Editing Antisense RNA

{{Short description|Single stranded RNA}}
[[File:Figure1_Definition_of_antisense_RNAs_(asRNAs)_.png|alt=This figure demonstrates an antisense RNA is complementary to its sense transcript.|thumb|AsRNA is transcribed from the lagging strand of a gene and is complementary to a specific mRNA or sense transcript.&nbsp;]]
'''Antisense RNA''' ('''asRNA'''), also referred to as antisense transcript,<ref name="Pelechano_2013">{{cite journal | vauthors = Pelechano V, Steinmetz LM | title = Gene regulation by antisense transcription | journal = Nature Reviews. Genetics | volume = 14 | issue = 12 | pages = 880–893 | date = December 2013 | pmid = 24217315 | doi = 10.1038/nrg3594 | s2cid = 2152962 }}</ref> [[natural antisense transcript]] (NAT)<ref name="Saberi_2016">{{cite journal | vauthors = Saberi F, Kamali M, Najafi A, Yazdanparast A, Moghaddam MM | title = Natural antisense RNAs as mRNA regulatory elements in bacteria: a review on function and applications | journal = Cellular & Molecular Biology Letters | volume = 21 | pages = 6 | date = 2016-07-28 | pmid = 28536609 | pmc = 5415839 | doi = 10.1186/s11658-016-0007-z | doi-access = free }}</ref><ref name="Magistri_2012">{{cite journal | vauthors = Magistri M, Faghihi MA, St Laurent G, Wahlestedt C | title = Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts | journal = Trends in Genetics | volume = 28 | issue = 8 | pages = 389–396 | date = August 2012 | pmid = 22541732 | pmc = 3768148 | doi = 10.1016/j.tig.2012.03.013 }}</ref><ref name="Wahlestedt_2013">{{cite journal | vauthors = Wahlestedt C | title = Targeting long non-coding RNA to therapeutically upregulate gene expression | journal = Nature Reviews. Drug Discovery | volume = 12 | issue = 6 | pages = 433–446 | date = June 2013 | pmid = 23722346 | doi = 10.1038/nrd4018 | s2cid = 288163 }}</ref> or antisense [[oligonucleotide]],<ref name="Kole_2012">{{cite journal | vauthors = Kole R, Krainer AR, Altman S | title = RNA therapeutics: beyond RNA interference and antisense oligonucleotides | journal = Nature Reviews. Drug Discovery | volume = 11 | issue = 2 | pages = 125–140 | date = January 2012 | pmid = 22262036 | pmc = 4743652 | doi = 10.1038/nrd3625 }}</ref> is a single stranded [[RNA]] that is complementary to a protein coding [[messenger RNA]] (mRNA) with which it hybridizes, and thereby blocks its [[Translation (biology)|translation]] into [[protein]]. The asRNAs (which occur naturally) have been found in both [[prokaryote]]s and [[eukaryote]]s,<ref name="Pelechano_2013" /> and can be classified into short (<200 nucleotides) and long (>200 nucleotides) [[non-coding RNA]]s (ncRNAs).<ref name="Wahlestedt_2013" /> The primary function of asRNA is regulating [[gene expression]]. asRNAs may also be produced synthetically and have found wide spread use as research tools for [[gene knockdown]]. They may also have therapeutic applications.<ref name="pmid10228554">{{cite journal | vauthors = Weiss B, Davidkova G, Zhou LW | title = Antisense RNA gene therapy for studying and modulating biological processes | journal = Cellular and Molecular Life Sciences | volume = 55 | issue = 3 | pages = 334–358 | date = March 1999 | pmid = 10228554 | doi = 10.1007/s000180050296 | s2cid = 9448271 | pmc = 11146801 }}</ref><ref name="Pelechano_2013" /><ref name="Wahlestedt_2013" />

