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Epitranscriptomics describes an aspect of molecular genetics, or a study thereof, that depends on biochemical modifications of RNA.[1][2] By analogy to the term epigenetics, described as "functionally relevant changes to the genome that do not involve a change in the nucleotide sequence", epitranscriptomics can be defined as a functionally relevant changes to the transcriptome that do not involve a change in the ribonucleotide sequence. The epitranscriptome, therefore, is defined as the ensemble of such functionally relevant changes.

There are several types of RNA modifications that impact gene expression. These modifications happen to all types of cellular RNA including, but not limited to, ribosomal RNA(rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and small nuclear RNA (snRNA)[3]. There are more than one hundred documented RNA modifications. A database maintained by the University of Albany details each modification[4]. The most common and well-understood mRNA modification at present is the N6-Methyladenosine (m6A), which has been observed to occur an average of three times in every mRNA molecule[5].

The relative youth of this field means there is still much progress to be made in characterizing all modifications to the transcriptome and elucidating their mechanisms of action. Once these questions are answered and biologists have a better sense of the amount of variation in RNA modification, the focus will turn to each modification’s biological function[6]. This has already been investigated in a select few proteins such as adenosine deaminase, which acts on RNA (ADAR). ADAR has been shown to affect antibody production and the innate immune system as well as transcripts encoding important receptors for the central nervous system. This plurality in function has caused some scientists to speculate that the epitransciptome may be even more expansive than the better defined epigenome[7].

N6-Methyladenosine (m6A )

m6A describes the methylation of the nitrogen at position 6 of the adenosine base within mRNA. Discovered in 1974,[1] m6A is the most abundant eukaryotic mRNA modification.[2] The term "epitranscriptome" was coined following transcriptome-wide mappings of m6A sites,[3][4] but does not necessarily exclude other post-transcriptional mRNA modifications.

m6A methylation regulates nuclear export of mature mRNA and mRNA stability.[5][6] How, and in response to what stimulus, the cell endogeneously regulates the level of m6A methylation remains unclear at present.

This m6A modification has a notable effect on translational dynamics[7]. As expected, because it is just a modified adenosine base, m6A base-pairs with uridine during decoding. However, the adenosine’s methylation hinders tRNA accommodation and translation elongation. When an m6A-modified codon interacts with its cognate tRNA, it acts more like a near-cognate codon interaction instead of the cognate codon interaction. This can be seen in that the delay in the tRNA accommodation, which is dependent upon both the position of the m6A in the mRNA codons and on how accurate the translation is. To summarize, translation-elongation dynamics are slower for codons with m6A and different locations of these modified nucleotides in the mRNA codons affect decoding dynamics in different ways.

The m6A modification affects tRNA incorporation’s distinct steps in different ways. It slows down GTP hydrolysis by EF-Tu 12-fold, delays tRNA accommodation two-fold, and it slows down peptidyl transfer two-fold. It also causes a 1.5-fold increase in the amount of GTP hydrolyzed per peptidyl transfer, which indicates that a lot of proofreading is required. Overall, this m6A modification leads to a kinetic loss of a factor of 18[7]. 

N6-Methyladenosine (m6A) on Alternative Splicing

Exons of the mRNA are shown in blue and introns (non-coding sequence) are in red. a) Alternative splicing involves the removal of introns from the transcript. b) Adenine is methylated to form m6A modification. c) m6A forms a uridine rich RNA stem loop near where the m6A modification was made. HNRNPC protein (involved in pre-mRNA processing) binds to the uridine rich region on the loop leading to the excision of intron.

The terms “eraser” and “reader” have been associated with RNA modification. “Eraser” is a general term to describe an enzyme that de-methylates m6A. Changes that mutate the gene encoding the “eraser” enzyme lead to obesity and cancer. “Reader” proteins are involved in gene expression where there are abundant m6A; “reader” proteins have a higher propensity to bind with greater affinity, while the de-methylated form has been reported to have a decreased binding affinity.

mRNA are subject to layers of regulatory gene expression. One known mechanism involves the formation of RNA stem-loops. Stem-loops occur when complementary bases within a single-stranded RNA molecule form Watson-Crick base pairs on the stem while forming an unpaired end or loop. Stem-loops do not have one definite function, but a plethora of functions. In the case of the m6A regulatory mechanism, it is involved in alternative splicing. These stem and loop structures are subject to alterations regarding changes in pH, temperature, ion concentrations, binding nature of proteins and also nucleic acids.

m6A has been observed to be located within the loops opposite of the HNRNPC binding site[8]. HNRNPC is single-stranded RNA binding protein where it participates in post-transcriptional regulation, specifically alternative splicing. HNRNPC protein binds to its site (uridine rich region on the stem loop) when methylated adenosine is present.The HNRNPC binding site on the mRNA consists of an abundance of uridine nucleotides. Studies have concluded that methylated adenosine residues destabilize the hairpin structure, elongating the uridine nucleotide stretch, causing the binding site to be more accessible for efficient HNRNPC protein binding.

