that are small and double-stranded. They infect epithelial cells in humans and are linked to numerous malignant and benign lesions of skin and mucosa . Even thCoronaviridae (CoV) family [The continuing COVID-19 pandemic is caused by a single-stranded RNA virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This positive sense RNA virus is part of the ) family .Previously stated evidence using bioinformatics analyses demonstrated several opposing pieces of evidence about the expression, strand preference, and abundance of CoV circRNAs. It was associated with datasets, circRNA analysis pipeline, and strategy Cai et al. used . A studyHuman cytomegalovirus (HCMV) replicates in the epithelial cells of the organ that it first infects and spreads through the blood to other organs. HCMV infections are the leading cause of birth defects. In the immunocompromised population, complications that are life-threatening and congenital anomalies can be caused if latent HCMV reactivates . A recenAG|GU was stated as the conservative splicing signatures of acceptor sequences and splicing donor that human circRNAs consisted of . BecauseLength, exon numbers, and strand preferences may vary in circRNAs between host and viruses. A recent study performed an in-depth analysis of viral and host circular RNAs to delineate these characteristics of circRNAs in hosts and viruses. The nucleotide length of circRNAs found in chickens, rabbits, mice, macaques, rats, and humans ranges from 250 to 500 nucleotides. CIRI-full, a de novo reconstruction of the entire length of circRNAs, was used to further define circRNAs in EBV, KSHV, and HCMV ,215. VarTo verify the number of exons enclosed in EBV, KSHV, HCMV, and host cells, Yang et al. used a statistical analysis of the circRNA exon numbers that were at full length and reconstructed. circRNAs that included a single exon made up about 80% of the circRNAs in KSHV and HCMV circRNAs. Multiple circular exons in circRNAs made up about 86% of EBVs. Host circRNAs also contain several circular exons .CircRNA’s fascinating biology allows it to have numerous functions in the cell and a critical role in numerous diseases ranging from cancer to pathogenic infections to DNA and RNA viruses. Notable functions of circRNA include acting as a miRNA and its role in cell proliferation. Here, we will take a deeper dive into the specific functions of cellular and viral circRNAs.When circRNAs bind to specific proteins in the cytoplasm, it may incite the induction of extrinsic stimuli and alter RNA translation, transportation, maturation, and transcription . TranscrIn neuronal tissue, evidence demonstrates that intronic sequences drive how quickly animal circRNAs are produced. Furthermore, canonical pre-mRNA splicing, and circularization of exons contend against each other, which are kept in animals and specific to the tissue. Muscle blind binding sites are only bound by MBL and are contained in both introns flanking circMbl and circMbl itself. MBL binding sites influence how circMbl biosynthesis is affected by how MBL levels modulate . SimilarcircZNF609, also known as circRNA zinc finger protein 609, is translated to a protein to regulate myoblast proliferation [CKAP5 mRNA interaction. The following several processes occurred: (1) the role of microtubule in cancer cells was controlled; (2) translation of CKAP5 was enhanced; (3) progression of cell-cycle was sustained; and (4) backsplicing junction overlapped [circZNF609-CKAP mRNA is regulated with other functions of circZNF609, including its role in translation [circZNF609 m6A modification that specifically selects methyltransferase-like 3, and promoting black-splicing with YTHDC1 [CircRNAs have the ability to directly bind to mRNA to affect the abundance of gene expression. feration ,232. Wheerlapped . Howevernslation , functionslation  or with h YTHDC1 . Furtherh YTHDC1 . RNA Polh YTHDC1 .PABPN1 translation is positively maintained by HuR, therefore suggesting that the binding of circPABPN1 and HuR can suppress HuR-promoted PABPN1 mRNA translation [FAM120A created circFAM120A. CircFAM120As are oncogenic RNA, mostly located in mono ribosomes that stimulate cell proliferation. They inhibit FAM120A mRNA from attaching to IGF2BP2 and the m6A reader. This causes FAM120A to efficiently translate, which is needed for the proliferation [FAM120A mRNA and circRAM120A to IGF2BP2 takes place in monoribosomes. Approximately one-third to one-fifth of the total FAM120A mRNA is the total amount of circFAM102. The binding affinity of circFAM120A to IGF2BP2 is heightened because of the m6A on circFAM120A [mRNA translation can be altered when circRNAs outperform mRNAs by interacting with proteins that are similar ,236,237.nslation . FurtherIn this review, we briefly described circRNA metabolism, specifically detailing the localization, regulation, biogenesis, degradation, and modification of circRNAs. Circular RNAs have sequences overlapped with their cognate linear RNA, which makes identification and quantification difficult. However, new biological methods have been developed to detect and study circular RNAs. Circular RNAs have been detected in cytoplasm and nucleus, thus playing important roles in regulating genetic and cellular processes. Although an animal model is lacking to evaluate the role of circular RNA in various diseases and pathological conditions, it is crucial to study the correlation between disease progression and the abundance of circRNAs. Identifying circRNA’s role under different pathological and physiological conditions can influence the development of new therapeutics for disease treatment. We summarize the mechanisms of (1) how circRNAs function as a miRNA, (2) how circRNAs function in cell proliferation, and (3) cirRNA’s roles in cancer, neurological diseases, autoimmune diseases , and pathogenic infection . We further delve into circRNA’s role in viral infections by discussing cellular circRNA during viral infection, viral circRNA in DNA/RNA viruses, characteristics of viral circRNA, and functions of cellular/viral circRNA. Recent studies have identified circRNAs in HCMV, SARS-CoV2, and other viruses, implying that circRNAs may function in virus replication, growth, and disease manifestation. It is imperative to identify circRNAs in other viruses and study their roles in disease progression. The presence of circRNAs in viruses created new opportunities for researchers to evaluate their biological functions and relevance to disease. Taken altogether, it is clear that circRNAs are critical in the pathological and physiological functions in human diseases and development due to their unique characteristics and functions as cellular and viral circRNA, which specifically include: (1) how protein binding produces biological effects, (2) how circRNAs act as protein sponges, (3) how binding to mRNAs affect circRNA abundance, and (4) how circRNAs compare to linear RNAs. In the future, we anticipate more roles of circRNA being discovered and intriguing circRNA-based applications being implemented in biomedical research and therapeutic approaches.


Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes. Cas12a is widely used in diagnostic platforms. Here the authors show that Cas12a can be programmed to directly detect RNA substrates, this is due to the 3’-end of the crRNA tolerating both RNA and DNA substrates: they use this to report a method, SAHARA, to detect RNA sequences. Upon infection, the invading DNA sequences are captured and integrated into the host genome between an array of repeat sequences. The captured DNA sequences are called ‘spacers’ and they provide a genetic memory of prior infections3. For prokaryotic immunity, the CRISPR locus is transcribed and processed to generate multiple mature CRISPR RNAs (crRNA), each encoding a unique spacer sequence. The crRNA forms a complex with an RNA-guided endonuclease, Cas (CRISPR-associated) protein. In the presence of nucleic acids complementary to the crRNA sequence, the CRISPR/Cas system enables the cleavage of nucleic acids. There are several diverse naturally occurring CRISPR/Cas systems found in prokaryotes5. Among these, Cas12a is a class 2, type V RNA-guided DNA endonuclease6. Since its discovery, it has been widely used for genome editing as well as molecular diagnostic applications11. Structural and biochemical studies have shown that Cas12a can catalyze the cleavage of DNA substrates14 but there are no reports of targeted RNA cleavage by Cas12a, without pre-processing steps such as reverse transcription. Recently there has been a report of a novel enzyme named Cas12a2 that sometimes co-occurs with Cas12a systems in bacteria and can use the Cas12a crRNA, but recognizes an RNA target instead of a double-stranded DNA15. A special characteristic of the type V Cas12 family of enzymes is their ability to initiate rapid and indiscriminate cleavage of any non-specific single-stranded DNA (ssDNA) molecules in their vicinity after target-specific recognition and cleavage17. This unique catalytic property, known as trans-cleavage has been harnessed to engineer CRISPR-based diagnostic tools that rely on the cleavage of FRET reporters upon target recognition18.CRISPR  is an adaptive immune system encoded within prokaryotes that has evolved to counter invasion by foreign nucleic acids from bacteriophages and plasmids21. Previously we have shown that CRISPR-Cas12a can tolerate DNA/RNA heteroduplexes, but only when RNA is located at the non-target strand22. This can be used to detect RNA targets with Cas12a by simply creating a heteroduplex using a reverse transcription step without amplification23. A reverse transcription step is inconvenient because it adds to the time, cost, error, and complexity of the assay. The other alternative is to use an RNA-targeting enzyme such as Cas12a215, Cas12g24, or Cas13a-d27; however, these systems can only detect RNA.So far, Cas12a-based tools have been limited to the detection of DNA substrates, unless they are coupled with additional steps involving reverse transcription or strand displacement6. It has been previously shown that DNA-cleaving enzymes such as Cas9 can be manipulated to also cleave RNA through the addition of a PAMmer sequence, a PAM-containing oligonucleotide, which is annealed to the target ssRNA to initiate cleavage28. However, similar approaches have not yet been investigated with trans-cleaving Cas enzymes like Cas12, primarily because the PAM recognition mechanism is different between Cas9 and Cas12 enzymes29. For instance, unlike Cas9, Cas12a can recognize and even trigger trans-cleavage activity using a PAM-less ssDNA. To date, there is no single CRISPR-Cas system identified that can innately tolerate both DNA and RNA substrates to trigger trans-cleavage.Cas12a orthologs are known to require a short protospacer adjacent motif (PAM) to be present on the target DNA to initiate recognition and cleavagetrans-cleavage activity. While the PAM-proximal seed region of the crRNA strictly tolerates DNA substrates, the PAM-distal end of the crRNA can tolerate both RNA and DNA substrates with multiple Cas12a orthologs. Thus, by merely supplying a short ssDNA or a PAM-containing dsDNA at the seed region of the crRNA we can detect RNA substrates at the 3’-end of the crRNA. This specific property is the