== Discovery and history in drug development ==
{{main|Antisense therapy}}
Some of the earliest asRNAs were discovered while investigating functional proteins. An example was [[MicF RNA|micF asRNA]]. While characterizing the outer membrane [[Porin (protein)|porin]] {{not a typo|ompC}} in [[Escherichia coli|''E.coli'']], some of the {{not a typo|ompC}} promoter clones observed were capable of repressing the expression of other membrane porin such as {{not a typo|ompF}}. The region responsible for this repression function was found to be a 300 base-pair locus upstream of the {{not a typo|ompC}} promoter. This 300 base-pair region is 70% homologous in sequence with the [[5' end]] of the {{not a typo|ompF}} mRNA and thus the transcript of this 300 base pair locus was complementary to the {{not a typo|ompF}} mRNA. Later on, this transcript, denoted micF, was found to be an asRNA of {{not a typo|ompF}} and capable of downregulating the expression of {{not a typo|ompF}} under stress by forming a duplex with the {{not a typo|ompF}} mRNA. This induces the degradation of the {{not a typo|ompF}} mRNA.<ref name="Saberi_2016" />

Unlike micF RNA being discovered by accident, the majority of asRNAs were discovered by genome wide searches for small regulatory RNAs and by [[transcriptome]] analysis. Conventionally, the first step involves computational predictions based on some known characteristics of asRNAs. During computational searches, the encoding regions are excluded. The regions that are predicted to have conserved RNA structures and act as orphan promoters and [[Rho independent termination|Rho independent terminators]] are preferenced during analysis. Because computational searches focuses on the [[intergenic region]], the asRNAs that are transcribed from the opposite strand of an encoding gene are likely to be missed using this method. To detect asRNA transcribed from the encoding region, [[oligonucleotide microarray]]s can be used. In this method, one or both strands of encoding genes can be used as probes. In addition to computational searches and microarrays, some asRNAs were discovered by sequencing cDNA clones as well as mapping promoter elements.<ref>{{cite journal | vauthors = Thomason MK, Storz G | title = Bacterial antisense RNAs: how many are there, and what are they doing? | journal = Annual Review of Genetics | volume = 44 | issue = 1 | pages = 167–188 | date = 2010 | pmid = 20707673 | pmc = 3030471 | doi = 10.1146/annurev-genet-102209-163523 }}</ref> Although many findings from the approaches mentioned above gave rise to a lot of possible asRNAs, only few were proven to be actual asRNAs via further functional tests. To minimize the number of false positive results, new approaches from recent years have been focusing on strand-specific transcription, [[chromatin]] binding noncoding RNAs and single cell studies.<ref name="Pelechano_2013" />

The idea of asRNAs as drug targets started in 1978 when [[Paul Zamecnik|Zamecnik]] and Stephenson found an antisense oligonucleotide to the viral RNA of Rous scarcoma virus that was capable of inhibiting viral replication and protein synthesis. Since then, much effort has been devoted to developing asRNAs as drug candidates. In 1998,  the first asRNA drug, [[fomivirsen]], was approved by FDA. Fomivirsen, a 21 base-pair oligonucleotide,  was developed to treat [[cytomegalovirus retinitis]] in patients with AIDS. It works by targeting the transcribed mRNA of the virus and consequently inhibiting replication of cytomegalovirus. Despite fomivirsen being discontinued in 2004 due to the loss of the market, it served as a successful and inspiring example of using asRNAs as drug targets or drug candidates.<ref name="Kole_2012" />

Another example of using an asRNA as a therapeutic agent is [[mipomersen]], which was approved by FDA in 2013. Mipomersen was developed to manage the level of [[low-density lipoprotein cholesterol]] (LDL) in patients with homozygous [[familial hypercholesterolemia]] (HoFH), which is a rare autosomal dominant genetic condition. Because of the high level of total cholesterol (650–1000&nbsp;mg/dL) and LDL receptor (above 600&nbsp;mg/dL) in HoFH, patients with HoFH has a high risk for coronary heart disease. Because the protein [[Apolipoprotein B|apo-B-100]] has been found to be required to produce [[very low-density lipoprotein]] (VLDL) and LDL, mipomersen complements with the mRNA of apo-B-100 and target it for [[RNAse H]] dependent degradation. Ultimately, mipomersen is able to reduce the level of LDL.<ref>{{cite journal | vauthors = Wong E, Goldberg T | title = Mipomersen (kynamro): a novel antisense oligonucleotide inhibitor for the management of homozygous familial hypercholesterolemia | journal = P & T | volume = 39 | issue = 2 | pages = 119–122 | date = February 2014 | pmid = 24669178 | pmc = 3956393 }}</ref>

