This study provides evidence of RNA microalgal viruses persisting in a CO2 remediation pilot project HRAP and uncovers the diversity of the RNA virosphere contained within it. Evidence shows that family Marnaviridae is cultured in the basin, alongside other potential microalgal infecting viruses . Finally, we demonstrate that the RNA viral diversity of the HRAP is temporally dynamic across two successive culturing seasons.Despite a surge of RNA virome sequencing in recent years, there are still many RNA viruses to uncover—as indicated by the relevance of viral dark matter to RNA virome studies . This study explores a unique site, a high-rate algal pond (HRAP), for culturing industrially microalgae, to elucidate new RNA viruses. The importance of viral-host interactions in aquatic systems are well documented, and the ever-expanding microalgae industry is no exception. As the industry becomes a more important source of sustainable plastic manufacturing, a producer of cosmetic pigments and alternative protein sources, and a means of CO RNA viruses persist in a range of environments from soils  to seas Dicistroviridae), similarly structured capsids and helicases, and an RNA dependent RNA polymerase (RdRp)  and single stranded negative sense [, as well as there being double stranded forms . Among te (RdRp) , howeverMarnaviridae, the virus HaRNAV was characterized and shown to infect the toxic bloom alga Heterosigma akashiwo [Rhizosolenia setigera [Chaetoceros spp. [Picornaviridae , which infect many economically important animals , and humans [Marnaviridae. Microalgae are an industrially and economically important group of organisms with a variety of uses, and potential uses, relating to cosmetics [2 remediation [Marnaviridae, have been labelled as a clear biological pollutant of microalgae culturing by cell infection [Among early studies of family akashiwo . HaRNAV setigera , and virros spp. ,28,29. Id humans , there aosmetics , food anosmetics , biopharosmetics , renewabosmetics , wastewaediation . These uediation . Microalediation . Given tediation  and geocediation  it has bediation , an impoediation . We shounfection , therebynfection , so knowMarnaviridae family, we carried out a study with the motive of characterizing new RNA viruses by sampling an industrial microalgal culturing basin; a high-rate algal pond (HRAP). Our goals are to (1) provide a complete view of the RNA viral diversity of the system, (2) identify new RNA viral populations, especially those that could be infecting microalgae, and (3) explore specific RNA viral population dynamics in the system to detect changes over time. We achieve goals (1) and (2) by a metagenomic study and (3) by a quantitative RT-PCR (RT-qPCR) approach.In consideration of the knowledge gaps within RNA viruses, especially in the 2 HRAP (for details see [2 remediation in the IFREMER (Institut Français de Recherche pour l’Exploitation de la Mer) marine station at Palavas-les-flots, France. Pure CO2 with a final pH of 7.5 was systematically pumped into the HRAP for the uptake of CO2 by these large-scale cultivations of microalgae as part of a green initiative for capturing carbon emissions expelled by industrial practices. This system featured natural colonization where seawater was pumped from the Mediterranean Sea (Plage du Prévost area) , and filtered through a 100 µm sand filter before entering into the HRAP. The system was restarted in the same way after each microalgal die-off . System restarts, termed a new “run”, after microalgal die-offs occurred on 17 July 2017 and May 28, July 5, and September 20 of 2018. Raw water samples (1 L) were taken for subsequent filtration and metagenomic sequencing  on April 17, May 17, July 5, September 11, and October 23 of 2018. Additional samples (50 mL) were also taken for subsequent filtration and nucleic acid extraction for RT-qPCR viral target tracking  throughout the basin culturing season (approximately spring to autumn) of 2017 and 2018  160mails see ) basin sg at room temperature (RT). The supernatant (800 mL) was serially filtrated at 5 µm with a Millipore Millex-SX filter, 1.2 µm with a Minisart NML Syringe Filter (Surfactant-free Cellulose Acetate) and 140 mL of the 0.2 µm eluate was concentrated into 5 mL  through 100 kDa TFF . Two subsamples of 1 mL were digested with a nuclease cocktail consisting in 100 µL 10× Turbo DNAase buffer , 40U Turbo DNase , 18U RNAse A, 125U benzonase, 100U exonuclease I, for one hour at 37 °C, followed by storage at −80 °C. For extraction, 3.75 mL of Trizol LS was added to 1.25 mL of the previously digested mixture, followed by homogenization and incubation for 5 min at RT. Chloroform (1 mL) was added and the mix was incubated at RT for 3 min. After centrifugation at 12,000× g  the aqueous phase was recuperated and another Trizol (3× volume)/chloroform (1.25× volume) was performed. The aqueous phase containing viral RNAs was then purified and cleaned with three columns of the Zymo Research RNA Clean & Concentrator Kit 25 using manufacturer’s instructions. The eluates (50 µL each) containing the purified RNAs were pooled  and 40U RNaseOUT  was added. RNAs were again cleaned with the Zymo Research RNA Clean & Concentrator Kit 25, 40U RNaseOUT  was added to the eluate (40 µL) and finally stored at −80 °C.Raw water samples (1 L) were clarified by centrifugation at 1500× To achieve a library of cDNA from low concentrations of RNA we carried out a reverse transcriptase and Klenow method ,45, withPaired-end Illumina MiSeq reads 2 × 250 bp) were preliminarily run through fastQC  and resu bp were −5. Each contig with a significant match was then attributed to the same putative taxonomic clade  as its best match. These results were then used to filter contigs by likely viral hits for inspection of the viral diversity of each sample.To study the diversity of each metagenomic sample we first performed a nucleotide-to-protein alignment  search u−5. Additionally, predicted proteins were assessed with InterProScan [Marnaviridae genomes accessed through NCBI GenBank based on best matches of putative ORFs from the said contigs.All assembled contigs were run through EMBOSS getorf  to extrarProScan  for furtrProScan  predicteContigs representing 99% of reads from the combined assembly of all sample reads were further investigated using the previous HISAT2  reads maAlpha diversity indices were calculated using all contigs above 250 bp individually for each month’s metagenome sample  and for the combined reads assembly. Contigs were processed through MerCat  using k-Discistroviridae possessing two), which are long proteins that undergo post translation modifications into subunits [To elucidate all useful RdRp proteins from the NCBI NR database (May 2020) all protein sequences assigned as order Picornavirales were downloaded using NCBI E-utilities commands  , collapssubunits , consequsubunits . Putativsubunits , gaps wesubunits   with emsubunits  with modPredicted proteins with viral functional annotations were used to select contigs assembled from metagenomic data as individual targets for population dynamics monitoring via RT-qPCR. Target sequences were used to produce primer pairs through a Primer3  Python sThe aforementioned raw water sample aliquots (including from samples destined for metagenomic sequencing) were filtered at 5.0 µm, then 0.2 µm in preparation for extraction. Nucleic acid extractions were carried out on 200 µL of filtrate using a High Pure Viral Nucleic Acid Kit  by manufacturer’s instructions except an elution volume of 40 µL was used. DNA was digested using a Turbo DNase Kit , with a reaction termination using a 0.5M EDTA. Reverse transcriptase to acquire cDNA was carried out using SuperScript™ VILO™ cDNA Synthesis Kit  by manufacturer’s instructions. RT-qPCR Reactions contained 0.2 µM of forward and reverse primer, 1 µL template, and 5 µL SsoAdvanced Universal SYBR 2x Green Supermix , supplemented with molecular biology grade (MBG) water to 10 µL total volume. The qPCR program was run as follows: 5 min at 95 °C, then 45 cycles of 10 s at each of 95 °C, 60 °C, and 72 °C, then finalized with a melt curve to check for number of products (starting at 60 °C and going stepwise at 5 s for each 0.5 °C to 97 °C). Amplicon sizes were verified on 2% agarose gels in 1% Tris-acetate-EDTA (TAE) buffer against a 100bp BenchTop pGEM DNA Marker ladder . Reactions were run in triplicate as on one 96 well plate  for each primer pair . Results were manually checked to confirm (1) amplification in the real-time visualization, (2) number of products as indicated by the melt curve, (3) cycle of quantification (Cq) values. A mean Cq value was calculated for each triplicate of sample reactions, the number of reaction cycles (45) minus the Cq value (termed the “inverse Cq”) and reported.Marnaviridae appear alongside them in two of these three months. These are potential hits of viruses infecting microalgae in our samples. Other notable hits include taxa that are known to infect fungi and oomycetes (family Narnaviridae [Totiviridae [Tombusviridae [Solemoviridae), family Birnaviridae infecting non-mammalian vertebrates and insects, most importantly including salmon and chickens [Yueviridae [Leviviridae [Narnaviridae, Totiviridae, and Yueviridae that were found in the HRAP [Phycodnaviridae, a dsDNA group of nucleocytoplasmic large DNA viruses (NCLDVs [Circoviridae (a single stranded (ss) DNA virus family [For April, May, July, September, October, and the all-reads  assembly the proportions of contigs with a database hit  were 72%, 82%, 81%, 32%, 44%, and 71% respectively. Of these hits the coverage of viral assignments was 14%, 8%, 10%, 31%, 54%, and 15%. Across the 2018 monthly metagenomic samples the proportion of database viruses labelled as “unclassified”  accounts for over 50% of the entire sample’s hits  in all months and our combined assembly . Of thesiviridae ), plantssviridae ; family chickens , a smalliviridae ). These the HRAP . In each (NCLDVs ) for whiCircoviridae hit recruits most (77%) of the reads, for May both unclassified Riboviria and “unclassified virus” hits recruit the majority of reads. For the months of July, September, and October, unclassified members of order Picornavirales dominate the read recruitments. This classification is based on only two putative full-length contigs (two viruses in theory) that recruits nearly half of the September reads each. Only one of these two contigs recruits reads in the July dataset (60%), whereas the other contig recruits most of the October reads (98%)  see .Marnaviridae (see references genomes), including their relative