RNA Editing or Modification

The molecular process of RNA editing, also known as RNA modification, allows some cells to make small modifications to particular nucleotide sequences inside an RNA molecule that has already been produced by RNA polymerase. The process of RNA editing, which includes the insertion, deletion, and base substitution of nucleotides within the molecule, is one of the most evolutionarily conserved features of RNAs. It happens in all living organisms. Common RNA processing steps including splicing, 5′-capping, and 3′-polyadenylation are often not thought of as editing, which contributes to the rarity of RNA editing. It has been associated with human disorders and has the potential to influence the activity, location, and stability of RNAs.

Evidence of RNA editing in various RNA molecules has been found in several organisms, including eukaryotic cells and viruses, archaea, and prokaryotes. RNA editing takes place in the nucleus of cells, mitochondria, and chloroplasts. Rarely occurring in vertebrates, editing often involves adding or removing a few base pairs from the sequence of the molecules in question. Extensive editing, also known as pan-editing, can happen in some creatures like squids. In extreme instances, editing can produce the majority of nucleotides in an mRNA sequence. To far, over 160 distinct RNA alterations have been documented.

There is a wide variety of molecular mechanisms involved in RNA editing, and some of these mechanisms seem to be separate acquisitions with a short evolutionary history. Among the many RNA editing processes are changes to nucleobases, such as adenosine (A) to inosine (I) deaminations and cytidine (C) to uridine (U) changes, and insertions and deletions of non-template nucleotides. By modifying messenger RNAs, it is possible to change the anticipated amino acid sequence of the encoded protein from that of the genomic DNA sequence. This process is known as RNA editing.

The Complex of the Editosome

Finding RNA editing changes

RNA Editing

The next generation of genomics

To identify diverse post-transcriptional modifications of RNA molecules and determine the transcriptome-wide landscape of RNA modifications by means of next generation RNA sequencing, recently many studies have developed conventional or specialised sequencing methods. Examples of specialised methods are MeRIP-seq, m6A-seq, PA-m5C-seq , methylation-iCLIP,  m6A-CLIP, Pseudo-seq, Ψ-seq, CeU-seq, Aza-IP and RiboMeth-seq. The foundation of several of these approaches is the targeted isolation of the RNA species that has undergone the desired alteration; this can be achieved, for instance, by combining antibody binding with read sequencing. Once the sequencing is complete, the reads are mapped against the entire transcriptome to determine their source. This method typically reveals the locations of the modifications and can even help identify consensus sequences for future mapping and identification. The PA-m5C-seq technique is an example of a specialized approach. Originally designed to detect N6-methyladenosine, this approach was refined from PA-m6A-seq to detect m5C alterations on messenger RNA. By simply changing the capturing antibody from m6A to m5C, it is easy to switch between different modifications as targets. These methods have been applied to identify various modifications, such as pseudouridine, m6A, m5C, and 2′-O-Me, within both coding and non-coding genes, at either single nucleotide or very high resolution.

Liquid Chromatography

An rise in mass for a particular nucleoside is a common result of alterations; mass spectrometry provides a means to subjectively and (relatively) quantify RNA modifications. Mass spectrometry also enables the study of modification kinetics in vivo by labeling RNA molecules with stable (non-radioactive) heavy isotopes, which yields a distinctive readout for both the nucleoside and its modified counterpart. Heavy isotope-labeled nucleosides may be differentiated from counterparts unlabeled isotopomeres using mass spectrometry, thanks to their specified mass increase. A number of methods may be employed to study the dynamics of RNA modifications using this technique, which is known as NAIL-MS (nucleic acid isotope labelling linked mass spectrometry).

Varieties of RNA

Modification of messenger RNA.

Experimental functional analysis has recently uncovered several previously unknown functional functions of RNA modifications. The majority of RNA modifications are located on transfer-RNA and ribosomal-RNA, while it has also been demonstrated that eukaryotic messenger RNA undergoes numerous modifications. There are 17 known naturally occurring mRNA modifications; the most common and extensively researched of them is N6-methyladenosine. mRNA modifications are associated with several cellular processes. They ensure the correct maturation and function of the mRNA, but also at the same time act as part of cell’s immune system. Certain modifications like 2’O-methylated nucleotides has been associated with cells ability to distinguish own mRNA from foreign RNA. For example, m6A has been predicted to affect protein translation and localization, mRNA stability, alternative polyA choice and stem cell pluripotency. Pseudouridylation of nonsense codons suppresses translation termination both in vitro and in vivo, suggesting that RNA modification may provide a new way to expand the genetic code. 5-methylcytosine on the other hand has been associated with mRNA transport from the nucleus to the cytoplasm and enhancement of translation. It is important to note that many modification enzymes are dysregulated and genetically mutated in many disease types. For instance, mutations in pseudouridine synthases cause mitochondrial myopathy, sideroblastic anemia (MLASA) , and dyskeratosis congenital . However, these functions of m5C in the cell are not fully known or proven.