basis of our method for RNA detection using Cas12a named Split Activators for Highly Accessible RNA Analysis (SAHARA).In this work, we show that Cas12a can tolerate RNA substrates at the PAM-distal end of the crRNA and initiate trans-cleavage activity of Cas12a, which are essential for SAHARA, a valuable and versatile tool that can simultaneously detect both DNA and RNA substrates.SAHARA achieves reverse transcription (RTx)-free detection of picomolar levels of DNA as well as RNA without amplification and can detect clinically relevant targets including hepatitis C virus (HCV) RNA and microRNA-155 (miR-155). We show that SAHARA works robustly at room temperature and has improved specificity for point mutation detection compared to conventional CRISPR/Cas12a systems.We demonstrate that its activity requires the seed region DNA which allows for multiplexed assays. We use this switch capability to simultaneously detect different DNA and RNA targets. We also show that SAHARA works with Cas13b to perform multiplexed detection of different RNA targets. These key findings provide insight into the substrate requirements for the Lachnospiraceae bacterium ND2006, Acidaminococcus sp. BVL36, and Eubacterium rectale and are simply referred to here as Lb, As, and Er, respectively33  activators , Moraxella bovoculi 237 (Mb), and Butyrivibrio sp.NC3005 (Bs) , we observed that all three orthologs displayed trans-cleavage activity only with Pp DNA but not Pp RNA. Suggesting that, unlike AsCas12a, these Cas12a also have a strict preference for DNA only at the PAM-proximal region.Interested in the ability of AsCas12a to tolerate RNA sequences at the Pp region, we wondered if there were other orthologs capable of the same. To investigate the tolerance of Pp RNA detection with other Cas12a, we tested three orthologs—s) Fig. S. We chosWhile a fully complementary RNA activator is not recognized by Cas12a, we have shown that RNA can be recognized at the 3’-end of the crRNA if DNA is bound at the seed region. Therefore, somewhere between the seed region and the 3’ spacer-end of the crRNA, RNA recognition starts being tolerated. To examine the exact position at which RNA detection begins, we designed increasingly long RNA activators of length ranging from 6-14 nt that are complementary to the Pd region of the crRNA, and their corresponding DNA activators of different lengths binding to the Pp region Fig. S. We test22. We were curious to check if these modified crRNAs (termed ENHANCE or EN) would have any significant effect on the trans-cleavage activity produced by the split-activator system. We analyzed the detection of ssDNA and ssRNA substrates using the split-activator system with both the wild-type (WT) and EN crRNAs. We observed that both ssDNA and ssRNA had significantly greater activity with the EN crRNA for LbCas12a, while AsCas12a and ErCas12a were unaffected by the different crRNA designs. This is consistent with previous observations with ENHANCE22. We observed ErCas12a to have the highest activity with the split-activator system irrespective of the crRNA design being used  or 8-nt of the Pd region of the crRNA to DNA (crRNA-12R8D)  Fig. S. HoweverD) Fig. S.Split Activators for Highly Accessible RNA Analysis) a tool for direct RNA detection with Cas12a. The design of SAHARA was such that a short (12-nt), synthetic PAM-containing dsDNA activator (named ‘S12’ due to its length) would bind to the Pp-end of the crRNA, while any target RNA sequence can be detected at the Pd-end. While it was clear that short RNA activators could be easily detected by SAHARA, this study aimed to aid in the optimization of detection for a variety of targets and lengths. Therefore, we designed two RNA targets: a short RNA (20-nt) and a longer RNA (~730-nt) containing the same 20-nt target sequence  DNA activators that are necessary for SAHARA to function, can bind in a more stable manner at temperatures lower than 37 °C, due to their lower Tm. To study this, we tested the detection of an ssDNA and ssRNA target each at 37 °C and room temperature (RT) Fig. S. We obse2+ ions are routinely used in Cas12a-based applications, Mn2+ ions have also been shown to work well38. To study the effect of different ions on the activity of SAHARA, we tested a range of different cations  and discovered that most cations severely inhibited the trans-cleavage activity of SAHARA , 3’-end (tail), or in the middle (mid)  Fig. .Fig. 4Ap41. Therefore, reliable detection of miR-155 is important for early diagnosis. The mature miR-155 is ~23-nt in length. We synthesized the mature miRNA target and designed two SAHARA guides targeting it at either 12-nt at the 5’-end or 11-nt at the 3’-end, keeping the S12-binding region constant  is known to play a crucial role in breast cancer progression and is overexpressed in breast cancer tissuesant Fig. .trans-cleavage, we observed significant variability in the activities produced by different guides. For miR-155, the head targeting guide showed an almost 5-fold higher activity than its tail targeting counterpart. Similarly, for HCV, the tail targeting guide showed as much as a 6-fold increase in activity as compared to head-targeting and the mid-targeting guide . However, we were able to detect this RNA upon increasing the concentration of crRNA, Cas12a, and S12 to 250 nM, 125 nM, and 105 nM respectively  that consists of fragments from 5’UTR and X-tail region, thus being a more complex sample as compared to the previously used 86-nt RNA. Initially, we found the detection of this RNA target with our typical protocol to be challenging due to the low concentration of this target RNA  we recommend