Evidence supporting this claim identified that decreased m6A levels in the transcriptome lead to significantly reduced HNRNPC binding, so alternative splicing is co-regulated by methylation and HNRNPC binding activity. However, the m6A modification does not directly cause protein binding. Rather, it alters the loop structure to regulate gene expression by acting as a switch that exposes the HNRNPC region. It is essentially a two-step, co-regulatory mechanism prevalent in biochemistry in controlling alternative splicing. Note that demethylase enzyme can indeed “erase” the methyl group, thus inhibiting alternative splicing: it is the reverse of the two-step regulation mechanism.

mRNA capping by NAD+, NADH & dpCoA

Cartoon schematic of NudC & RppH cleavage of NCIN-capped and ATP-capped RNAs respectively forming 5'-monophosphate RNA product.

mRNA capping in eukaryotes have shown to exist and have been studied extensively regarding the 5’ 7-methylguanylate cap and its poly-A tail.[9] It provides RNA stability, processing, localization and translational efficiency. Recently, researchers have proven a similar mechanism in RNA pre-processing in prokaryotes. In prokaryotic (E.coli) mRNA, the 5’ is capped with nicotinamide adenine dinucleotide (NAD+ or NADH) or 3’-desphospho-coenzyme A (dpCoA). It was previously thought that the NAD+,NADH, dpCoA modification occurs after transcription analogously with the 7-methylguanylate cap; however, recently it has been shown that modifications are incorporated during transcription initiation by acting as a non-canonical initiating nucleotides (NCIN) for de novo transcription by cellular RNA polymerase. RppH and NudC pyrophosphohydrolases cleaves specific phosphate bonds to eliminate the cap modification. ATP was initially known to cap an RNA product in prokaryotes while NAD+, NADH and dpCoA was still being studied. RppH specifically cleaves 5’-triphosphate and 5’-diphosphate RNAs to 5’-monophosphate RNA products which only targets the ATP capped RNAs. On the other hand, NudC cleaves 5’-(NADH,NAD+,dpCoA) capped RNAs to 5’-monophosphate RNAs but did not cleave ATP capped RNA. This discovery of two specific hydrolases that targets specific products lead to the discovery of NCIN-mediated transcription. Both prokaryotes and eukaryotes transcripts have been observed with x-ray crystallography to have a NCIN capped RNA product in addition to being observed under in vivo condition. The efficiency of NCIN capping is largely influenced by promoter sequence especially the -35 box and -10 box upstream of the transcription start site. Studies so far have defined the mechanism and structural basis of NCIN mediated capping but the function of these caps are still to be discovered. Recent consensus is that NCIN-mediated ab initio capping occurs in all organisms.

Pseudo-seq and the regulation of pseudouridyltaion in yeast and human cells

Pseudouridine is a modified nucleoside found within non-coding RNAs (ncRNA).[10] It increases the function of tRNA and rRNA by stabilizing the structure. Though mRNAs are not known for containing pseudouridine, the artificial process of pseudouridylation has an affect on the function of mRNA: it changes the genetic code by making non-canonical base pairing possible in the ribosome decoding center.

A certain paper looks at pseudouridylation in yeast and human RNAs using pseudo-seq[11], a process that utilizes a single-nucleotide-resolution method for pseudouridine identification. It identifies the known modification sites as well as other sites in ncRNAs in addition to the many pseudouridylated sites in mRNA.

There are more than 100 classes of RNA modifications that have been found, a majority of which are in tRNA and rRNA while only three modified nucleotides have been discovered inside a coding sequence of mRNA: m6A, 5-methylcytosine (m5C), and inosine. Research has shown, however, that pseudouridines are quite scarce in yeast. Nonetheless, much of the regulation in regards to pseudouridylation is regulated through the environment. In yeast this may be nutrient deprivation and in humans it is the serum starvation. When looking at yeast, research has utilized perturbing pseudouridine synthases deletion strains grown to high density and identified mRNA targets for each PUS protein. Results came back showing that most mRNA targets showed increase modification during post-diauxic growth. The pseudo-seq method identified 96 pseudouridines in 89 mRNAs, similar to yeast the growth of pseudouridine was regulated by cellular growth state. This approach provides an analysis of RNA pseudouridylation with single-nucleotide resolution and shows endogenous mRNAs are specifically pseudouridylated in a highly regulated manner in yeast and human cells. mRNA pseudouridyltaion could also bring a change in translation initiation efficiency, RNA localization, and other processes all cause pseuduridine  stabilizes RNA structure.

Epitrancriptome and modulating sections of RNA

The concept of epitranscriptomics has been seen to have an effect not only on RNA but also on protein synthesis. RNA methylase NSun2 methylates mRNAs. This methylation has an effects the components of the postsynaptic neurons. The RNA modification sites are seen to occur at adenosine to create methyl-6-adenosine found around stop codons. Not much is known about the purpose of this methylation but it was found that human patients  lacking NSun2 are characterized by intellectual disability and neural.