== Examples across species ==
The initial asRNAs discovered were in prokaryotes including [[plasmid]]s, [[bacteriophage]] and [[bacteria]]. For example, in plasmid ColE1, the asRNA termed RNA I plays an important role in determining the plasmid copy number by controlling replication. The replication of ColE1 relies on the transcription of a primer RNA named RNA II. Once RNA II is transcribed, it hybridizes to its DNA template and later cleaved by RNase H. In the presence of the asRNA RNA I, RNA I and RNA II forms a duplex which introduces a conformational change of RNA II. Consequently, RNA II cannot  hybridize with its DNA template which results in a low copy number of ColE1. In bacteriophage P22, the asRNA sar helps regulate between lytic and lysogenic cycle by control the expression of Ant.<ref name="pmid2468573">{{cite journal | vauthors = Simons RW | title = Naturally occurring antisense RNA control—a brief review | journal = Gene | volume = 72 | issue = 1–2 | pages = 35–44 | date = December 1988 | pmid = 2468573 | doi = 10.1016/0378-1119(88)90125-4 }}</ref> Besides being expressed in prokaryotes, asRNAs were also discovered in plants. The most well described example of asRNA regulation in plants is on  [[Flowering Locus C|Flowering Locus C (FLC)]] gene. FLC gene in [[Arabidopsis thaliana]]  encodes for a transcription factor that prevent expression of a range of genes that induce floral transition. In cold environment, the asRNA of FLC gene, denoted COOLAIR, is expressed and inhibits the expression of FLC via chromatin modification which consequently allows for flowering.<ref>{{cite journal | vauthors = Ietswaart R, Wu Z, Dean C | title = Flowering time control: another window to the connection between antisense RNA and chromatin | journal = Trends in Genetics | volume = 28 | issue = 9 | pages = 445–453 | date = September 2012 | pmid = 22785023 | doi = 10.1016/j.tig.2012.06.002 }}</ref> Another well studied example is DOG1 (Delay of Germination 1) gene. Its expression level is negatively regulated by the antisense transcript (asDOG1 or 1GOD) acting in cis. <ref>{{cite journal |last1=Fedak |first1=Halina |last2=Palusinska |first2=Malgorzata |last3=Krzyczmonik |first3=Katarzyna |last4=Brzezniak |first4=Lien |last5=Yatusevich |first5=Ruslan |last6=Pietras |first6=Zbigniew |last7=Kaczanowski |first7=Szymon |last8=Swiezewski |first8=Szymon |title=Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript |journal=Proc Natl Acad Sci USA |date=2016 |volume=113 |issue=48 |pages=E7846–E7855 |doi=10.1073/pnas.1608827113 |pmid=27856735 |pmc=5137729 |doi-access=free }}</ref> In mammalian cells, a typical example of asRNA regulation is X chromosome inactivation. [[XIST|Xist]], an asRNA, can recruit [[PRC2|polycomb repressive complex 2]] (PRC2) which results in heterochromatinization of the X chromosome.<ref name="Magistri_2012" />

== Classification ==
Antisense RNAs can be classified in different ways. In terms of regulatory mechanisms, some authors group asRNAs into RNA-DNA interactions, RNA-RNA interactions either in [[cell nucleus|nucleus]] or [[cytoplasm]] and RNA-protein interactions ([[epigenetic]]).<ref name="Magistri_2012" /> Antisense RNAs can be categorized by the type of the promoters that initiate expression of asRNAs: independent promoters, shared bidirectional promoters or cryptic promoters. In terms of length, although asRNA in general is classified as [[Long non-coding RNA|lncRNAs]], there are short asRNAs with length of less than 200 base pairs. Because the regulatory mechanism of asRNAs are found to be species specific, asRNAs can also be classified by species.<ref name="Pelechano_2013" /> One of the common ways of classifying asRNAs is by where the asRNAs are transcribe relatively to their target genes: [[cis-acting]] and [[trans-acting]].{{cn|date=February 2023}}