order of appearance along the contig  instead of taxonomic species, therefore freeing the data from the limitations of database dependent classification where sequencing gaps in RNA classification exist and an abundance of viruses remain as “unclassified”. A consequence of this limitation is that diversity is only quantified if the virus is “known” through a database hit, whereas using k-mers species permits an interpretation of diversity that is free from information loss when there are no hits to a database for a contig. In theory, k-mer based alpha diversity uses reoccurring k-mers as an indication of shared homology among contigs, and thusly a potential shared taxonomy.Four intra-community diversity  indices were run on k-mer based data from our assembled contigs . Using kThe Shannon diversity index A indicatAn inter-community species diversity  comparison using Bray-Curtis dissimilarity on k-mer species A indicatPicornaviridae, Polycipiviridae, Solinviviridae, Secoviridae, and Marnaviridae. Recovered RdRp domains from our data include nine unique sequences clustering with Marnaviridae, providing further evidence that this group of RNA viruses infecting microalgae not only persists in the HRAP where cultured microalgae would serve as hosts, but also as multiple Marnaviridae species.By processing nearly 39,000 sequences of published Picornavirale polyproteins and our assembled contigs we were able to form a fairly robust tree in terms of taxonomy . From ouMarnaviridae, with targets 1, 2, and 4 having a hit to a virus with an Aurantiochytrium sp. host, target 3 with a hit to a virus with Cylindrotheca closterium as a host, and target 5 with an unknown host. Additionally, target 6 has a top hit with a “Beihai noda-like virus”, and target 7 with a member of family Birnaviridae known to infect a member of phylum Rotifera and has been previously isolated before in the HRAP area in an unrelated study [Marnaviridae virus targets share a similar pattern where infections appear to occur between the microalgae die-offs of 5 July 2018 and 20 September 2018, with persistence before, during, and after the die-offs associated with 22 October 2018. Target 2 appears only on 23 October 2018 and beyond, and finally target 1 exhibits a completely distinct pattern compared to the other potential Marnaviridae in which it sustains a relatively low presence during April to mid-May of 2018 and appears once thereafter. Interestingly, targets 3, 4, and 5 are all of potential Marnaviridae, have the most similar pattern (to a lesser extent target 3), and do not share the same host associated with their top hit. Target 6, the “Beihai noda-like virus” shows a sudden appearance and slow disappearance before the late May 2018 crash, and finally target 7, the likely Rotifera sp. virus comes and goes with low levels of amplification, appearing a couple of times with a relatively higher number of copies, but disappears just as suddenly. Interestingly, there are instances in 2017 dates where targets (exclusively sequenced by metagenomics from 2018 samples) are amplifying in 2017 samples also, often with a Cq implying a relatively high copy number.In total, seven RNA viruses of interest (see ed study . Two (taMarnaviridae targets (1–5) we do see variation in viral dynamics. Indeed, targets 3 to 5 are present from late September to the end of our sampling in late October, but they are not necessarily present in the same periods (with the exception of targets 4 and 5 that are quite similar). As an example, target 3 appears in October of 2018, only, but is also present in May and June of 2017, although we do not have coverage of October 2017, we can at least see that the dynamics have not repeated exactly in 2018 as they were in 2017, and therefore are not annual in our study. Target 1 has a different pattern than other putative Marnaviridae viruses being primarily present only in April and May of 2018.Specifically, to the putative Marnaviridae through a phylogenetic study, and (3) measuring dynamics of select viral targets appearing in the system over time. This study offers a unique view of an HRAP RNA virome over time that has not yet been conducted before, and can offer new insights for future microalgal cultivation, specifically in an industrial context, and in cultures which are exposed to the environment .Our study documents the RNA viral community of an HRAP during periods of microalgae growth over two years by (1) exploring the taxonomic diversity to infer potential hosts, (2) using the RdRp gene to uncover Classification of viruses, in general, using metagenomics is made difficult by inconsistent coverage of taxonomic groups in databases causing what is known as “viral dark matter” where viral sequences return no hits  to major databases hosting viral sequences . In the Sclerophthora macrospora (Oomycete) virus, which is a chimeric virus (RNA-DNA hybrid) found only in our October sample . With these data we observe that the HRAP is hosting a wealth of viral diversity contained in unclassified and understudied taxonomies. This includes the potential of interesting chimeric viruses that are considered a rare event and with a plausible association to microalgae as they are found in samples enriched for Bathycoccus [Marnaviridae, and with hits to members of this microalgae-infecting family appearing in September and October metagenomes (2018) alongside assembled RNA contigs from the HRAP resembling the domain inclusion and domain order of known Marnaviridae and “marna-like” viruses  through metagenomics alone, there are many RNA viruses contained within the study samples that could be infecting microalgae. Overall, our taxonomic results reflect that of other RNA metagenomic studies and reviews in that members of order Picornavirales are common in aquatic settings [With the above points in mind, we can draw some conclusions from our taxonomic analyses. Foremost, it is clear that the unclassified viruses of Riboviria, and order Picornavirales are important to this HRAP community throughout the culturing period of 2018. Of course, in regard to Riboviria, it simply means that the viruses contain either RdRp, or the RdDp (RNA dependent DNA polymerase) of retroviruses. Many of these hits are to an invertebrate focused virus study , which ihycoccus . Of couruses see , we can me study  the authsettings .Circoviridae hit accounting for 77% of read recruitment in April, lake metagenomic studies have previously identified Circoviridae hits in RNA viromes, but concluded it was a contamination based on the presence of Circoviridae also in the DNA viromes [Astroviridae appearing in May, are known to infect both mammals and birds [By taking a normalized read mapping approach to investigate each of the 2018 metagenomic samples we can observe what viral contigs are recruiting a substantial number of reads, however, we should take into account sequencing biases produced by the process of sequencing. Although there are less studies on the subject of RNA virome sequencing biases, there is evidence of ssDNA viruses being recovered at higher proportions than dsDNA viruses ,89 for e viromes . An alte viromes , given t viromes . Additiond birds , therefoMarnaviridae. These infections have been confirmed by isolation in laboratory cultures [Marnaviridae  to occur in this basin. This domain searching method was both useful in extracting RdRp domains, and in identifying putative members of Marnaviridae.Finally, an RdRp tree of families within the order Picornavirales were chosen for our focus because one of the seven families of Picornavirales has ample evidence of infecting microalgae, the family cultures , and notDue to the presence of viral dark matter in our study, we chose to use a k-mer based approach for measuring the alpha and beta diversity of our samples. Specifically, we chose k-mer species created from contigs and not reads to avoid library preparation and sequencing related biases, and sequencing errors contained in raw read data. Since these are k-mer species, and not taxonomic units, we cannot easily compare our results with taxonomy based alpha diversity indices of other studies but can make observations between and among our own samples. Shannon diversity is a measure of entropy/disorder in a sample that is relatively sensitive to the number of rare species present, whereas Simpson diversity has less bias in reference to the number of species sampled  . In otheMyoviridae) that was linked to host seasonality [In the context of our beta diversity results, the diversity is different between the samples, with the dissimilarity being especially apparent between October and other months except September. There is a similar pattern when assessing similarities between samples using reads mapping, however, July also recruits over ~50% to September and October, whereas with beta diversity it’s most similar  to May. Based on this study’s RT-qPCR results (discussed more below), there is evidence of viruses appearing and disappearing over time, so we cannot assume viruses appear according to season/month alone, and furthermore we cannot exclude the fact that die-offs and restarts, therefore many different runs, of the culture are occurring in the HRAP. In fact, a study focusing on a subset of dsDNA viruses (NCLDVs) showed temporal variation that did not follow a year-to-year seasonality in viral diversity , and anosonality . Althougsonality , etc. Thn number of nucleotides to be different) on contig based k-mers could improve it, and also looking at protein-based data where k-mers are created from amino acid sequences because substitutions could occur less frequently at the amino acid level than at the nucleotide level. Additionally, an obstacle of protein-based k-mer species is deciding what proteins to use and what not to use. For example, should proteins included have a specified degree of certainty in their prediction, should only taxonomically informative proteins  be included exclusively? These adjustments to the process are not mutually exclusive and conducting them in parallel might be necessary. These approaches suggested above are not available as computational tools at the time of writing, and if made available a variety of testing would need to occur for determining best practices, which is outside the scope of this study.Results from these database independent approaches to studying diversity in metagenomics must be taken with caution. Using reads based approaches we are no doubt affected by biases introduced before and during sequencing, and with k-mer species counting we are using a relatively new method that