Modifications found on messenger RNA (mRNA) are insignificant as compared to those found on transfer RNA (tRNA) and ribosomal RNA (rRNA). The lack of adequate research methods is a major contributor to the general ignorance around mRNA alterations. Other RNA species lag behind us not just in terms of documented modifications but also in terms of understanding of related proteins. Enzymes interact with RNA in unique ways, which causes alterations to occur. When it comes to mRNA modifications, the majority of the associated enzymes are writer enzymes, meaning they add the modification to the mRNA. Due to the fact that the extra sets of enzymes, readers and erasers, are either poorly understood or completely unknown for most of the alterations, there has been a tremendous amount of interest in understanding these modifications and their function over the past ten years.

Genomic RNA editing

The most frequently modified RNA type is transfer RNA, or tRNA.[35] tRNA modifications are essential for efficient translation because they support structure, interact with enzymes, and prevent anticodon-codon interactions.

In order to decode messenger RNA correctly, anticodon alterations are critical. Anticodon alterations are essential for correct mRNA decoding due to the degeneracy of the genetic code. The anticodon’s wobble location is crucial in determining the codon reading. In eukaryotic organisms, for instance, the conversion of adenosine to inosine can occur at position 34 of the anticodon. Inosine may form base pairs with uridine, cytosine, and adenine.[37]

The nucleotide immediately after the anticodon is another frequent target of tRNA modifications. Hypermodification at position 37 with heavy chemical changes is common. Frameshifting is prevented and anticodon-codon binding stability is increased by stacking interactions by these modifications.

Alterations to ribosomal RNA

At each stage of ribosome synthesis, alterations are made to ribosomal RNA. In order to preserve translational efficiency, modifications mostly function in the rRNA structure.

Various transitions

Changing by adding or removing

What happens to pre-mRNA transcripts when uracils are inserted

RNA editing by uracil addition and deletion has been discovered in trypanoplasts from Trypanosoma brucei mitochondria. This process is frequently referred to as “pan-editing” to differentiate it from localized editing of a few sites, as it can include a substantial proportion of a gene’s sites.

Base-pairing the unaltered main transcript with a guide RNA (gRNA)—which has complementary sequences to the areas surrounding the insertion/deletion points—is the first step in pan-editing. The editosome, a huge complex of proteins that catalyzes editing, encases the freshly generated double-stranded area. It begins inserting uridines into the transcript at the first mismatched nucleotide. By base-pairing with the guide RNA, the inserted uridines will keep inserting until they reach a C or U. This causes a frameshift, which in turn produces a translated protein that is different from its gene.

When the guide RNA and the unedited transcript are at a mismatch, the editing machinery makes an endonucleolytic cut. The following step is carried out by a terminal U-transferase, an enzyme in the complex. It transfers Us from UTP to the 3′ end of the messenger RNA. Other proteins in the complex keep the opened ends in place. The unpaired Us is removed by an additional enzyme called a U-specific exoribonuclease. An RNA ligase links the ends of the edited mRNA transcript after editing has rendered it complementary to gRNA. So, the editosome can only edit in the 3′ to 5′ direction along the initial RNA transcript. There is a temporal limit on how many guide RNAs the complex may interact with. Thus, many guide RNAs and editosome complexes will be required for a transcript of RNA that requires significant editing.

Using deamination for editing

Editing from C to U

The ApoB gene in humans and the impact of C-to-U RNA editing

A cytidine base is converted into a uridine base by the editing process by use of cytidine deaminase. The apolipoprotein B gene in humans is an instance of C-to-U editing.It is in the intestines that apo B48 is expressed, while the liver is responsible for apo B100 expression. The shorter B48 version is produced in the intestines by editing the mRNA with a CAA sequence to UAA, a stop codon. The mitochondrial RNA of blooming plants frequently undergoes C-to-U editing. In some plants, such as the moss Funaria hygrometrica, there are only eight C-to-U editing events in the mitochondria, while in others, like the lycophytes Isoetes engelmanii, there are more than 1,700. Members of the pentatricopeptide repeat (PPR) protein family are responsible for this process. The PPR families of angiosperms are enormous, and they serve as trans-factors for cis-elements that do not have a consensus sequence. For example, the PPR family of Arabidopsis contains about 450 members. Numerous PPR proteins in plastids and mitochondria have been found.