=== Cis-acting ===
{{See also|Cis-natural antisense transcript}}
[[Cis-acting]] asRNAs are transcribed from the opposite strand of the target gene at the target gene locus. They often show high degree or complete complementarity with the target gene. If the cis-acting asRNA regulates gene expression by targeting mRNA, it can only target individual mRNA. Upon interactions with the targeting mRNAs, cis-acting asRNAs can either block ribosome binding or recruit [[Ribonuclease|RNAase]] to degrade the targeting mRNAs. Consequently, the function of these cis-acting asRNAs is to repress translation of the targeting mRNAs.<ref name="Saberi_2016" /> Besides cis-acting asRNAs that target mRNAs, there are cis-acting epigenetic [[Silencer (genetics)|silencers]] and [[Activator (genetics)|activators]]. Antisense RNA has been shown to repress the translation of LINE1-ORF2 domain of [[Entamoeba histolytica]]. However it is not confirmed yet whether its cis-acting or trans.<ref>{{cite journal|doi=10.1016/j.plasmid.2021.102560|pmid=33482228|title=Transcriptomic analysis of Entamoeba histolytica reveals domain-specific sense strand expression of LINE-encoded ORFs with massive antisense expression of RT domain|year=2021|last1=Kaur|first1=Devinder|last2=Agrahari|first2=Mridula|last3=Singh|first3=Shashi Shekhar|last4=Mandal|first4=Prabhat Kumar|last5=Bhattacharya|first5=Alok|last6=Bhattacharya|first6=Sudha|journal=Plasmid|volume=114|page=102560}}</ref>

In terms of epigenetic modification, cis-acting refers to the nature of these asRNAs that regulate epigenetic changes around the [[Locus (genetics)|loci]]  where they are transcribed. Instead of targeting individual mRNAs, these cis-acting epigenetic regulators can recruit chromatin modifying enzymes which can exert effects on both the transcription loci and the neighboring genes.<ref name="Magistri_2012" />

=== Trans-acting ===
[[Trans-acting]] asRNAs are transcribed from loci that are distal from the targeting genes. In contrast to cis-acting asRNAs, they display low degree of complementarity with the target gene but can be longer than cis-acting asRNAs. They can also target multiple loci. Because of these properties of trans-acting asRNAs, they form less stable complexes with their targeting transcripts and sometimes require aids from RNA [[chaperone protein]] such as Hfq to exert their functions. Due to the complexity of the trans-acting asRNAs, they are currently considered to be less druggable targets.<ref name="Saberi_2016" />

=== Function ===
[[File:Figure_2_Mechanism_of_gene_repression_by_asRNAs._.png|thumb|'[[Epigenetic regulation]]:' a) AsRNAs can induce DNA methylation by recruiting a DNA methyltransferase (DNMT). b) AsRNAs can induce histone methylation by recruiting of a histone methyltransferase (HMT). 'Co-transcriptional regulation:' c) AsRNAs can cause RNA polymerase (Pol) collision and stop the transcription. d) AsRNAs can preference translation of a specific splice variant (mRNA V1) by blocking the other splice variant (mRNA V2). '[[Post-transcriptional regulation]]:' e) AsRNA-mRNA duplex can either block ribosome from binding to the mRNA or recruit RNase H to degrade the mRNA. By this mechanism, asRNAs directly inhibit translation of the mRNAs.]]

==== Epigenetic regulation ====
Many examples of asRNAs show the inhibitory effect on transcription initiation via epigenetic modifications.