is quite strict (k-mer species are considered different if one out of 21 nucleotides are different) and in this case it may be currently difficult to apply to metagenomes targeting taxa with high mutation rates—such are the case for RNA viruses . LogicalIn general, it is not surprising that the diversity of the HRAP overall  is higher than that of each month alone due to the changes witnessed in the HRAP overtime, but it does suggest that the diversity of the HRAP is better captured over time and not as a single time point. Conclusively, we insist the RNA viral diversity using metagenomics is better captured temporally in newly explored environments than as single time points.Marnaviridae because of their host relationship with microalgae. However, the association between the appearance of these targets and the die-offs is not clear. We are also limited by our sampling, where the die-offs in May and July of 2018 lack the coverage that we gained by sampling done in September and October 2018 around their specific die-offs. We cannot completely rule out the impact of viruses on the system, however because our tracking was limited and other viruses  could also be contributing. The effects of viral infection on microalgae may be that of several populations and other viruses in unison. Regardless, there is evidence of viruses potentially infecting microalgae appearing and disappearing temporally in the HRAP. Interestingly, three of the potential Marnaviridae targets appear in the 2017 samples , however we do not see a gradual increase and decrease in the relative population (inferred by RT-qPCR Cq), that appears in the 2018 dynamics. These sporadic appearances could be amplification of similar strains or related viruses of the 2018 Marnaviridae targets, so-called “viral quasispecies”. Overall, these Picornavirale dynamics results are not unlike results obtained in a study of two Picornavirale strains among a three-lake system in America , where the strains exhibited different dynamics when compared to each other and also compared across the three lakes at the same sampling times [Our RT-qPCR methods in coordination with microalgae die-off dates has the potential to inform us of what viruses could be contributing or be responsible for these sudden die-offs. We used this technique to follow viruses of interest within the HRAP. We primarily focused on the ng times . MembersRotifera virus are quite interesting. The relatively common Brachionus sp. are known predators in HRAP/microalgal culturing practices that are open faced and outdoors [Rotifera spp. are being reintroduced, infected, and dying off or their populations is being dramatically changed by new HRAP runs . We assume that the amplicon is a reflection of the relative amount of the viral copies/viruses and therefore the Rotifera spp. host(s) appearing in the basin, with this we might conclude a brief outbreak of Rotifera spp. occurred in early September 2018, and reoccurred and persisted in October of 2018. We have also assumed that this virus is a component in the regulation of the Rotifera spp. likely appearing in the HRAP, but we cannot exclude the importance of factors like intra-species competition and abiotic factors [Rotifera spp. play a role in the die-offs in the HRAP because tracking of this microalgal grazer is not reported in this study.Aside from the targets potentially infecting microalgae, the dynamics of the outdoors  and are outdoors . Given t factors ,107. AltMarnaviridae, which has been uncovered through putative taxonomic classification, a phylogenetic study, and tracked in the HRAP using RT-qPCR. We cannot conclude that RNA viruses are alone responsible for the die-offs experienced in the HRAP considering we did not investigate other factors , however we have provided evidence of viruses likely infecting the microalgae being cultured in the HRAP. We have provided an overview of the taxonomic diversity of viruses contained in the HRAP, albeit limited by database completeness, alongside database independent approaches to quantifying the basin diversity. Lastly, we glimpsed into the dynamics of some viruses of interest.In this study we have identified several putative RNA viruses alongside evidence to suggest that they could be infecting microalgae, specifically in the case of In a broad sense, from our work we can conclude that future studies of RNA viruses should aim to classify environmental viruses more thoroughly alongside efforts to improve database-independent methods of studying diversity in viruses, specifically in environments not previously sampled. Advancing viral taxonomy and classification is on-going and will take time, thus improving our database independent approaches in the near future could be quite informative for viral studies similar to this. Finally, we have approached studying viral dynamics with a RT-qPCR approach, highlighting the array of viral dynamics patterns that can occur temporally and concluding that individual viruses can behave quite differently.


Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues. Multiplexed RNA profiling in single cells, in their native spatial contexts, holds great promise to reveal the composition, function, regulation, and interaction of the different cell types in complex biological systems . MicroarTo enable multiplexed in situ RNA profiling, combinatorial labeling ,9,10, reHere, we report a highly sensitive and multiplexed in situ RNA profiling approach. In this approach, RNA targets are stained with horseradish peroxidase (HRP), conjugated oligonucleotides, and cleavable fluorescent tyramide (CFT) with both a high sensitivity and specificity. After imaging, the fluorophores are chemically cleaved, and the oligonucleotide probes are stripped. Through cycles of target staining, fluorescence imaging, fluorophore cleavage, and probe stripping, highly sensitive and multiplexed RNA analysis can be achieved in single cells in situ. We demonstrate that the staining signals can be very efficiently removed by a mild chemical reaction and that the oligonucleotide probes can also be stripped by DNase and formamide solutions with a high efficiency. These two processes maintain RNA integrity. In applying this approach, we show that multiple RNA targets in FFPE and fixed frozen tissues can be unambiguously detected by our approach. With these multiplexed single-cell RNA profiling data, we study the varied cell type compositions and their spatial organizations within tissues. We also demonstrate that this RNA analysis approach can be combined with multiplexed protein imaging technology, which allows multiple RNA and protein molecules to be profiled together in the same tissue in situ.Chemicals and solvents were purchased from Sigma-Aldrich  or Ambion , and were used without further purification, unless otherwise indicated. Bioreagents were purchased from Invitrogen , unless otherwise noted.2O2, 0.1% Triton-X 100 in 1× phosphate buffered saline (PBS)) at room temperature, followed by 3 times 1× PBS wash at room temperature, each for 5 min. After blocking, the slide was incubated with the antigen retrieval buffer  at 100 °C for 15 min. Finally, the slide was incubated with RNAscope® Protease Plus  for 30 min and washed with 1× PBS 3 times at room temperature, each for 5 min.The mouse lung formalin-fixed paraffin-embedded (FFPE) tissue slide was baked for 1 h at 60 °C. Subsequently, the deparaffinization of the slide was performed by 3 times xylene wash, each for 5 min. Then, the slide was immersed 3 times in 100% ethanol at room temperature, each for 1 min. Afterwards, HRP blocking was conducted via a 10 min incubation of the slide in HRP blocking buffer  for 30 min and washed with 1× PBS 3 times at room temperature, each for 5 min.® Multiplex Fluorescent Reagent Kit v2 , following the manufacturer’s instructions. Signal development was subsequently performed by incubating the slide with 0.25 pmol/uL tyramide-N3-Cy5 in amplification buffer  at 40 °C for 30 min. Afterwards, the slide was washed 3 times with 1× RNAscope Wash Buffer  at 40 °C, each for 5 min. After imaging, the slide was incubated with cleavage solution , pH 9.5) at 40 °C for 30 min to remove fluorescence signal. Oligonucleotide probes were then stripped by incubating the slide with 0.5 unit/µL DNase I  at room temperature for 1 h. DNase was subsequently quenched by washing the slide with DNase quenching buffer (30% lithium dodecyl sulfate and 30% formamide in Tris-EDTA buffer) at room temperature, each for 10 min. In order to fully strip of the remaining oligonucleotide probes, the slide was then incubated with 70% formamide in 2× SSC at 40 °C for 30 min, followed by 3 times 1× PBS wash at room temperature. The slide was then ready for the next cycle of RNA detection.After pretreatment, mouse lung FFPE tissue or mouse spinal cord fixed frozen tissue was first incubated with RNAscope mRNA probe  for 2 h at 40 °C. Then, the slide was treated with RNAscope3-Cy5 in an amplification buffer at room temperature for 7 min. After imaging, the slide was incubated with cleavage solution at 40 °C for 30 min to remove fluorescence signal. The slide was then ready for the next cycle of protein detection or mRNA detection. The primary antibodies used include HRP-conjugated rabbit anti-HMGB1  and HRP-conjugated mouse anti-hnRNP K .The mouse spinal cord fixed frozen tissue, after pretreatment, was blocked in 1× blocking buffer  at room temperature for 1 h. Then, the slide was incubated with 5 µg/mL of HRP-conjugated primary antibody in 1× blocking buffer at room temperature for 45 min, and subsequently washed 3 times with PBT (0.1% Triton-X 100 in 1× PBS) at room temperature, each for 5 min. Afterwards, signal development was performed by incubating the slide with 10 pmol/uL tyramide-NThe slide to be imaged was first incubated with GLOX buffer (0.4% glucose and 10 mM Tris HCl in 2× SSC) for 1–2 min at room temperature. Then, the slide was imaged in GLOX solution .https://www.c2b2.columbia.edu/danapeerlab/html/cyt.html, accessed on 5 January 2020) [Both the FFPE slides and the fixed frozen slides were imaged under a Nikon Ti-E epifluorescence microscope equipped with a 20× objective. Images were taken using a CoolSNAP HQ2 camera . DAPI images were taken using the C-FL DAPI HC HISN filter. Cy5 images were taken using the Chroma 49009 filter. Imaging, processing, and pseudo-color assignment were processed by ImageJ . Cell sery 2020) .2O2. Fifth, step two to four may be repeated to stain other RNA targets in the first cycle  in the same tissue.The two factors that determine the multiplexing capacity of this approach include the number of analysis cycles and the number of RNA profiled in each cycle. We have demonstrated that the RNA integrity is maintained after the treatment with TCEP for 12 h, DNase for 24 h, and 70% formamide for 12 h. These results suggest that at least 12 cycles can be carried out on the same specimen. In each cycle, four or five different RNA targets can be stained with varied fluorophores and imaged simultaneously. Therefore, we anticipate that this technology could profile over 50 distinct RNA species in the same specimen if different RNA targets are stained in different cycles. When the same set of RNA species is stained throughout all the cycles, this approach has the potential to quantify thousands of varied RNA targets within eight cycles (4In addition to RNA and protein analysis, our reiterative staining approach can also be applied to highly sensitive in situ DNA  and meta


