RNA editing
This article is missing information about pseudouracil.(December 2020) |
Part of a series on |
Genetics |
---|
RNA editing (also RNA modification) is a molecular process through which some cells can make discrete changes to specific
RNA editing has been observed in some
RNA-editing processes show great molecular diversity, and some appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing phenomena includes
Detection of RNA editing
Next generation sequencing
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[9] or specialised sequencing methods.[1][2][3] Examples of specialised methods are MeRIP-seq,[10] m6A-seq,[11] PA-m5C-seq [12], methylation-iCLIP,[13] m6A-CLIP,[14] Pseudo-seq,[15] Ψ-seq,[16] CeU-seq,[17] Aza-IP[18] and RiboMeth-seq[19]). Many of these methods are based on specific capture of the RNA species containing the specific modification, for example through antibody binding coupled with sequencing of the captured reads. After the sequencing these reads are mapped against the whole transcriptome to see where they originate from.[20] Generally with this kind of approach it is possible to see the location of the modifications together with possible identification of some consensus sequences that might help identification and mapping further on. One example of the specialize methods is PA-m5C-seq. This method was further developed from PA-m6A-seq method to identify m5C modifications on mRNA instead of the original target N6-methyladenosine. The easy switch between different modifications as target is made possible with a simple change of the capturing antibody form m6A specific to m5C specific.[12] Application of these methods have identified various modifications (e.g. pseudouridine, m6A, m5C, 2′-O-Me) within coding genes and non-coding genes (e.g. tRNA, lncRNAs, microRNAs) at single nucleotide or very high resolution.[4]
Mass Spectrometry
Mass spectrometry is a way to quantify RNA modifications.[21] More often than not, modifications cause an increase in mass for a given nucleoside. This gives a characteristic readout for the nucleoside and the modified counterpart.[21] Moreover, mass spectrometry allows the investigation of modification dynamics by labelling RNA molecules with stable (non-radioactive) heavy isotopes in vivo. Due to the defined mass increase of heavy isotope labeled nucleosides they can be distinguished from their respective unlabelled isotopomeres by mass spectrometry. This method, called NAIL-MS (nucleic acid isotope labelling coupled mass spectrometry), enables a variety of approaches to investigate RNA modification dynamics.[22][23][24]
Types of RNA
Messenger RNA modification
Recently, functional experiments have revealed many novel functional roles of RNA modifications. Most of the RNA modifications are found on transfer-RNA and ribosomal-RNA, but also eukaryotic mRNA has been shown to be modified with multiple different modifications. 17 naturally occurring modifications on mRNA have been identified, from which the N6-methyladenosine is the most abundant and studied.[25] mRNA modifications are linked to many functions in the cell. They ensure the correct maturation and function of the mRNA, but also at the same time act as part of cell's immune system.[26] Certain modifications like 2’O-methylated nucleotides has been associated with cells ability to distinguish own mRNA from foreign RNA.[27] For example, m6A has been predicted to affect protein translation and localization,[1][2][3] mRNA stability,[28] alternative polyA choice [14] and stem cell pluripotency.[29] 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.[30] 5-methylcytosine on the other hand has been associated with mRNA transport from the nucleus to the cytoplasm and enhancement of translation. These functions of m5C are not fully known and proven but one strong argument towards these functions in the cell is the observed localization of m5C to translation initiation site.[31] Importantly, many modification enzymes are dysregulated and genetically mutated in many disease types.[1] For example, genetic mutations in pseudouridine synthases cause mitochondrial myopathy, sideroblastic anemia (MLASA) [32] and dyskeratosis congenital.[33]
Compared to the modifications identified from other RNA species like tRNA and rRNA, the amount of identified modifications on mRNA is very small. One of the biggest reasons why mRNA modifications are not so well known is missing research techniques. In addition to the lack of identified modifications, the knowledge of associated proteins is also behind other RNA species. Modifications are results of specific enzyme interactions with the RNA molecule.[25] Considering mRNA modifications most of the known related enzymes are the writer enzymes that add the modification on the mRNA. The additional groups of enzymes readers and erasers are for most of the modifications either poorly known of not known at all.[34] For these reasons there has been during the past decade huge interest in studying these modifications and their function.[20]
Transfer RNA modifications
Transfer RNA or tRNA is the most abundantly modified type of RNA.[35] Modifications in tRNA play crucial roles in maintaining translation efficiency through supporting structure, anticodon-codon interactions, and interactions with enzymes.[36]