using a healthy matrix as a control to account for background activity arising from non-specific interactions within the matrix, and then making use of statistical methods to detect the presence or absence of target RNA based on the differences in the fluorescence intensity with respect to the control.trans-cleavage, we designed three different S12 activators each containing either a TTTA, AAAT, or VVVN PAM respectively  sequence, and VVVN encompasses the space of all the PAM sequences that are not tolerated by Cas12a. We tested the detection of a short 20-nt RNA with these S12 activators. As expected, the TTTA-PAM S12 was able to mediate RNA detection with all three Cas12a orthologs , with unique targets and S12 activators, and pooled them all together  were active, whereas the reactions without target A  were inactive despite the Cas, crRNA-b, crRNA-c, and their respective targets being mixed. The data here shows strong evidence that the S12 DNA can be used to selectively activate specific crRNAs and can be used to control the trans-cleavage activity of CRISPR-Cas12a, thereby enabling simultaneous and multiplexable detection of both DNA and RNA targets.In the no S12 control, there was no 26. We used DNA and RNA reporters consisting of orthogonal dyes to differentiate the signal obtained from SAHARA and Cas13. This is similar to the multiplexed detection with Cas13 and Cas12 demonstrated before26, but here we aimed to detect RNA substrates with both Cas12 and Cas13, and not DNA. To test this, we used Lb, As, and Er Cas12a orthologs in conjunction with PsmCas13b to detect two distinct RNA targets (T1 and T2). We designed Cas12 and Cas13 guide RNAs such that the guide for Cas12 was complementary to only the T1 target while the guide for Cas13 was complementary to only T2  and miRNA-155. We found that the secondary structure of the target played an important role in the RNA detection activity of Cas12a. Increased secondary structure in the RNA target makes it more inaccessible to bind to the crRNA and reduces the activity. We also observed that pooling together multiple crRNAs targeting different regions of the activator enhanced the detection capability, as previously reported for Cas13. Applying this, we were able to detect picomolar levels of both miRNA-155 as well as HCV with SAHARA.While SAHARA works well with synthetic RNA targets, a key limitation of our study is the lack of validation with respect to detecting RNA samples with more complex material in a clinical setting. A potential drawback of SAHARA is its specificity in more complex samples. Since SAHARA is designed to only bind to 12-nt of the target RNA, the odds of finding similar RNA sequences in a complex material are high, potentially resulting in false positives. Upon testing the detection of an HCV target spiked in nucleic acids extracted from healthy serum samples, we observed an increase in the background activity resulting from SAHARA as compared to the detection of the same sample in water. However, we also observed that the on-target activity increased proportionally.In a diagnostic setting, depending on the kind of matrix being tested  we recommend using a healthy matrix as a control to account for background activity arising from non-specific interactions within the matrix and then making use of statistical methods to detect the presence or absence of target RNA based on the differences in the fluorescence intensity with respect to the control.51 that can be game-changing for the field of Cas12a-based diagnostics.Developing a robust, clinically applicable, diagnostic test with SAHARA will require additional efforts, especially with regards to improving not just the specificity but also the sensitivity of detection. SAHARA in its current form is limited to only picomolar levels of detection without amplification, which may not be good enough for the detection of clinical samples. However, a promising outlook lies in combining SAHARA with the plethora of amplification-free detection techniquesFor this study, de-identified human serum collection and processing were approved as a non-human study by the University of Florida Institutional Review Board (IRB202003085), and all ethical regulations were followed.Serum samples from healthy patients were obtained from a commercial vendor, Boca Biolistics, procured from their network of CAP/CLIA accredited partner laboratories across the United States as remnant (leftover) samples. These samples were delinked under the IIRB delinking protocol SOP 10–00114 Rev E.35. Briefly, plasmids expressing LbCas12a and AsCas12a were obtained from Addgene (a gift from Zhang lab and Doudna lab) and directly used for protein expression. For ErCas12a, a plasmid containing the human codon-optimized Cas12a gene was obtained from Addgene, then was PCR amplified using Q5 Hot Start high fidelity DNA polymerase , and subcloned into a bacterial expression vector . The product plasmids were then transformed into Rosetta™(DE3)pLysS Competent Cells  following the manufacturer’s protocols.Plasmids expressing Lb, As, and ErCas12a enzymes were constructed as described in Nguyen et al.For protein production, bacterial colonies containing the protein-expressing plasmid were plated on an agar plate and grown at 37 °C overnight. Individual colonies were then picked and inoculated for 12 h in 10 mL of LB Broth . The culture was subsequently scaled up to a 1.5 L TB broth mix and grown until the culture reached an OD = 0.6 to 0.8. The culture was then placed on ice before the addition of Isopropyl β- d-1- thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The culture then continued to grow overnight