Recent advances in the generation, purification and cellular delivery of RNA have enabled development of RNA-based therapeutics for a broad array of applications. RNA therapeutics comprise a rapidly expanding category of drugs that will change the standard of care for many diseases and actualize personalized medicine. These drugs are cost effective, relatively simple to manufacture, and can target previously undruggable pathways. It is a disruptive therapeutic technology, as small biotech startups, as well as academic groups, can rapidly develop new and personalized RNA constructs. In this review we discuss general concepts of different classes of RNA-based therapeutics, including antisense oligonucleotides, aptamers, small interfering RNAs, microRNAs, and messenger RNA. Furthermore, we provide an overview of the RNA-based therapies that are currently being evaluated in clinical trials or have already received regulatory approval. The challenges and advantages associated with use of RNA-based drugs are also discussed along with various approaches for RNA delivery. In addition, we introduce a new concept of hospital-based RNA therapeutics and share our experience with establishing such a platform at Houston Methodist Hospital. RNA Therapeutics comprise a rapidly expanding category of drugs that will speed solutions to the clinic; will actualize personalized medicine; and will make the term “undruggable” obsolete. The first RNA drugs have been approved, and many more are in development. We are in the midst of a therapeutic revolution, the likes of which have not been seen since the advent of recombinant protein technology almost 50 years ago in Silicon Valley. Accordingly, we will review recent developments in RNA Therapeutics, and their promise to alter the landscape of the pharmaceutical industry.Conventional drug strategy relies on the ability of small molecule drugs to target active sites of proteins so as to inhibit or alter their function. It is well known that only ∼1.5% of the human genome encodes proteins . FurtherDNA drugs generate therapeutic proteins when delivered to the nuclei of the patient’s cells. DNA drugs may be delivered as plasmids or integrated into a viral vector.DNA plasmids are high molecular weight, double-stranded (ds), circular DNA molecules that encode a therapeutic protein. Such proteins could replace defective or missing proteins in the patient . DNA plaDNA-based drugs may be directed toward replacing defective or missing protein(s) . HoweverThe Chinese food and drug regulatory agency approved the first DNA therapy drug, Gendicine, in 2003 to treat head and neck squamous cancer. Gendicine is an adenoviral vector encoding the wild type (wt) p53 gene which restores the expression of this tumor suppressor function to treat cancer . ApproxiDNA-based vaccines encode specific antigen(s) to induce a protective immune response . Imlygicex vivo to alter cell phenotype or function, and then these cells are expanded and delivered into the patient . The CD34+ cells are then transduced with a gammaretrovirus vector carrying the gene for human adenosine deaminase (ADA), then reinfused into the patient. These cells home to the patient’s bone marrow where CD34+ cells replicate and generate normal ADA protein to correct the deficiency . Strimvelis uses the patient’s own CD34ficiency . Zyntelgmoglobin . A simil-infused . As desctrans-activating CRISPR RNA (tracrRNA), serves as a scaffold and binds to Cas9, a DNA endonuclease. The other molecule, called CRISPR RNA (crRNA), has sequence homology to the foreign DNA  is an early stage clinical CRISPR medicine to treat patients suffering from Leber Congenital Amaurosis type 10 (LCA10). LCA10 is a genetic blindness caused by an A to G point mutation within intron 26 in the CEP290 gene involved in phototransduction. This mutation results in a splicing defect to include a 128 bp cryptic exon in CEP290 transcript, thereby leading to expression of non-functional mutant protein. EDIT-101 is designed to deliver function .A few other CRISPR-Cas9-based drugs are also in clinical trials now. For instance, CTX001, CTX110, CTX120, and CTX130 from CRISPR Therapeutics  antisense RNA (RNAi), where short oligonucleotides recognize and hybridize to complementary sequences in endogenous RNA transcripts and alter their processing ; and (2)These hurdles have been substantially overcome with recent advancements in RNA biology, bioinformatics, separation science and nanotechnology thereby facilitating the recent rapid development of RNA therapeutics. Advantages of RNA-based drugs that are driving development include: (1) their ability to act on targets that are otherwise “undruggable” for a small molecule or a protein; (2) their rapid and cost effective development, by comparison to that of small molecules or recombinant proteins; (3) the ability to rapidly alter the sequence of the mRNA construct for personalized treatments or to adapt to an evolving pathogen. Below, we review the different classes of RNA therapeutics see , the chaAntisense oligonucleotides are short single-stranded DNA, phosphorothioate DNA, RNA analogs, conformationally restricted nucleosides , or morpholino phosphorodiamidate oligonucleotides complementary to a certain region of RNA that they are meant to target. The modifications in backbone, and sugar molecules give antisense oligos more affinity and stability . ZamecniThere are two classes of ASOs: RNase H-dependent ASO , and RNaO-2-methoxyethyl ASOs that were complementary to the 5′ cap region of the intercellular adhesion molecule 1 (ICAM-1) transcript in human umbilical vein endothelial cells. These ASOs inhibited protein expression of the targeted transcript by interfering with the formation of the 80S translation initiation complex  nusinersen  ; (2) eteon 7 see , and, thon 7 see .Eteplirsen  is used to treat Duchenne muscular dystrophy (DMD). DMD is caused by mutations in the DMD gene that result in a premature stop codon and a non-functional dystrophin protein, with a mutation in exon 51 being most frequent single exon mutation . EteplirInotersen  is used to treat familial amyloid polyneuropathy caused by the mutation in the transthyretin (TTR) gene. The mutated gene leads to translation and accumulation of the abnormal TTR protein in different organs, including heart, kidney, eyes, and nerves. Tegsedi is a 20-mer chemically modified RNA molecule that hybridizes to TTR mRNA, mimicking a DNA/RNA complex, and thereby interferes with TTR protein production .Volanesorsen  was recently approved in Europe . This is an antisense oligonucleotide indicated for treatment of familial chylomicronemia syndrome . It recoC. elegans. They showed that dsRNA are more effective than ssRNA to artificially induce RNAi and destroy an mRNA target  are small non-coding RNA duplexes that originate from precursor siRNAs. The latter are either transcribed or artificially introduced and range from 30 bp to more than 100 bp. The precursor siRNA duplex is processed by the endogenous Dicer enzyme into 20–30 bp long siRNA with two base overhangs in the 3′ region, which interacts with the endogenous RNA-induced silencing complex (RISC) to elicit RNA interference (RNAi). The endonuclease argonaute 2 (AGO2) component of the RISC cleaves the sense strand, leaving intact the antisense strand, which guides the active RISC to its target mRNA. Then AGO2 cleaves the phosphodiester backbone of the target mRNA. The antisense strand is usually fully complementary to the coding region of the target mRNA, therefore siRNA knocks down one specific target gene . Fire etA target . PatisirA target . It is uA target . RecentlA target  for the A target .C. elegans  are small non-coding RNA molecules that regulate the expression of multiple mRNAs by blocking translation or promoting degradation of the target mRNAs. They were first discovered in  elegans . This cl elegans . The miR elegans . Cobomar elegans . Remlars elegans , MRG-229 elegans , while M elegans . InteRNA elegans . Regulus elegans . Besides elegans . Miravir elegans . Another elegans . OverallAptamers are short single-stranded nucleic acids that can bind to variety of targets, such as proteins, peptides, carbohydrates, and other molecules, by virtue of the tertiary structure of the aptamer, rather than its sequence. Aptamers were first described in 1990 independently by two groups, Although aptamers hold promise as therapeutics, there is only one FDA-approved aptamer-based drug on market. Pegaptanib  is a 27 base RNA aptamer see  selectedAptamers have great potential to replace monoclonal antibodies in therapeutic and diagnostic  applicatin vivo. They injected synthetic mRNA encoding luciferase, chloramphenicol acetyltransferase, or beta-galactosidase into mouse skeletal muscle and detected proteins translated from these RNAs at the injection site. Importantly, the extent of expression from RNA was comparable to that obtained from injection of a DNA vector encoding the same proteins (Messenger RNAs (mRNAs) serve as the intermediates between the coding genomic DNA and the encoded proteins . Essentiproteins . Jirikowproteins .in vitro-translated mRNA is engineered to mimic naturally occurring mRNA , CureVac , and BioNTech . Together, they have a diverse portfolio of gene therapy products in the pipeline, covering metabolic diseases, heart diseases, and immunomodulators for applications in immuno-oncology. For instance, Moderna’s AZD8601 is a naked mRNA encoding vascular endothelial growth factor (VEGF-A) that is intended to be delivered through epicardial injection during coronary artery bypass surgery. The intent is to enhance local angiogenesis, thereby reducing myocardial ischemia and improving left