Anticodon modifications are important for proper decoding of mRNA. Since the genetic code is degenerate, anticodon modifications are necessary to properly decode mRNA. Particularly, the wobble position of the anticodon determines how the codons are read. For example, in eukaryotes an adenosine at position 34 of the anticodon can be converted to inosine. Inosine is a modification that is able to base-pair with cytosine, adenine, and uridine.[37]
Another commonly modified base in tRNA is the position adjacent to the anticodon. Position 37 is often hypermodified with bulky chemical modifications. These modifications prevent frameshifting and increase anticodon-codon binding stability through stacking interactions.[37]
Ribosomal RNA modification
Ribosomal RNA (rRNA) is essential to the makeup of ribosomes and peptide transfer during translation processes.[38] Ribosomal RNA modifications are made throughout ribosome synthesis, and often occur during and/or after translation. Modifications primarily play a role in the structure of the rRNA in order to protect translational efficiency.[38] Chemical modification in rRNA consists of methylation of ribose sugars, isomerization of uridines, and methylation and acetylation of individual bases.[39]
Methylation
Methylation of rRNA upholds structural rigidity by blocking base pair stacking and surrounds the 2’-OH group to block hydrolysis. It occurs at specific parts of eukaryotic rRNA. The template for methylation consists of 10-21 nucleotides.[38] 2'-O-methylation of the ribose sugar is one of the most common rRNA modifications.[40] Methylation is primarily introduced by small nucleolar RNA's, referred to as snoRNPs. There are two classes of snoRNPs that target methylation sites, and they are referred to box C/D and box H/ACA.[39][40] One type of methylation, 2′-O-methylation, contributes to helical stabilization.[38]
Isomerization
The isomerization of uridine to pseudoridine is the second most common rRNA modification. These pseudoridines are also introduced by the same classes of snoRNPs that participate in methylation. Psuedouridine synthases are the major participating enzymes in the reaction.[41] The H/ACA box snoRNPs introduce guide sequences that are about 14-15 nucleotides long.[39] Pseudouridylation is triggered in numerous places of rRNAs at once to preserve the thermal stability of RNA.[39] Pseudouridine allows for increased hydrogen bonding and alters translation in rRNA and tRNA.[40][41] It alters translation by increasing the affinity of the ribosome subunit to specific mRNAs.[38]
Base Editing:
Base editing is the third major class of rRNA modification, specifically in eukaryotes. There are 8 categories of base edits that can occur at the gap between the small and large ribosomal subunits.[38] RNA methyltransferases are the enzymes that introduce base methylation.[38] Acetyltransferases are the enzymes responsible for acetylation of cytosine in rRNA. Base methylation plays a role in translation. These base modifications all work in conjunction with the two other main classes of modification to contribute to RNA structural stability. An example of this occurs in N7-methylation, which increases the nucleotide's charge to increase ionic interactions of proteins attaching to the RNA before translation.
Editing by insertion or deletion
RNA editing through the addition and deletion of uracil has been found in kinetoplasts from the mitochondria of Trypanosoma brucei.[42] Because this may involve a large fraction of the sites in a gene, it is sometimes called "pan-editing" to distinguish it from topical editing of one or a few sites.
Pan-editing starts with the base-pairing of the unedited primary transcript with a
The mechanism of the
Editing by deamination
C-to-U editing
The editing involves cytidine deaminase that deaminates a cytidine base into a uridine base. An example of C-to-U editing is with the
A-to-I editing
Adenosine-to-inosine (A-to-I) modifications contribute to nearly 90% of all editing events in RNA. The deamination of adenosine is catalyzed by the double-stranded RNA-specific adenosine deaminase (ADAR), which typically acts on pre-mRNAs. The deamination of adenosine to inosine disrupts and destabilizes the dsRNA base pairing, therefore rendering that particular dsRNA less able to produce siRNA, which interferes with the RNAi pathway.
The
The development of high-throughput sequencing in recent years has allowed for the development of extensive databases for different modifications and edits of RNA. RADAR (Rigorously Annotated Database of A-to-I RNA editing) was developed in 2013 to catalog the vast variety of A-to-I sites and tissue-specific levels present in humans, mice, and flies. The addition of novel sites and overall edits to the database are ongoing.[56] The level of editing for specific editing sites, e.g. in the filamin A transcript, is tissue-specific.[57] The efficiency of mRNA-splicing is a major factor controlling the level of A-to-I RNA editing.[58][59] Interestingly, ADAR1 and ADAR2 also affect alternative splicing via both A-to-I editing ability and dsRNA binding ability.[60][61]
Alternative mRNA editing
Alternative U-to-C mRNA editing was first reported in
RNA editing in plant mitochondria and plastids
It has been shown in previous studies that the only types of RNA editing seen in the plants' mitochondria and plastids are conversion of C-to-U and U-to-C (very rare).