at 16 °C for 14–18 hours.g for 30 min. The cell lysate was filtered through a 0.22 µm syringe filter  and then ran through into 5 mL Histrap FF  pre-equilibrated with Wash Buffer A  connected to BioLogic DuoFlow™ FPLC system (Bio-rad). The column was eluted with Elution Buffer B . The eluted fractions were pooled together and transferred to a 10–14 kDa MWCO dialysis bag. Homemade TEV protease (44) was added to the bag, submerged in Dialysis Buffer  and dialyzed at 4 °C overnight.The overnight culture was pelleted by centrifuging at 10,000 × g for 5 min. The cells were then resuspended in lysis buffer . The cell mixture was then subjected to sonication followed by centrifugation at 39,800 × The protein mixture was taken out of the dialysis bag and concentrated down to around 10 mL using a 30 kDa MWCO Vivaspin® 20 concentrator. The concentrate was then equilibrated with 10 mL of Wash Buffer C  before injecting into 1 mL Hitrap Heparin HP column pre-equilibrated with Wash Buffer C operated in the BioLogic DuoFlow™ FPLC system (Bio-rad). The protein was eluted from the column by running a gradient flow rate that exchanges Wash Buffer C and Elution Buffer D . Depending on how pure the protein samples were, additional size-exclusion chromatography may have been needed. In short, the eluted protein from the previous step was run through a HiLoad® 16/600 Superdex® . Eluted fractions with the highest protein purity were selected, pooled together, concentrated using a 30 kDa MWCO Vivaspin® 20 concentrator, snap-frozen in liquid nitrogen, and stored at –80 °C until use.All DNA and RNA oligos as well as the chimeric DNA/RNA hybrid crRNAs were obtained from Integrated DNA Technologies (IDT). Single-stranded oligos were diluted in 1xTE buffer . Complementary oligos for synthesizing dsDNA were first diluted in nuclease-free duplex buffer  and mixed in a 1:5 molar ratio of target:non-target strand. Both strands were then subjected to denaturation at 95 °C for 4 mins and gradient cooling at a rate of 0.1–25 °C.For generating the 730-nt long GFP target sequence, Addgene plasmid pCMV-T7-EGFP (BPK1098)  was obtained and PCR amplified using Q5 Hot Start high fidelity DNA polymerase  from position 376-1125. The PCR amplified product was in-vitro transcribed using the HiScribe T7 High Yield RNA synthesis kit (NEB #E2040S) following the manufacturer’s protocol. The transcribed product was treated with DNase I for 30 min at 37 °C and then purified using RNA Clean and Concentrator Kit (Zymo Research #R1016).4+, Rb+, Mg2+, Zn2+, Co2+, Cu2+, Ni2+, Ca2+, Mn2+, and Al3+) were diluted to a final concentration of 10 mM. The pH of the buffer was adjusted to 7.9 by adding 1 M NaOH.The different metal ion buffers were prepared by first creating a master mix of the following components: 50 mM NaCl, 10 mM Tris-HCl, and 100 µg/ml BSA. To this master mix, chloride salts of different monovalent, divalent, and trivalent cations . Synthetic RNA resembling a fraction of the HCV 5-UTR gene was spiked to a final concentration of 1 nM and tested with SAHARA using the fluorescence-based detection method.5-μL of HCV-ATCC RNA (Cat# VR-3233SD) was combined with 5-μL of CRISPR-Cas mix consisting of 250 nM crRNA, 125 nM Cas12a, 105 nM S12, and 500 nM of FQ reporter. The reaction was transferred to a 384-well plate and then incubated in a BioTek Synergy fluorescence plate reader at 37 °C for 1 h. Fluorescence intensity measurements for a FAM reporter were measured at the excitation/emission wavelengths of 483/20 nm and 530/20 nm every 2.5 min.trans-cleavage assay was carried out with several different dilutions of the activator. The LoD calculations were based on the following formula:52To find the limit of detection (LoD), the No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource Data


Transcriptome complexity is emerging as an unprecedented and fascinating domain, especially by high-throughput sequencing technologies that have unveiled a plethora of new non-coding RNA biotypes. This review covers antisense long non-coding RNAs, i.e., lncRNAs transcribed from the opposite strand of other known genes, and their role in hepatocellular carcinoma (HCC). Several sense–antisense transcript pairs have been recently annotated, especially from mammalian genomes, and an understanding of their evolutionary sense and functional role for human health and diseases is only beginning. Antisense lncRNAs dysregulation is significantly involved in hepatocarcinogenesis, where they can act as oncogenes or oncosuppressors, thus playing a key role in tumor onset, progression, and chemoradiotherapy response, as deduced from many studies discussed here. Mechanistically, antisense lncRNAs regulate gene expression by exploiting various molecular mechanisms shared with other ncRNA molecules, and exploit special mechanisms on their corresponding sense gene due to sequence complementarity, thus exerting epigenetic, transcriptional, post-transcriptional, and translational controls. The next challenges will be piecing together the complex RNA regulatory networks driven by antisense lncRNAs and, ultimately, assigning them a function in physiological and pathological contexts, in addition to defining prospective novel therapeutic targets and innovative diagnostic tools. As the result of pervasive transcription of the mammalian genome, non-coding RNAs (ncRNAs) represent the majority of the transcriptome. High-throughput sequencing technologies unveiled an unprecedent and fascinating picture of transcriptome complexity, where less than 2% of transcription encodes proteins, and a plethora of ncRNA biotypes are found, in addition to the well-known