ventricular systolic function  in patieBioNTech, in collaboration with Sanofi, is evaluating an mRNA-based intratumoral immunomodulator BNT131 (SAR441000) as a monotherapy in patients with advanced melanoma. This immunomodulator is a combination of IL-12sc, IL-15sushi, IFNα, and GM-CSF mRNAs, increased concentration of which in the local tumor microenvironment may promote natural killer cell activation and induce cytotoxic T-cell responses, resulting in an immune-mediated destruction of tumor cells . The mRNAlthough the aforementioned pharmaceutical companies were pioneers in commercialization of mRNA therapeutics, there are many other research groups and biotech start-ups worldwide entering the field. For instance, in vivo (mRNAs encoding antigens and/or adjuvants can be used as vaccines to evoke protective immunity against infectious diseases (prophylactic vaccines), or to harness the immune system to fight cancer (therapeutic vaccines). The COVID-19 pandemic has showcased the utility and advantages of RNA technology for vaccination, as out of all COVID-19 vaccines under development, the first two to have received emergency use authorization by the FDA were RNA-based. The first authorized vaccine was developed by BioNTech in collaboration with Pfizer. Initial clinical trials conducted in Germany helped to identify two leading vaccine candidates, BNT162b1 and BNT162b2, which were then tested further in the US , b. BNT1in vivo  and are in vivo . A largein vivo .The Moderna RNA vaccine against COVID-19 received emergency use authorization shortly thereafter  , based oThe biotechnology response to the COVID-19 pandemic has highlighted the speed and flexibility of mRNA vaccines , and revin vivo by immunizing animals with subcutaneous injection of liposome-encapsulated mRNA, which encoded the nucleoprotein of the influenza virus. RNA vaccines can activate both cell-mediated and humoral immunity. For instance, in vivo  is a great example of mRNA therapeutics encoding human monoclonal neutralizing antibodies (mAb). This drug is designed to provide passive protection against chikungunya infection. The ultrapotent antibodies were isolated from the B cells of a survivor of natural infection, and their sequences were encoded into mRNA molecules, encapsulated in lipid nanoparticles and delivered by infusion into mice. After mRNA delivery, one human mAb, CHKV-24, was found to be expressed at immunologically relevant levels, and its protective capacity was evaluated in mouse models of chikungunya. Treatment with CHKV-24 mRNA reduced viremia to undetectable levels at 2 days after inoculation and protected mice from mortality. Further studies with non-human primates also demonstrated a long-lasting immunogenic effects of the drug . OverallAnother interesting RNA-based approach to vaccination is that with self-amplifying RNA vaccines. The backbone sequence for the latter is adapted from an alphavirus, a positive-sense single-stranded RNA virus with high capacity for replication . Such mRThere are multiple RNA cancer vaccines in clinical trials such as CV9202 (CureVac), which is a self-adjuvating RNA vaccine targeting six antigens commonly expressed in non-small cell lung cancer . The mRNex vivo with mRNA encoding the desired proteins . Then the mRNA-enhanced cells are re-infused into the patient to treat the disease , together with mRNA encoding the transposase enzyme. The plasmid DNA encoding the transposon is co-delivered along with the mRNA transposase enzyme in a single electroporation reaction. The transposase that is translated from the mRNA then binds to the inverted repeats and cuts the DNA, to release the transposon. After that, the transposon binds a strand of genomic DNA with a TA dinucleotide, where the transposase creates a double-stranded break, allowing the transposon to integrate . Using tTargeted delivery is a major hurdle for effective RNA Therapeutics, a hurdle that must be overcome to broaden the application of clinical translation of this type of therapeutic. Because mRNA is inherently unstable it requires delivery vehicles that will protect the cargo from RNAase degradation. There is a need for novel delivery vehicles that will deliver the RNA drug to the site of therapeutic action facilitating the entry of the RNA drug into the cytoplasm where it may exert its effect. In the following paragraphs we provide a brief review of the advances made in RNA delivery vehicles.Liposomes are formed when materials containing polar head groups and non-polar tails (phospholipids) are dispersed in aqueous phase. They are spherical vesicles consisting of at least one phospholipid bilayer enclosing an aqueous core. They are a flexible drug delivery particle which may have various surface modifications see  capable O-octadecenyl-3-trimethylammonium-propane) and DOTAP  readily form complexes with negatively charged RNA. The use of cholesterol modified lipid makes the resulting complex more stable and improves transfection. Liposomes have an inherent advantage in that they mimic cell membrane composition and can encapsulate mRNA when combined with cationic lipids. Positively charged lipids can electrostatically interact with negatively charged mRNA to form complexes of RNA and liposomes. In this way, RNA is encapsulated within liposomes. Cationic lipids such as, DOTMA  made of cationic and other lipids, cholesterol, and polyethylene glycol (PEG) with a hydrophilic inner core retain the capacity to carry anionic RNA, protect it from degradation and prolong its circulation. An example of the efficacy of the LNP platform is patisiran , the siRNA-based therapeutic against hereditary transthyretin-mediated amyloidosis discussed above. In patisiran, the dsRNA is encapsulated inside four lipid excipients : DSPC , PLA (polylactic acid), chitosan, gelatin, polycaprolactone, and poly-alkyl-cyanoacrylates. These materials have the virtue of a long shelf life; the ability to encapsulate hydrophilic and hydrophobic compounds and proteins; and the capability for tuned delivery of therapeutic compounds . The fleThe ease of encapsulation extends to nucleic acids. Cationic hydrophilic polymers with hydrophobic modification can self-assemble in aqueous phase to encapsulate RNA. An example of this was employed by Consideration must also be given to the intrinsic tendency of polymers to induce inflammation within the immediate microenvironment, due either to lack of degradation such as in the case of PLA or through byproducts. In the case of PLGA, degradation into its basic monomers, lactic acid and glycolic acid leads to a decrease in pH at sites of degradation, promoting inflammation . This isMesoporous silica nanoparticles (MSNPs) have gained more attention for their therapeutic applications. The nanoparticles consist of an amorphous silica (silicon dioxide) matrix with ordered porosity in the mesoporous range. The peculiar feature of this nanoparticle includes large surface areas with large pore volumes, ease of modification, and established silanol chemistry. The surface of the nanoparticles can be modified by positively charged moieties to transport negatively charged RNA. The MSNPs pore sizes are tunable with a broad range and can be quite uniform. The particles have high loading capacity for nucleic acids and efficient delivery. RNA can be loaded inside pores for their transport and the surface can be modified with cancer specific ligands and antibodies to deliver the RNA to the target site. Particle aggregation is a hurdle that must be overcome so that the product is safe for injection. Aggregation can cause thrombosis or induce tissue injury. However, PEGylation of the surface of the nanoparticles greatly decreases aggregation and tissue damage. These nanoparticles degrade into non-toxic products and are safely excreted . Meka etGold nanoparticles, quantum dots, nanographene oxide, carbon nanotubes are each synthesized nanostructures that have the capacity to harbor RNA, protecting it from degradation and delivering it to the targeted disease site. GalNAc is a trivalent ligand that binds to asialoglycoprotein (ASGPR) receptors in hepatocytes. Clinical studies suggest that GalNAc conjugated siRNAs are very efficient to knockdown gene expression in the liver. We are witnessing the dawn of a new era of biopharmacotherapy. The promise of mRNA therapeutics has galvanized the pharmaceutical companies and investment sector, as evidenced by the current market caps of Biontech ($22.4B), Curevac ($18.6B), and Moderna ($52B), as well as the rapid proliferation of biotech start-ups in this field . Each ofNotably, mRNA therapeutics are a disruptive therapeutic technology, as small biotech startups, as well as academic groups, can rapidly develop new and personalized mRNA constructs. Our group has long-standing expertise in designing and manufacturing RNA therapeutics for the scientific community. Initially we were funded by the NHLBI to serve as an mRNA core for stem cell investigators in the Progenitor Cell Biology Consortium. Subsequently, the Cancer Prevention and Research Institute of Texas funded us to address an unmet need for cancer researchers in Texas who use mRNA as a research tool or a therapeutic. The demand for our services has increased substantially within the last 5 years, while we strengthened our expertise through extensive collaboration with cancer biologists, immunologists, RNA biologists, bioinformaticians, and nanomedicine scientists. During this time we established our hospital-based cGMP-compliant manufacturing capability and quality control methods see . We liceDuring these years we learned that many small biotech companies and academic groups, who have innovative ideas for disruptive RNA therapeutics, lack key competencies to reach the clinic, such as manufacturing capabilities, delivery technologies, or regulatory expertise, while our group has been operating in the environment established to accelerate novel therapeutics from conception to the clinic. Houston Methodist Research Institute is adjacent to the Houston Methodist Hospital, which is ranked by US News and World Report as the No. 1 hospital in Texas. The Institute was established with the focus on clinical translation. As such, the Institute has the infrastructure necessary to support pre-clinical and clinical development of novel therapeutics. We have clean rooms and calibrated equipment for cGMP manufacturing of RNA constructs, as well as the equipment to synthesize and analyze lipid nanoparticles for RNA encapsulation. We have an Office of Translational Production and Quality that provides guidance and quality oversight