RNA editing is essential for the normal functioning of the plant's translation and respiration activity. Editing can restore the essential base-pairing sequences of tRNAs, restoring functionality.[83] It has also been linked to the production of RNA-edited proteins that are incorporated into the polypeptide complexes of the respiration pathway. Therefore, it is highly probable that polypeptides synthesized from unedited RNAs would not function properly and hinder the activity of both mitochondria and plastids.
C-to-U RNA editing can create start and stop
RNA editing in viruses
Viruses (i.e.,
Additionally, the RNA modifications are shown to have both positive and negative effects on the replication and translation efficiency depending on the virus. For example, Courtney et al.[12] showed that an RNA modification called 5-methylcytosine is added to the viral mRNA in infected host cells in order to enhance the protein translation of HIV-1 virus. The inhibition of the m5C modification on viral mRNA results in significant reduction in viral protein translation, but interestingly it has no effect on the expression of viral mRNAs in the cell. On the other hand, Lichinchi et al.[87] showed that the N6-methyladenosine modification on ZIKV mRNA inhibits the viral replication.
Origin and Evolution of RNA editing
The RNA-editing system seen in the animal may have evolved from mononucleotide deaminases, which have led to larger gene families that include the apobec-1 and adar genes. These genes share close identity with the bacterial deaminases involved in nucleotide metabolism. The adenosine deaminase of E. coli cannot deaminate a nucleoside in the RNA; the enzyme's reaction pocket is too small for the RNA strand to bind to. However, this active site is widened by amino acid changes in the corresponding human analog genes, APOBEC1 and ADAR, allowing deamination.[88][89] The gRNA-mediated pan-editing in
Thus, RNA editing evolved more than once. Several adaptive rationales for editing have been suggested.[93] Editing is often described as a mechanism of correction or repair to compensate for defects in gene sequences. However, in the case of gRNA-mediated editing, this explanation does not seem possible because if a defect happens first, there is no way to generate an error-free gRNA-encoding region, which presumably arises by duplication of the original gene region. A more plausible alternative for the evolutionary origins of this system is through constructive neutral evolution, where the order of steps is reversed, with the gratuitous capacity for editing preceding the "defect".[94]
Therapeutic mRNA Editing
Directing edits to correct mutated sequences was first proposed and demonstrated in 1995.[95] This initial work used synthetic RNA antisense oligonucleotides complementary to a pre-mature stop codon mutation in a dystrophin sequence to activate A-to-I editing of the stop codon to a read through codon in a model xenopus cell system.[95] While this also led to nearby inadvertent A-to-I transitions, A to I (read as G) transitions can correct all three stop codons, but cannot create a stop codon. Therefore, the changes led >25% correction of the targeted stop codon with read through to a downstream luciferase reporter sequence. Follow on work by Rosenthal achieved editing of mutated mRNA sequence in mammalian cell culture by directing an oligonucleotide linked to a cytidine deaminase to correct a mutated cystic fibrosis sequence.[96] More recently, CRISPR-Cas13 fused to deaminases has been employed to direct mRNA editing.[97]
In 2022, therapeutic RNA editing for Cas7-11 was reported.[98][99] It enables sufficiently targeted cuts and an early version of it was used for in vitro editing in 2021.[100]
Comparison to DNA editing
Unlike DNA editing, which is permanent, the effects of RNA editing − including potential off-target mutations in RNA − are transient and are not inherited. RNA editing is therefore considered to be less risky. Furthermore, it may only require a guide RNA by using the ADAR protein already found in humans and many other eukaryotes' cells instead of needing to introduce a foreign protein into the body.[101]
See also
- DNA editing
- Epigenome editing
- NcRNA therapy
References
- ^ PMID 24898039.
- ^ PMID 23138310.
- ^ PMID 24713629.
- ^ PMID 26464443.
- S2CID 11283810.
- ^ "New genetic editing powers discovered in squid". phys.org. Retrieved 2020-04-05.
- PMID 29106616.