Editing from A to I

Also referenced: adenine deamination

A-to-I alterations account for approximately 90% of all RNA editing events. The enzyme ADAR, which is specific to double-stranded RNA, normally targets pre-mRNAs and catalyzes the deamination of adenosine. When adenosine is deaminated to inosine, it disturbs and destabilizes the base pairing of double-stranded RNA. As a result, that specific double-stranded RNA is less capable of producing siRNA, which impedes the RNA interference pathway.

Deaminated RNA has a distinct structure due to the jiggling of base pairs, which could be associated with the suppression of the RNA translation start process. Research has demonstrated that methylases, which are involved in the formation of heterochromatin, are recruited by I-RNA (RNA with many repeats of the I-U base pair), and that this chemical modification significantly disrupts miRNA target sites. The significance of A-to-I modifications and their function are being investigated in the emerging field of epitranscriptomics. One well-established result of A-to-I in mRNA is the functional A-to-G substitution, which occurs, for example, when ribosomes interpret the genetic code. More recent research, however, has cast doubt on this association by demonstrating that the ribosome can interpret I’s as well as A’s and U’s, albeit to a lower degree. Is also known to cause ribosome stalling on I-rich messenger RNA, according to previous research [52].

Extensive databases for various RNA modifications and edits have been developed thanks to the advancement of high-throughput sequencing in recent years. The extensive range of A-to-I sites and tissue-specific amounts found in humans, mice, and flies were cataloged in 2013 by the Rigorously Annotated Database of A-to-I RNA editing (RADAR). New sites are being added to the database and overall edits are being made. The amount of editing at specific editing sites, like in the filamin A transcript, varies from one tissue to another. One factor that controls the level of A-to-I RNA editing is the efficiency of mRNA-splicing. It is interesting to note that ADAR1 and ADAR2 affect alternative splicing through their A-to-I editing ability and dsRNA binding ability.

Mutant messenger RNA editing

Alternative U-to-C mRNA editing was first reported in WT1 (Wilms Tumor-1) transcripts, and non-classic G-A mRNA changes were first observed in HNRNPK (heterogeneous nuclear ribonucleoprotein K) transcripts in both malignant and normal colorectal samples. The latter changes were also later seen alongside non-classic U-to-C alterations in brain cell TPH2 (tryptophan hydroxylase 2) transcripts. Although the reverse amination might be the simplest explanation for U-to-C changes, transamination and transglycosylation mechanisms have been proposed for plant U-to-C editing events in mitochondrial transcripts. A recent study reported novel G-to-A mRNA changes in WT1 transcripts at two hotspots, proposing the APOBEC3A (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3A) as the enzyme implicated in this class of alternative mRNA editing. It was also shown that alternative mRNA changes were associated with canonical WT1 splicing variants, indicating their functional significance.

Modifying plant RNA in plastids and mitochondria

Previous research has demonstrated that the only RNA editing observed in plant mitochondria and plastids is the extremely rare conversion of C-to-U and U-to-C. RNA-editing sites are mainly located upstream of mitochondrial or plastid RNAs, as well as introns and other non-translated regions. In fact, RNA editing can restore the functionality of tRNA molecules. There has been a lot of research on where C to U RNA editing events occur in mitochondria and plastids, but nobody knows what proteins make up the editosome or how they are organized. Proper editing at several sites also requires certain members of the MORF (several Organellar RNA editing Factor) family, and members of the expanding PPR protein family have been demonstrated to operate as trans-acting factors for RNA sequence recognition. The fact that some MORF proteins have been found to interact with PPR family members suggests that MORF proteins could be part of the editosome complex. As of now, we don’t know which enzyme is responsible for trans- or deamination of the RNA transcript, but PPR proteins could be one of them.

Plant translation and respiration cannot take place normally without RNA editing. Editing can bring back the functional essential base-pairing sequences of tRNAs. It has also been associated with making RNA-edited proteins that are part of the respiration pathway’s polypeptide complexes. Consequently, it’s quite likely that plastid and mitochondrial activity would be impaired by polypeptides produced from unprocessed RNAs.

Editing RNA from C to U can add new start and stop codons but remove them from existing ones. When the codon ACG is changed to AUG, it creates a cryptic start codon.