ribosomal RNAs . The finBased on their length, ncRNA molecules can be broadly classified into small or short ncRNAs (from few to 200nt) and long ncRNAs ((lncRNAs) longer than 200nt, with a size up to several kilobases (up to 100 kb)) .Short ncRNAs comprise: tRNA (transfer RNA), engaged in translation of mRNA; snRNA , involved in splicing; snoRNA , involved in ribosomal RNA modification; piRNA (Piwi-interacting RNA), mainly implicated in transposon repression; microRNAs (miRNAs), the most studied group of small ncRNAs, acting as post-transcriptional regulators of gene expression . miRNAs LncRNAs, the largest class of ncRNAs in the mammalian genome, can be further classified into subclasses, depending on their genomic locations, origins, and transcription directions : long inThe specific name of antisense lncRNAs is derived from their sense gene, with the addition of “-AS”; they can be further categorized according to their localization with respect to sense transcripts, i.e., tail to tail (sense and antisense transcripts overlapping in the 3′ region), internal (antisense lncRNA covering the sense transcript), and head to head (sense and antisense transcripts overlapping the 5′ region) .Sense–antisense gene pairs have been found in a significant proportion in the genome/transcriptome of different species, from prokaryotes to mammals ; furthermore, sense–antisense gene pairs are generally organized into one coding and one non-coding transcript ,13,14. IIt is becoming increasingly clear that antisense lncRNAs play key roles, not only in physiological states, but also in pathological conditions, especially cancer. They can contribute to the onset and progression of different type of tumors, acting as oncogenes, oncosuppressors, or both, depending on the type of cancer. lncRNAs have also been implicated in chemotherapy and radiation therapy resistance.Antisense lncRNAs are also emerging as novel players in hepatocellular carcinoma (HCC); in particular, one study found that they represent 16% of the newly assembled lncRNAs from HCC patients . Viral hThis review aims to explore antisense lncRNA biology, mechanisms of action shared with lncRNAs and unique to antisense lncRNAs, their contribution to HCC hallmarks, and their possible exploitation as innovative biomarkers and potential novel therapeutic targets.Antisense lncRNAs and, generally, lncRNA biogenesis, share various features with coding RNAs and precursor transcripts of miRNAs: they are transcribed, generally, by RNA polymerase II, subjected to 5′-capping, 3′-polyadenylation, and splicing, since they are mainly composed of two exons . In contIn the nucleus and cytoplasm, antisense lncRNAs exploit all the mechanisms of gene regulation known for other lncRNA biotypes. However, antisense lncRNAs can also reroute these mechanisms onto their sense genes. In addition, due to sequence complementary, antisense lncRNAs can play a special role regarding sense genes . As a coIn the nucleus, interaction with DNA, chromatin-modifying complexes, histone-modifying enzymes, and/or various transcriptional regulators determines antisense lncRNAs function as epigenetic and transcriptional regulators a–c. In pIn the cytoplasm, by binding proteins and RNA molecules, lncRNAs can regulate gene expression at the post-transcriptional level by sponging miRNAs, regulating mRNA translation and degradation; short open reading frames hidden in the lncRNA sequence could even serve as templates for the synthesis of so-called “micropeptides” up to 100 amino acids long ,35,36 Fd–g. CurrFinally, sequence complementary allows antisense lncRNAs to have a specific effect on their sense gene. In fact, at the post-transcriptional level, antisense lncRNAs can bind their sense transcript, generating an RNA duplex and affecting the stability of the sense transcript via an RNA interference mechanism; probably the best-known example of this is represented by the pair XIST and TSIX (XIST spelled in reverse order) involved in the X chromosome inactivation, but other examples have been subsequently reported ,43,44 Fd. An oppThe above mechanisms have been distilled from many studies, detailed in the next section as related to HCC.https://lncipedia.org (accessed on 3 April 2023) [Antisense lncRNAs are increasingly recognized as mediators of human cancers  and, depil 2023) .Here, we first discuss the role and molecular mechanisms of different oncogenic antisense lncRNAs involved in pathways driving HCC, focusing on the best characterized ones  and dediAlteration of different molecular pathways is known to be involved in HCC development and progression. Among them, the Wnt/β-catenin signaling pathway is a well-known oncogenic pathway, and its increased activation frequently occurs in HCC. The expression status of β-catenin is a key feature that affects Wnt/β-catenin signaling and plays a key role in signal transduction to activate the transcription of Wnt target genes that are involved in tumor onset and progression . InteresAnother pathway that plays a crucial role in HCC is the Akt/mTOR pathway. The Akt/mTOR signaling pathway regulates crucial cellular processes in the physiological setting as well as most hallmarks of cancer, including cell cycle, survival, metabolism, motility, and angiogenesis . SeveralHypoxia is a crucial feature in all solid tumors, especially HCC. Although hypoxia itself is toxic to cancer cells, a hypoxic environment leads HCC cells to induce a series of adaptive, “pro-survival” changes, which subsequently leads to elevated angiogenesis, adapted metabolic alteration, tumor invasion, and metastasis ,153. He Beyond the involvement of antisense lncRNAs in