- PMID 10371035.
- PMID 29228294.
- PMID 22608085.
- S2CID 3517716.
- ^ PMID 31415754.
- PMID 23871666.
- ^ PMID 26404942.
- PMID 25192136.
- PMID 25219674.
- PMID 26075521.
- PMID 23604283.
- PMID 25417815.
- ^ PMID 32257049.
- ^ PMID 26501195.
- PMID 28488916.
- PMID 30328661.
- PMID 30395967.
- ^ PMID 32301288.
- S2CID 135425191.
- ^ PMID 28219769.
- PMID 24284625.
- S2CID 206562941.
- PMID 21677757.
- PMID 28418038.
- PMID 15108122.
- S2CID 205342127.
- PMID 27313037.
- S2CID 6727707.
- PMID 28375166.
- ^ PMID 17187822.
- ^ PMID 27911188.
- ^ PMID 35796987, retrieved 2024-02-29
- ^ PMID 32870730.
- ^ PMID 32870730.
- PMID 7513284.
- ^ PMID 8652667.
- PMID 9380494.)
{{cite journal}}
: CS1 maint: DOI inactive as of February 2024 (link - S2CID 19656609.
- PMID 9175474.
- S2CID 4634304.
- PMID 1713359.
- ^ Hajduk SL, Sabatini RS (1998). "Mitochondrial mRNA editing in kinetoplastid protozoa". In Grosjean H, Benne R (eds.). Modification and Editing of RNA. Washington, DC.: ASM Press. pp. 377–394.
- PMID 24732437.
- PMID 25585161.
- PMID 20192758.
- PMID 26148686.
- PMID 27044895.
- PMID 30462291.
- PMID 24163250.
- PMID 24025532.
- PMID 27112566.
- PMID 31427386.
- PMID 32727871.
- PMID 32034135.
- PMID 7926762.
- PMID 16404425.
- PMID 20126463.
- S2CID 2174535.
- PMID 25807502.
- S2CID 4373041.
- S2CID 19402913.
- ^ PMID 2480644.
- S2CID 4303733.
- ^ S2CID 30396182.
- ^ PMID 1365399.
- ^ Grienenberger JM (1993). "RNA editing in plant organelles". RNA Editing (Benne, R., Ed.), Ellis Harwood, New York.
- PMID 8635473.
- PMID 9177209.
- PMID 8915541.
- PMID 9326494.
- S2CID 26005486.
- ^ Marchfelder A, Binder S, Brennicke A, Knoop V (1998). "Preface". In Grosjean H, Benne R (eds.). Modification and Editing of RNA. Washington, DC: ASM Press. pp. 307–323.
- PMID 24274753.
- PMID 24471833.
- PMID 23818871.
- ^ Price DH, Gray MW (1998). "Editing of tRNA". In Grosjean H, Benne R (eds.). Modification and Editing of RNA. Washington, DC: ASM Press. pp. 289–306.
- PMID 1655410.
- PMID 1629949.
- ^ a b Kolakofsky D, Hausmann S (1998). "Chapter 23: Cotranscriptional Paramyxovirus mRNA Editing: a Contradiction in Terms?". In Grosjean H, Benne R (eds.). Modification and Editing of RNA. Washington, DC: ASM Press. pp. 413–420.
- PMID 27773536.
- ^ Carter CW (1998). "Nucleoside deaminases for cytidine and adenosine: comparisons with deaminases acting on RNA". In Grosjean H, Benne R (eds.). Modification and Editing of RNA. Washington, DC: ASM Press. pp. 363–376.
- PMID 8379005.
- PMID 8415006.
- ^ Bachellerie JP, Cavaille J (1998). "Small nucleolar RNAs guide the ribose methylations of eukaryotic rRNAs". In Grosjean H, Benne R (eds.). Modification and Editing of RNA. Washington, DC: ASM Press. pp. 255–272.
- S2CID 205470421.
- S2CID 1743092.
- ^ PMID 7545300.
- PMID 24108353.
- PMID 29070703.
- ^ Williams S. "Neuroscientists expand CRISPR toolkit with new, compact Cas7-11 enzyme". Massachusetts Institute of Technology. Retrieved 22 June 2022.
- S2CID 249103058.
- S2CID 237432753.
- ^ Cross R (25 March 2019). "Watch out, CRISPR. The RNA editing race is on". Chemical & Engineering News. 97 (12). Retrieved 30 September 2020.