The Multiple Uses of RNA Editing Summarized

VIRUS RNA editing

There is evidence that viruses with an RNA genome, such as parainfluenza, measles, or mumps, have evolved to employ RNA alterations in a variety of ways when they infect host cells. A virus-encoded RNA-dependent RNA polymerase is known to pause and “stutter” at certain nucleotide combinations while transcribing viral RNAs, which the virus uses for a variety of purposes throughout its infection cycle, including immune evasion and protein translation enhancement. RNA editing is employed for stability and the generation of protein variants. Furthermore, polymerase adds hundreds of non-templated A’s to the 3′ end of nascent mRNA, which helps stabilize the mRNA. In addition, the RNA polymerase can include one or two Gs or As upstream of the translational codon due to its stuttering and pausing. The inclusion of non-templated nucleotides changes the reading frame, resulting in the production of a new protein.

Furthermore, it has been demonstrated that, depending on the virus, the RNA changes can have either beneficial or detrimental effects on the efficiency of replication and translation.As an example, Courtney et al. demonstrated that infected host cells boost HIV-1 protein translation by adding a change to the viral mRNA termed 5-methylcytosine. Curiously, virus mRNA expression is unaffected by blocking the m5C alteration, which leads to a significant decrease in viral protein translation. Conversely, Lichinchi et al. demonstrated that the N6-methyladenosine alteration on ZIKV mRNA hinders viral replication.

Where RNA editing came from and how it developed

Animal RNA-editing systems may have descended from mononucleotide deaminases, which in turn gave rise to more extensive gene families, such as apobec-1 and adar. Bacterial deaminases, which are involved in nucleotide metabolism, are highly similar to these genes. Enzyme adenosine deaminase from E. Due to the insufficiency of the enzyme’s reaction pocket, coli is unable to deaminate RNA nucleosides. Nevertheless, deamination is made possible by expanding this active site by amino acid alterations in the related human analogue genes, APOBEC1 and ADAR. The gRNA-mediated pan-editing in trypanosome mitochondria, which comprises the templated insertion of U residues, is a completely distinct biochemical process. While previous research has shown that the enzymes in question can originate from a variety of places, the specificity with which gRNA and mRNA interact to insert nucleotides is reminiscent of tRNA editing processes in animal and amoeba mitochondria. Similarly, eukaryotic ribose methylation of rRNAs by guide RNA molecules is a type of modification that shares many similarities with these processes.

So, RNA editing changed throughout time. There are a number of proposed adaptive rationales for editing. One common description is that editing is a way to fix or fix defective gene sequences. Nevertheless, this theory doesn’t appear to work for gRNA-mediated editing, as there’s no way to produce an error-free gRNA-encoding area unless the original gene region is duplicated. The constructive neutral evolution theory offers a more convincing explanation for the system’s evolutionary background, in which the “defect” comes after the gratuitous ability for editing, in the reverse order of events.

RNA degradation might require RNA editing.

The role of RNA editing in RNA degradation was investigated in a study. The researchers zeroed in on the ADAR-UPF1 interaction, which is an enzyme in the NMD pathway (nonsense-mediated mRNA decay). Researchers discovered that the suprasliceosome contains ADAR and UPF1, which together inhibit gene expression. At now, we do not know the precise process or routes in which these two are engaged. Their ability to down-regulate particular genes is the sole piece of information that has been uncovered by this study.

Therapeutic Modulation of mRNA

Notable references: CRISPR-Cas13 (formerly known as C2c2)

In 1995, the idea of directing edits to fix mutated sequences was first proposed and shown to work. The researchers used synthetic RNA antisense oligonucleotides that were complementary to a dystrophin sequence that had a premature stop codon mutation. They used these to activate A-to-I editing, which changed the stop codon to a read-through codon in a model xenopus cell system. However, this process also caused nearby accidental A-to-I transitions. A-to-I (read as G) transitions can fix all three stop codons, but cannot generate a stop codon. The result was a >25% correction of the intended stop codon, which allowed the read-through to a downstream luciferase reporter gene, thanks to the modifications. Some time ago, researchers used CRISPR-Cas13 fused to deaminases to direct mRNA editing. This was a step forward from the work done by Rosenthal, who successfully edited a mutated cystic fibrosis sequence in mammalian cell culture by directing an oligonucleotide linked to a cytidine deaminase.

In 2022, Cas7-11 was introduced, which is an improvement over Cas13 for therapeutic RNA editing. It allows for cuts that are adequately focused and, in 2021, an early version of it was employed for in vitro editing.

Analogy with DNA editing

The consequences of RNA editing, including the possibility of off-target alterations in RNA, are temporary and not passed down through generations, in contrast to DNA editing, which is permanent. Because of this, RNA editing is thought to be safer. In addition, it could be possible to use the ADAR protein that is already present in the cells of humans and other eukaryotic organisms as a guide RNA rather than introducing a foreign protein into the body.