the well-known oncogenic pathways described above, many other dysregulated antisense lncRNAs in HCC have been identified in several studies. Among them, LEF1-AS1 is highly expressed in HCC tissues and cells, promoting proliferation and invasion by increasing EZH2 expression via the CEBPB-CDCA7 signaling pathway . MoreoveHOX transcript antisense RNA (HOTAIR), which is transcribed from the HOXC locus and interacts with both the polycomb repressive complex 2 (PRC2) and lysine-specific histone demethylase 1A complex to enhance histone H3 lysine trimethylation and histone H3 lysine 4 demethylation, leading to the silencing of multiple genes [One of the most well studied antisense lncRNA in HCC is the le genes . Recentlle genes . At the cellular level, HOTAIR is involved in proliferation, cell motility, invasion, cell cycle progression, apoptosis, and autophagy through a plethora of molecular mechanisms summarized here. Upregulation of HOTAIR can promote the proliferation, migration, and invasion of human HCC cells by regulating ATG3 and ATG7 expression, which are involved in autophagosome formation and activation . Ding anHOTAIR has also been reported to exploit the ceRNA mechanism to regulate tumor progression . In thisantisense non-coding RNA from the INK4 locus (ANRIL), also known as CDKN2B-AS. ANRIL is transcribed as a 3.8 kbp lncRNA that contains 19 exons in the antisense direction of the INK4B-ARF-INK4A gene cluster at chromosome 9p21 and is involved in its repression. ANRIL was found significantly upregulated in HCC tissues and cells, suggesting that it may play an important role in HCC progression [Another well-studied antisense lncRNA is the gression , with muHuang and colleagues proposed that ANRIL could regulate cell growth, both in vitro and in vivo, via epigenetic silencing of Kruppel-like factor 2 (KLF2) by binding to PRC2 . ANRIL kThe vast majority of antisense lncRNAs with a role in HCC have been characterized as oncogenes; a few other antisense lncRNAs have been reported as tumor suppressors. One example of this is represented by the lncRNA MAGI2-AS3. In vitro, MAGI2-AS3 is able to impede cell proliferation, invasiveness, and migration and enhance cell apoptosis; in vivo, MAGI2-AS3 remarkably restrained tumorigenesis in an HCC mouse model. Mechanistically, MAGI2-AS3 can decrease the expression of the oncogene RACGAP1 by inducing histone demethylation of its promoter through recruitment of the demethylase KDM1A, as demonstrated by Chip in RIP and RNA pull-down assays, . MAGI2-ALikewise, WWOXA-S1 is downregulated in HCC tissues and cell lines, and is involved in the proliferation, apoptosis, migration, and EMT processes. Interestingly, WWOX, the sense gene of WWOX-AS1, has been confirmed to exert an anticancer role in various cancers and is found to be downregulated in HCC. Xu and colleagues demonstrated that WWOX-AS1 may function as a ceRNA by sequestering miR-20b-5p, thus upregulating WWOX expression . In addition, the lncRNA ADORA2A-AS1 may exert a tumor-suppressive role in HCC. Low expression of ADORA2A-AS1 in HCC tissues correlates with advanced stage, invasion, and poor survival rate. At the cellular level, ADORA2A-AS1 can repress proliferation, migration, and invasion; it is also able to restrict HCC xenograft growth and metastasis in vivo. Mechanistically, ADORA2A-AS1 can interact with HuR and suppress the binding of HuR to its mRNA target, FSCN1, resulting in the inhibition of the Akt pathway . Abnormal activation of the hedgehog signaling pathway is one pivotal cause of cancer initiation and progression . HHIP isAs stated in the Introduction, antisense lncRNAs can interact with other RNA molecules  and proteins, but they can also interact with DNA sequences to exert their regulatory role. One example of this, found in HCC, is represented by the tumor suppressor WT1-AS, which has a binding site in the promoter region of WT1; this binding, demonstrated by reporter constructs and WT1-AS overexpression, downregulated the expression of WT1, thus promoting cell apoptosis by suppressing the JAK/STAT3 signaling pathway . The knowledge of the molecular mechanisms underlying the role of antisense lncRNA in HCC may pave the way to new perspectives on their clinical use, both as innovative diagnostic/prognostic biomarkers and as new therapeutic targets.In this section, we will first discuss their possible use as biomarkers, providing some relevant examples; then, we will focus on their potential as targets for HCC treatment by providing an overview of possible antisense lncRNA-based therapies.Despite the recent progress in HCC diagnosis, the overall prognosis still remains unsatisfactory, and there is an urgent need to search for new biomarkers with high efficacy for the early detection of HCC and prediction of tumor progression . The ideFor instance, HOTAIR, an oncogenic lncRNA significantly overexpressed in HCC tissues, was found to be associated with an increased risk of recurrence after hepatic resection; this risk was significantly higher in patients with lymph node metastasis . AnotherSimilarly, high expression levels of ANRIL , HOXB-ASThe real future challenge may be the design of a panel combining the dosage of the molecules independently identified in the various studies to further validate them with the same methodologies in large cohorts of patients and, ultimately, select those antisense lncRNAs endowed with diagnostic/prognostic potentiality and clinical utility.Although the clinical treatment of HCC has vastly improved, the five-year survival rate of HCC patients is still low because of high refractoriness to chemotherapy. This is due to the interaction