===== DNA methylation =====
[[DNA methylation]] can result in long term downregulation of specific genes. Repression of functional proteins via asRNA induced DNA methylation has been found in several human disease. In a class of [[alpha-thalassemia]], a type of blood disorder that has reduced level of hemoglobin leading to insufficient oxygen in the tissues,<ref>{{cite web | url = https://ghr.nlm.nih.gov/condition/alpha-thalassemia | title = alpha thalassemia |date =14 November 2017 | website = Genetics Home Reference | publisher = NIH U.S. National Library of Medicine}}</ref> [[Hemoglobin, alpha 1|hemoglobin alpha1]] gene (HBA1) is downregulated by an abnormal transcript of putative RNA-binding protein Luc7-like (LUC71) that serves as an asRNA to HBA1 and induces methylation of HBA1's promoter.<ref name="Pelechano_2013" /> Another example is silencing of a tumor suppressor gene p15INK4b, also called CDKN2B, in acute lymphoblastic leukemia and acute myeloid leukemia. The asRNA that is responsible for this silencing effect is antisense non-coding RNA in the INK locus ([[CDKN2BAS|ANRIL]]), which is expressed in the same locus that encodes for p15INK4b.<ref name="Magistri_2012" />

===== Histone modification =====
{{main|Histone modification}}
In eukaryotic cells, DNA is tightly packed by [[histone]]s. Modification on histones can change interactions with DNA which can further induce changes in [[gene expression]]. The biological consequences of [[histone methylation]] are context dependent. In general, histone methylation leads to gene repression but gene activation can also be achieved.<ref>{{cite book | title = Handbook of Cell Signaling | edition = Second | chapter = Histone Methylation | last = Whetstine | first = Johnathan R. | name-list-style = vanc | pages = 2389–2397 | doi = 10.1016/b978-0-12-374145-5.00287-4 | isbn = 978-0-12-374148-6 | year = 2010 }}</ref> Evidence has shown histone methylation can be induced by asRNAs. For instance, ANRIL, in addition to the ability to induce DNA methylation, can also repress the neighboring gene of [[CDKN2B]], [[CDKN2A]], by recruiting [[PRC2|polycomb repressive complex 2]] (PRC2) which leads to histone methylation (H3K27me). Another classic example is X chromosome inactivation by [[XIST]].<ref name="Pelechano_2013" />

ANRIL induced epigenetic modification is an example of cis acting epigenetic regulation.<ref name="Magistri_2012" /> In addition,  Antisense RNA-induced chromatin modification can be both trans-acting. For example, in mammals, the asRNA [[HOTAIR]] is transcribed from [[homeobox]] C (HOXC) locus but it recruits [[PRC2]] to HOXD which deposits H3K27 and silences HOXD. HOTAIR is highly expressed in primary breast tumors.<ref name="Pelechano_2013" />

==== Co-transcriptional regulation ====
Epigenetic regulations such as DNA methylation and histone methylation can repress gene expression by inhibiting initiation of transcription. Sometimes, however, gene repression can be achieved by prematurely terminating or slowing down transcription process. AsRNAs can be involved in this level of gene regulation. For example, in bacterial or eukaryotic cells where complex RNA polymerases are present, bidirectional transcription at the same locus can lead to polymerase collision and results in the termination of transcription. Even when polymerase collision is unlikely during weak transcription, polymerase pausing can also occur which blocks elongation and leads to gene repression. One of the examples is repression of [[IME4]] gene by its asRNA [[RME2]]. Another way of affecting transcription co-transcriptionally is by blocking splicing. One classic example in human is [[zinc-finger]] E-box binding homeobox 2 gene ([[ZEB2]]) which encodes E-cadherin, a transcriptional repressor. Efficient translation of ZEB2 mRNA requires the presence of an [[internal ribosome entry site]] (IRES) in intron of the mRNA at the [[5' end]]. With the asRNA of ZEB2 being expressed, it can mask the splicing site and maintain the IRES in the mRNA which results in an efficient synthesis of E-cadherin. Lastly, depending on the level of asRNA expression, different isoforms of the sense transcript can be produced. Therefore, asRNA dependent regulation is not limited to on/off mechanism; rather, it presents a fine tone control system.<ref name="Pelechano_2013" />