for manufacturing, QC testing and release. In addition, our Office of Regulatory Affairs assists with regulatory roadmaps and translational research planning. Our Comparative Medicine Program supports GLP animal studies for IND applications, while our Cockrell Clinical Trial Center designs and implements first-in-man clinical trials. This infrastructure has facilitated our development of the first fully integrated hospital-based RNA therapeutics program.Our RNA core is the central pillar, as the RNA core team develops new constructs and manufactures research or clinical grade RNA. The RNA core is complemented by RNA biologists, bioinformaticians, and nanomedicine experts. This program offers a single entry point with consultation to ensure a seamless transition between development stages. This system facilitates the development of RNA therapeutics by academic groups and small biotechs and dramatically shortens the time “from bench to bedside.” Furthermore, it promotes collaboration between our RNA faculty and clinician-scientists at the hospital to identify unmet clinical needs and develop mRNA-based solutions.The success of hospital-based programs to generate novel nucleic acid drugs has already been demonstrated. For instance, the investment of Nationwide Children’s hospital into cGMP facilities and a critical mass of gene therapy experts led to the creation of transformative gene therapies for lethal childhood diseases: SMA  and DMD . Golodirsen is a phosphorodiamidate morpholino oligomer that hybridizes to exon 53 of dystrophin pre-mRNA, and restores the mRNA reading frame in patients with confirmed DMD mutations amenable to exon 53 skipping . BesidesIL2RG was designed to include insulators to block activation of genes adjacent to where IL2RG is inserted into the patients’ DNA. The goal was to prevent the gene therapy from inadvertently causing leukemia by switching on an oncogene in the patient’s blood stem cells. Vector production and gene therapy treatment were streamlined using a stable producer cell line and cryopreservation. Combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1, the therapy had only low-grade adverse effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months  to correct the misplicing and restore MFSD8 expression. Soon after pre-clinical studies to confirm efficacy and assess toxicity studies, the drug was administered to the patient with clinical improvement observed . NotablyRNA Therapeutics is a rapidly emerging field of biotherapeutics. These therapies are based upon powerful and versatile platform which has nearly unlimited capacity to address unmet clinical needs. RNA Therapeutics are destined to change the standard of care for many diseases. The number of RNA drugs under development, and in clinical trials, is growing rapidly. The rapid growth of RNA therapeutics has been due to solving the problems of stability, delivery and immunogenicity. Whereas there is room for further improvement and innovation in each of these areas, the solutions have advanced to the point that RNA Therapeutics are now feasible. Although several dominating players in the RNA biopharma sector have emerged, new small biotech startups as well as academic groups with transformative ideas are propagating. In addition, hospital-based RNA therapeutics programs will facilitate RNA-based drug development and accelerate translation of transformative therapies from lab bench to patient’s bedside.TRD: first draft and illustrations. RS, CB, FT, and RIP: subsequent revisions of manuscript. JPC: revisions of the manuscript and funding of effort. All authors contributed to the article and approved the submitted version.Houston Methodist Hospital has been assigned intellectual property related to the synthesis, purification, validation, and delivery of nucleic acid therapeutics. JPC is an inventor on issued patents related to mRNA telomerase therapy, which have been assigned to Stanford University and licensed to his company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA methylation is considered a significant epigenetic modification, a process that does not alter gene sequence but may play a necessary role in multiple biological processes, such as gene expression, genome editing, and cellular differentiation. With advances in RNA detection, various forms of RNA methylation can be found, including N6-methyladenosine (m6A), N1-methyladenosine (m1A), and 5-methylcytosine (m5C). Emerging reports confirm that dysregulation of RNA methylation gives rise to a variety of human diseases, particularly hepatocellular carcinoma. We will summarize essential regulators of RNA methylation and biological functions of these modifications in coding and noncoding RNAs. In conclusion, we highlight complex molecular mechanisms of m6A, m5C, and m1A associated with hepatocellular carcinoma and hope this review might provide therapeutic potent of RNA methylation to clinical research. Hepatocellular carcinoma (HCC) is a common global disease. It has a poor prognosis and has become the third cause of cancer death . AlthougRNA methylation is commonly regarded as posttranscriptional modification with multiple forms . AlthougA prior survey revealed that abnormal regulation of these RMPs would give rise to incidence of various malignant tumors . For insMethylation refers to epigenetic transformations to influence gene expression but does not alter gene sequence, which can be mainly found in DNA, RNA, and protein . As apprm6A is methylated adenosine at the nitrogen-6 position and was identified as a posttranscriptional modification in 1974 . Previoum5C is defined as the accession of methyl group on the fifth carbon atom of cytosine . Abundanm1A is methylation at the N1 position of adenosine and is capable of altering RNA secondary structure. A previous study identified m1A in tRNA, rRNA, mRNA, and mitochondrial RNA. Affluent m1A modification is observed in tRNA and rRNA, while the level of m1A remains low in mRNA. The occurrence of m1A methylation in mRNA is represents a six-fold reduction compared to that of m6A methylation ; howeverRNA methylation takes place in various RNAs, which give rise to different outcomes to influence RNA function . The detThe stability of mRNA is mainly regulated by modification m6A. YTHDF2, an m6A “reader”, might recruit mRNA into processing bodies and participate in the process of degradation to stabilize mRNA . NumerouIn mRNA, modification m5C can have a significant impact on metabolism. ALYREF was considered to enhance the efficiency of nuclear-cytoplasmic export by forming a complex with mRNA. Previous experiments confirmed that NSUN2 meditates mRNA transport, which facilitates ALYREF binding to mRNA . TherefoModification m1A is found predominantly in structured regions of the 5′-UTR and near alternative start codons, indicating that m1A is significantly involved in stabilizing mRNA structure. The accomplishment of m1A methylation also exerts an indispensable effect on translation efficiency. For instance, m1A in the CDS region of mRNA has been considered to block the productivity of protein synthesis because it disrupts Watson-Crick base pairing. The presence of m1A might be vital to regulate the structural thermostability of tRNAs. It was reported that m1A together with other post-transcriptional modifications is capable of enhancing the melting temperature of tRNAs. ALKBH1 deficiency improves the cellular level of tRNA-Met to maintain the functional effect of m1A, stabilizing tRNA-Met . On the Recently emerging evidence has demonstrated that RNA methylation plays a dramatic role in tumorigenesis, invasion, and migration of HCC and elucidated complex mechanisms. We present the evidence for regulators and the effect of m6A, m5C, and m1A related to initiation and progression of HCC in ID2 and GATA3 mRNA (Zeb1 mRNA, which participates in HCC tumorigenesis. The lifetime of Zeb1 gain improved via the effect of m6A modification (Numerous studies have recently probed the relationships between m6A methylation and HCC pathogenesis. Wang et al. demonstrated that circ-KIAA1429 is expressed at a higher level in HCC cells than in normal cells, and the patients generally have shorter survival times . In addiTA3 mRNA . Previoufication . It was fication .EGFR mRNA via posttranscriptional modification of m6A in Previous survey unraveled HBXIP and METTL3 maintained high level in HCC patients. HBXIP could stimulate the occurrence of HCC cell malignant behaviors through the upregulation of METTL3 , catalyzMYC mRNA to advance the effect of degradation (MYC mRNA. When H19 lncRNA is poorly methylated, binding to G3BP1 will be further attenuated. Interestingly, MYC was found to accelerate H19 lncRNA transcription (A recent survey demonstrated that m5C modification has effects on distribution in HCC tissues and normal tissues. Compared with adjacent non-tumor tissue, high expression of m5C was shown in HCC tissue, which indicated that m5C methylation is closely associated with HCC pathogenesis . NSUN2, radation . H19 lnccription . Therefocription . Moreovecription .As a burgeoning discussion hotspot, research on m1A modification links to multiple cancers is also gradually becoming the basis of extensive concern. The understanding of m1A function related to HCC still requires further exploration. Shi et al. illustrated that TP53 mutations were primarily correlated with regulators mediating m1A methylation . TP53 isRNA methylation has emerged as the post-transcriptional modification to significantly affect a variety of genes expression processes, which not only has a broad influence on RNA metabolism but alters the function of various RNAs. Numerous proteins regulate methylation, demethylation, and specifically bind to diverse RNAs to promote or inhibit the biological functions, and are referred to, respectively, as “writers,” “erasers,” and “readers”. Prior research found aberrant expression of these regulators might lead to increasing disease. We summarize the distribution and functional consequences of m6A, m5C, and m1A modifications to further understand the role of RNA methylation and corresponding physiological mechanisms in HCC. For instance, overexpression of NSUN2 could promote malignant behaviors of HCC. METTL14, the “writer” of m6A, was proved to prevent metastasis of HCC. In this review, we found that RNA methylation may potentially serve as a novel marker and make valuable contributions to diagnosis and treatment in HCC, providing a promising future for a great many patients. Simultaneously, many studies are necessary to further explore and testify for clinical application.