of very complex and diverse mechanisms of chemoresistance (MOC) that protect tumor cells from the chemotherapeutic . RecentlSorafenib is a multitarget kinase inhibitor that suppresses tumor cell proliferation and angiogenesis. Its mechanism of action was also related to ncRNAs in HCC, e.g., to the oncosuppressive miR-125a . SorafenCisplatin (CDDP) is a drug extensively administered for the treatment of various cancers, including HCC. Thus, it is imperative to determine the molecular mechanisms underlying HCC chemoresistance to aid the diagnosis and treatment of patients with HCC. Multidrug resistance (MDR) could be caused by the overexpression of ATP-binding cassette subfamily B member 1 (ABCB1), which pumps anticancer agents out of cells, contributing to a reduced intracellular drug concentration and cytotoxicity. Inhibition of ABCB1 expression is able to reduce the resistance of HCC cells to anticancer agents . LncRNA Similarly, radiation therapy is a very common treatment for many types of cancer, either alone or in combination with other therapeutic methods. Its effectiveness greatly depends on the radiosensitivity of the cancer cells. Some studies reported that dysregulation of antisense lncRNAs might be involved in this process. For instance, in HCC, lncRNA TP73-AS1 is highly expressed and increases after X-ray irradiation. Song and colleagues demonstrated that TP73-AS1 overexpression may induce pAkt/Akt expression, leading to a reduction of PTEN expression. Thus, lncRNA TP73-AS1 silencing may repress the development of HCC and promote radiosensitivity in HCC via the PTEN/Akt signaling pathway, improving HCC therapy .As discussed, antisense lncRNAs have critical roles in HCC onset and progression. Thus, their modulation may be a promising tool for HCC treatment. In recent years, great progress was achieved in developing biological drugs that target RNA molecules in cells, providing the basis for lncRNA-based cancer therapies. These therapies are essentially based on the silencing of oncogenic antisense lncRNA or, ideally, on restoring the expression of tumor suppressor lncRNAs b. Of theIn this regard, RNA interference (RNAi) has been studied and developed extensively and represents a powerful method to target RNA molecules in cells. RNAi can be triggered experimentally by exogenous introduction in the cells of double-stranded RNAs (dsRNAs) or constructs which express short-hairpin RNA (shRNAs). Small interfering RNAs (siRNAs) are double-stranded RNAs that are usually approximately 20 base pairs long. They can interfere with the expression of specific genes by degrading target RNA and/or preventing translation. Once introduced into the cells, siRNAs are bound by RISC (RNA-induced silencing complex), the RNA duplex is separated, and the strand with the less stable 5′-end is loaded onto the active RISC complex. Then, the RISC complex can recognize the target transcript, leading to its cleavage, and resulting in gene silencing . Thus, sAntisense oligonucleotides (ASOs) are small-sized, single-stranded nucleic acids that can target both nuclear and cytoplasmic-located antisense lncRNAs. Usually, RNaseH1 recognizes the DNA:RNA heteroduplex and catalyzes the RNA cleavage . As mentThe above strategies exploit the versatility and increasing knowledge of ncRNA biology by using different RNA-based tools targeting other RNA molecules. The discovery that some drugs may also exert their anticancer activity by regulating antisense lncRNAs could suggest additional therapeutic interventions, e.g., based on the combination of therapeutic RNAs and drugs. For instance, Tian and colleagues demonstrated that curcumol could downregulate HOTAIR expression, which induces the suppression of EZH2, leading to the inhibition of tumor growth . AnotherOverall, the studies reported above show the great potential of antisense lncRNA-targeted HCC therapies, encouraging further studies for deepening the molecular mechanisms and ultimately assessing specificity, tolerable side effects, safety, formulations, and delivery. Recent insights into RNA biology induced a paradigm shift towards the recognition of RNAs—beyond their role of serving as messengers for protein-encoding genes—as functional molecules. In this new perspective, DNA can be envisaged as the “hardware”, and RNA can be viewed as the “software” managing multiple “biological apps” through its structural/functional versatility. Among the different ncRNA biotypes, antisense lncRNAs are emerging as new players in gene expression regulation by exploiting different molecular mechanisms shared with other ncRNA molecules, and by exploiting special molecular mechanisms on their sense gene, through epigenetic, transcriptional, post-transcriptional, and translational modulations. High-throughput sequencing technologies are allowing the annotation of an increasing number of new antisense lncRNAs, found in normal and/or tumor tissues, unveiling various sense–antisense pairs transcribed from several loci. These pairs are no longer considered an oddity but are viewed as a possible evolutionary opportunity to increase complexity and lineage-specific regulatory outcomes. Antisense lncRNAs dysregulation has been shown to be deeply involved in hepatocarcinogenesis, where they can act as an oncogenes or an oncosuppressors, thus playing a key role in tumor onset, progression, and chemoradiotherapy response. The next challenges will be to piece together the entire RNA regulatory networks of antisense lncRNAs and assign them a function in physiological and pathological contexts, and to define prospective novel therapeutic targets and innovative diagnostic tools.