==== Post-transcriptional regulation ====
The direct post transcriptional modulation by asRNAs refers to mRNAs being targeted by asRNAs directly; thus, the translation is affected. Some characteristics of this type of asRNAs are described in the cis- and trans- acting asRNAs. This mechanism is relatively fast because both the targeting mRNA and its asRNA need to be present simultaneously in the same cell. As described in the cis-acting asRNAs, the mRNA-asRNA pairing can result in blockage of ribosome entry and RNase H dependent degradation. Overall, mRNA-targeting asRNAs  can either activate or inhibit translation of the sense mRNAs with inhibitory effect being the most abundant.<ref name="Pelechano_2013" />

=== Therapeutic potential ===
As a regulatory element, asRNAs bear many advantages to be considered as a drug target. First of all, asRNAs regulate gene expression at multiple levels including transcription, post-transcription and epigenetic modification. Secondly, the cis-acting asRNAs are sequence specific and exhibits high degree of complementarity with the targeting genes.<ref name="Pelechano_2013" /> Thirdly, the expression level of asRNAs is very small compared to that of the targeting mRNAs; therefore, only small amount of asRNAs is required to produce an effect. In terms of drug targets, this represents a huge advantage because only a low dosage is required for effectiveness.<ref name="Wahlestedt_2013" />

Recent years the idea of targeting asRNAs to increase gene expression in a locus specific manner has been drawing much attention. Due to the nature of [[drug development]], it is always easier to have drugs functioning as downregulators or inhibitors. However, there is a need in developing drugs that can activate or upregulate gene expression such as tumor suppressor genes, neuroprotective growth factors and genes that are found silenced in certain Mendelian disorders. Currently, the approach to restore deficient gene expression or protein function include enzyme replacement therapies, [[microRNA]] therapies and delivery of functional cDNA. However, each bears some drawbacks. For example, the synthesized protein used in the enzyme replacement therapies often cannot mimic the whole function of the endogenous protein. In addition, enzyme replacement therapies are life-long commitment and carry a large financial burden for the patient.  Because of the locus specific nature of asRNAs and evidences of changes in asRNA expression in many diseases, there have been attempts to design single stranded oligonucleotides, referred as antagoNATs, to inhibit asRNAs and ultimately to increase specific gene expression.<ref name="Wahlestedt_2013" />

Despite the promises of asRNAs as drug targets or drug candidates, there are some challenges remained to be addressed.<ref>Weiss, B. (ed.):  Antisense Oligodeoxynucleotides and Antisense RNA : Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, FL, 1997.</ref> First of all, asRNAs and antagoNATs can be easily degraded by RNase or other degrading enzymes. To prevent degradation of the therapeutic oliogoneucleotides, chemical modification is usually required. The most common chemical modification on the oligonucleotides is adding a [[phosphorothioate]] linkage to the backbones.<ref name="Kole_2012" /> However, the phosphrothioate modification can be proinflammatory. Adverse effects including fever, chills or nausea have been observed after local injection of phosphrothioate modified oligonucleotides. Secondly, off target toxicity also represents a big problem. Despite the locus-specific nature of the endogenous asRNAs, only 10–50% synthesized oligonucleotides showed expected targeting effect. One possible reason for this problem is the high requirement on the structure of the asRNAs to be recognized by the target sequence and RNase H. A single mismatch can result in distortion in the secondary structure and lead to off target effects.<ref name="Wahlestedt_2013" /> Lastly, artificial asRNAs have been shown to have limited intracellular uptake.<ref name="Kole_2012" /> Although neurons and glia have been shown to have the ability to freely uptake naked antisense oligonucleotides, a traceable carriers such as virus and lipid vesicles would still be ideal to control and monitor the intracellular concentration and metabolism.<ref name="Wahlestedt_2013" />

== See also ==
* [[Cis-natural antisense transcript]]
== References ==
{{Reflist}}

{{natural antisense transcripts}}
{{nucleic acids}}

[[Category:Antisense RNA| ]]
[[Category:Non-coding RNA]]