To produce the proteins needed for the cell to survive, the information in the DNA is converted to a mobile form known as messenger RNA, which exits the cell nucleus and binds to machines that convert RNAs into proteins in a process referred to as translation. Importantly, the RNA message can be altered at any point in its journey prior to translation. These alterations constitute changes to the chemical nature of the messenger RNA and modulate the ability of mRNAs to be converted into proteins and even lead to the production of proteins with different functionalities than those encoded by their original forms. Here we provide a conceptual framework for the integration of these mRNA maturation steps with translation with a focus on the proteins that escort these mRNAs through these steps and into the translation machines. We discuss the relevance to cancer and therapeutic strategies to target these in malignancy.7G cap on the 5′ end of RNAs, splicing, as well as cleavage and polyadenylation (CPA). Importantly, each of these steps can be coopted to modify the transcript signal. Factors that bind the m7G cap escort these RNAs through different steps of maturation and thus govern the physical nature of the final transcript product presented to the translation machinery. Here, we describe these steps and how the major m7G cap-binding factors in mammalian cells, the cap binding complex (CBC) and the eukaryotic translation initiation factor eIF4E, are positioned to chaperone transcripts through RNA maturation, nuclear export, and translation in a transcript-specific manner. To conceptualize a framework for the flow and integration of this genetic information, we discuss RNA maturation models and how these integrate with translation. Finally, we discuss how these processes can be coopted by cancer cells and means to target these in malignancy.The translation of RNA into protein is a dynamic process which is heavily regulated during normal cell physiology and can be dysregulated in human malignancies. Its dysregulation can impact selected groups of RNAs, modifying protein levels independently of transcription. Integral to their suitability for translation, RNAs undergo a series of maturation steps including the addition of the m The translation of RNAs into proteins is a fundamental step in the conversion of DNA signals (in the form of coding transcripts) into their active form (proteins). Most focus has been on the impact of modulating the translation of a given RNA on the ultimate levels of the corresponding protein produced. While this is clearly important, it is equally critical to consider the processes that chemically modify the transcripts which ultimately impact the composition of the final protein product, as well as the processes that regulate the availability of these transcripts to the ribosome. In this regard, many of the major RNA metabolism steps can physically alter the composition of transcripts, which can lead to altered abilities of RNAs to access the ribosome and/or the production of different functional forms of the resulting proteins. In this way, RNA metabolism provides a means to reprogram the transcriptional signals which are the ultimate substrates of the ribosomes. For instance, splice variants and alternatively polyadenylated transcript variants can produce proteins with opposing or modified functions ,2. Eleva7G “cap” modification on the 5′end of coding RNAs [A fundamental feature central to the control of RNA maturation, export, and translation is the ming RNAs . Factorsing RNAs ,7,8,9,10ing RNAs ,12,13. Ting RNAs , can alsing RNAs ,19,20,21ing RNAs ,23,24,25ing RNAs   a description of RNA maturation, RNA export, and translation focusing on the role of cap-chaperones; (2) models for RNA maturation based on these observations and the interplay with translation; and (3) relevance to cancer.7G capping, splicing, and CPA [One of the most highly held tenets in gene expression is that during transcription, coding as well as many non-coding RNAs, undergo a series of processing steps that physically modify the chemical structure of the RNA in order to generate mature transcripts that are substrates of the ribosome . There a and CPA . This is and CPA ,28. In s7G capping [7G cap is multifold. It enables the engagement of specific cap-binding factors such as CBC and eIF4E and, furthermore, it protects RNAs from degradation by 5′exonucleases [The first maturation step for most transcripts is referred to as m capping ,29,30. Tucleases ,32. Cappucleases ,34,35, tucleases ,39,40,41ucleases . The resucleases ,43,44. Rucleases ,45,46. Iucleases ,48,49. Fucleases . Thus, aucleases ,52,53  so that eIF4E tips the balance to favor specific transcripts. These studies also revealed that in human cells the capping of specific RNAs at steady-state was lower than anticipated, positioning this as an important regulatory step in terms of the functionality of RNAs. eIF4E does not simply provide protection against decapping by directly binding to m7G caps of its target RNAs [LacZ reporter upon eIF4E overexpression [Recently, eIF4E was shown to play a role in steady-state capping . eIF4E eget RNAs . Indeed,get RNAs . An RNA pression  and coulSplicing is the removal of introns and the joining of flanking exons in pre-messenger RNAs (pre-mRNAs) and some non-coding RNAs . This prRecently, the location of splicing has been shown to be more diverse than first thought. Approximately 80% of splicing occurs co-transcriptionally and ~20% post-transcriptionally in nuclear speckles ,61. Feat7G cap with the CBC. CBC is most often associated with removal of the first intron in a co-transcriptional manner [The possibility that splicing occurs in different locations, co-transcriptionally and post-transcriptionally, leads to the question of whether different cap-chaperones would be used in different contexts. It is generally considered that RNAs are recruited to the spliceosome through interactions of their ml manner , where il manner . For exal manner . Additiol manner ,66.cis-elements influence the CBC-dependent control of AS. Finally, CBC may have an indirect influence on AS through the regulation of snRNAs synthesis and export [In addition to facilitating the constitutive splicing of first introns, CBC regulates AS through its co-factor, the positive transcription elongation factor P-TEFb, supporting a transcription-dependent regulation of AS by CBC . Indeed,d export ,71,72. Id export . This ocd export . These fLargely considered to be a co-transcriptional event, CPA is a two-step process required for maturation of the majority of coding RNAs and also for some non-coding transcripts . This maCTN RNA (Cat2 transcribed nuclear RNA) showcased the notion of nuclear post-transcriptional APA [CTN RNA undergoes a first round of CPA, likely co-transcriptionally, but then the polyadenylated mRNA remains stored in the nucleus until specific signaling events trigger the cleavage of another PAS within the CTN RNA to produce the CAT2 RNA, which is followed by poly(A) tail addition and is then exported to the cytoplasm and translated into protein [Interestingly, even though CPA is mainly a co-transcriptional nuclear process, transcripts can undergo additional CPA either post-transcriptionally in the nucleus or in the cytoplasm ,78,79. Aonal APA . Here, t protein . Cytopla protein .Cap-chaperones are involved in PAS selection and can influence cleavage efficiency. CBC, for instance, specifically regulates the 3′end processing of structured transcripts from intronless genes such as replication-dependent histones (RDHs) and pre-snRNAs. CBC promotes the selection of proximal PAS through two mutually exclusive co-factors: the negative elongation factor (NELF)  and the APA contributes to disorders ranging from cancer to neurodegeneration ,76,77. I7G cap of mRNAs and also with the 2,2,7-trimethylguanosine cap on snRNAs during export, albeit in different complexes [7G cap, albeit with less affinity than NCBP2 [To reach the cytoplasm, mRNAs do not diffuse freely through the nuclear pore complex (NPC). Instead, mRNAs transit to the cytoplasm through the NPC escorted by NPC receptors and an array of export co-factors including cap-chaperones. Once in the cytoplasm, the mRNA cargoes are released from these factors so that they can be ultimately translated by the ribosome. NPC receptors and export factors are then reimported into the nucleus to act in future rounds of export. The addition of the cap enhanced the export of both snRNAs and mRNAs, highlighting the importance of cap-chaperones to this process . Consistomplexes ,88. CBC-omplexes ,89 whichomplexes ,91,92. Nomplexes , and it omplexes ,95. In Complexes , NCBP1 iomplexes . Howeveromplexes , openingan NCBP2 , was alsan NCBP2 . Additioan NCBP2 . Howeveran NCBP2 ,99,100. 7G cap on the target RNA) and with CRM1/XPO1 [An alternative nuclear export path for selected mRNAs employs CRM1/XPO1 to transit through the NPC rather than NXF1/NXT1 ,28. ThisRM1/XPO1 ,101. TheRM1/XPO1 ,102 whicRM1/XPO1 . AdditioRM1/XPO1 . Indeed,RM1/XPO1 . MoreoveRM1/XPO1 . IntriguRM1/XPO1 , which aRM1/XPO1 . In thisRM1/XPO1 , and thu7G cap of the RNA [7G-capped mRNA [GAPDH or ACTN [Translation can be regulated at many steps and has been recently reviewed ,104,105; the RNA . The eIFped mRNA ,108. Aft or ACTN . In addi or ACTN , two exp or ACTN ,111.Interestingly, CBC is also implicated in translation via its cap-binding activity . The besCBC translation complexes are distinct form those employed by eIF4E. For example, CBC–RNA complexes recruit the CBC-dependent translation initiation factor (CTIF), which acts as an assembly platform protein similar to eIF4G. CBC–CTIF associates with eIF3 through eIF3g to recruit the small ribosomal subunit and to access the start codon . In thiseIF3d plays a well-established role in ribosome recruitment during translation initiation . HoweverAs described above, the combinatorial outcomes of RNA maturation define the functional proteome. Here, we describe classic notions for how these events are coordinated and discuss the possibility of parallel RNA maturation networks governed by cap-chaperones.In this model, the CBC binds RNAs during the early stages of transcription, shortly after they have been capped ,129 Fig. The CBCThis model is built upon the classical linear model but incorporates recent observations that capping, splicing, and CPA can sometimes occur post-transcriptionally as well as co-transcriptionally . Indeed,There are several observations that support the possibility that chaperoning capped RNAs through maturation steps is not limited to CBC but includes eIF4E, and by inference, other cap-binding proteins. These observations include (1) there are substantial amounts of both eIF4E and CBC found in the nucleus where most maturation occurs ,24,131; CCND1 and MYC transcripts are found to immunoprecipitate with both NCBP1 and eIF4E in the nucleus [GAPDH is restricted to associations with NCBP1 with no binding of this RNA with eIF4E detected in the nucleus [CCND1 and MYC transcripts to exit via eIF4E-dependent export rather than bulk export mediated by CBC, while GAPDH is not an eIF4E-dependent RNA export target [It is likely that RNAs are not restricted to one cap-chaperone path. In this case, we propose that RNAs can undergo cap-chaperone switching during their maturation and translation in a process we refer to as interleaving . In this nucleus . By cont nucleus . Furthert target . These ot target ,134,135.Recent studies provide one possible example for how cap-chaperone switching might occur . In this7G cap is a USER code for both CBC and eIF4E, other elements within the RNA are needed to confer further selectivity. In this model, USER codes in RNAs will engage certain steps such as splicing which can in turn modulate the USER code content to favor specific outcomes in terms of the functional protein  in the nuclear matrix fraction; however, in the nucleoplasmic fraction, LacZ-4ESE only bound to eIF4E (and not CBC) while LacZ only bound to CBC (and not eIF4E) [LacZ-4ESE but not LacZ RNAs. Consistently, LacZ-4ESE was exclusively bound to eIF4E-dependent RNA export factors and was exported solely by that pathway [LacZ did not bind to eIF4E or its associated export co-factors in the nucleus and was exported via the CBC-dependent bulk pathway [There is evidence for cap-chaperone switching and the influence of USER codes in this process ,17. For t eIF4E) . Thus, c pathway . By cont pathway . In the  pathway . In all, pathway . In this pathway . While tOther examples of cap-chaperone switching have been observed as for the Staufen1 example above. Indeed, RNAs passed from the pioneer round of translation by CBC to steady-state translation by eIF4E in a step referred to as the “hand off” is a well characterized example of cap-chaperone switching ,117. Als7G caps. However, a small percentage of coding RNAs have FAD, NAD, UDP-glucuronic acid, UDP-glucosamine, m3G, or even potential protein caps such as the potyviral VPg protein [It is also important to consider that the status of the cap itself will be key for assigning which cap-chaperone(s) is favored. In this review, we focus on m protein ,140. Whi protein , the capGle1, DDX19) through increased RNA export of their corresponding transcripts as well as by reducing levels of the NPC component RanBP2 indirectly [RNMT, RAM, and RNGTT), splicing, and CPA  machineries [Notably, eIF4E has an additional impact on RNA maturation that has not been reported for CBC. eIF4E impacts the production of components of the capping, splicing, CPA, and export machinery ,16,19,21hineries ,19,21. TThere is substantial evidence that RNA maturation, export, and translation can be dysregulated in many human malignancies. In cell lines, eIF4E overexpression promotes foci formation, growth in soft agar, invasion, migration, tumor formation, and apoptotic rescue from a variety of stimuli ,143,144.ClinicalTrials.gov (accessed on 25 November 2021)). Clearly, inhibition of eIF4E activity is likely to be potent because it impacts nuclear and cytoplasmic activities and also the capacity of eIF4E to produce relevant RNA maturation and export machinery.eIF4E levels are increased in many cancers, where its elevation generally correlates with poor prognosis ,157,158.ClinicalTrials.gov NCT02040558 (accessed on 25 November 2021)), the results of which are not yet known. eIF4E phosphorylation by MNK impacts its nuclear RNA export function [eIF4E has also been targeted with antisense oligonucleotides (ASOs) in animal models and patients. Unfortunately, in patients, there were no responses better than short-term stable disease despite the promising results of ASOs in prostate mouse models . This fafunction ,176, butfunction ,178,179,function ,181,182.https://www.proteinatlas.org/ENSG00000136937-NCBP1/pathology (access on 25 November 2021)) as a favorable prognostic marker for prostate cancer and unfavorable for pancreatic cancer, while NCBP2 (https://www.proteinatlas.org/ENSG00000114503-NCBP2/pathology (access on 25 November 2021)) has been identified as an unfavorable prognostic marker for liver, pancreatic, and prostate cancer. Further investigations are needed to more deeply investigate the involvement of the different CBC components in oncogenesis and whether they constitute biomarkers and/or targets.By contrast, CBC is relatively understudied in terms of its role in malignancies. The depletion of NCBP2 or NCBP1 is not lethal in mammalian cells . However7G cap, is known for favoring eIF4E-dependent nuclear RNA export and CPA; however, further decoding is necessary to properly dissect the USER codes that will underpin cap-chaperone choice and the subsequent decision tree of information flow. This information flow can have substantive impacts on cell physiology, even leading to structural and functional changes to the cell by altering the functionality of the proteome.Above, we discussed the information flow that generates the transcripts that will ultimately underpin the functional proteome. There remain several open questions to further probe and build on this model. For instance, does eIF4E or other cap-binding proteins aside from CBC interact with RNAs co-transcriptionally and act in maturation at that site? Does eIF4E act in RNA maturation in the cytoplasm beyond translation? Does CBC act in post-transcriptional CPA or splicing in the nucleus or cytoplasm? What are the USER code complements that provide enrichment for specific cap-chaperones? To date, the 4ESE, in combination with the mOur model of non-linear, parallel, interleaved RNA maturation provides a molecular basis to subvert the flow of genetic information and thus the functional proteome . In prin