Greater Implications of the Epitranscriptome

The concept of epitranscriptomics has been seen to have an effect not only on RNA but also on protein synthesis. RNA methylase, NSun2, methylates mRNAs. This methylation has an effects on the components of the postsynaptic neurons. The RNA modification sites are seen to occur at adenosine to create methyl-6-adenosine found around stop codons. Not much is known about the purpose of this methylation but it was found that human patients  lacking NSun2 are characterized by intellectual disability and neural defects

The Epitrancriptome has many diverse components however only a few have been analyzed in the detail required to explain these modifications. The modifications occur both on coding and non-coding RNA and studies have suggested multiple roles for the epitranscriptome modifications many involving protein-synthesis control. Some other biological controls are the regulation of the circadian rhythms in the suprachiasmatic nucleus of a rat hypothalamus.

An important hypothesis is that it seems that the epitrancriptomic modifications can be dynamically regulated. Chuan He laboratory showed that m6A modifications in mRNAs promoted binding of a related reader, YTHFD1, this binding speeds up the rate of translation in HeLa cells. RNA modifications appear to offer many layers of control that allow protein synthesis to occur in a signal dependent manner. mRNA translation to participate directly in complex cellular functions. This theory is also supported by the fact that mRNA translation levels correlate poorly with cellular mRNA levels.

See also

Further reading

  • Dominissini, Dan (2014). "Roadmap to the epitranscriptome". Science. 346 (6214): 1192. doi:10.1126/science.aaa1807.
  • Zumbo, Paul; Christopher E. Mason (2014). "Methods for RNA Isolation, Characterization, and Sequencing". In Maria S. Poptsova (ed.). Genome Analysis: Current Procedures and Applications. Horizon Scientific Press. p. 31. ISBN 978-1-908230-29-4. {{cite book}}: External link in |chapterurl= (help); Unknown parameter |chapterurl= ignored (|chapter-url= suggested) (help)

References

  1. ^ Desrosiers, Ronald; Friderici K.; Rottman F. (Oct 1974). "Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells". Proc Natl Acad Sci U S A. 71 (10): 3971–5. doi:10.1073/pnas.71.10.3971. PMC 434308. PMID 4372599.
  2. ^ Bokar, Joseph A. (January 2005). "The biosynthesis and functional roles of methylated nucleosides in eukaryotic mRNA". In H. Grosjean (ed.). Topics in Current Genetics, Vol. 12, Fine-Tuning of RNA functions by Modification and Editing. Springer-Verlag Berlin Heidelberg. pp. 141–177.
  3. ^ Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR (May 2012). "Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3' UTRs and near Stop Codons". Cell. 149 (7): 1635–46. doi:10.1016/j.cell.2012.05.003. PMC 3383396. PMID 22608085.
  4. ^ Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G (May 2012). "Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq". Nature. 485 (7397): 201–6. doi:10.1038/nature11112. PMID 22575960.
  5. ^ Wang, X.; Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C. (2 Jan 2014). "N6-methyladenosine-dependent regulation of messenger RNA stability". Nature. 505 (7481): 117–20. doi:10.1038/nature12730. PMC 3877715. PMID 24284625.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  6. ^ Fustin JM, Doi M, Yamaguchi Y, Hayashi H, Nishimura S, Yoshida M, Isagawa T, Morioka MS, Kakeya H, Manabe I, Okamura H (November 2013). "RNA-Methylation-Dependent RNA Processing Controls the Speed of the Circadian Clock". Cell. 155 (4): 793–806. doi:10.1016/j.cell.2013.10.026. PMID 24209618.
  7. ^ a b Choi, Junhong; Ieong, Ka-Weng; Demirci, Hasan; Chen, Jin; Petrov, Alexey; Prabhakar, Arjun; O'Leary, Seán E; Dominissini, Dan; Rechavi, Gideon. "N6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics". Nature Structural & Molecular Biology. 23 (2): 110–115. doi:10.1038/nsmb.3148. PMC 4826618. PMID 26751643.
  8. ^ Theler, Dominik; Allain, Frédéric H.-T. (2015-02-26). "Molecular biology: RNA modification does a regulatory two-step". Nature. 518 (7540): 492–493. doi:10.1038/518492a. ISSN 1476-4687. PMID 25719665.
  9. ^ Bird, Jeremy G.; Zhang, Yu; Tian, Yuan; Panova, Natalya; Barvík, Ivan; Greene, Landon; Liu, Min; Buckley, Brian; Krásný, Libor. "The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA". Nature. 535 (7612): 444–447. doi:10.1038/nature18622. PMC 4961592. PMID 27383794.
  10. ^ Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V. (2014-11-06). "Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells". Nature. 515 (7525): 143–146. doi:10.1038/nature13802. ISSN 0028-0836. PMC 4224642. PMID 25192136.
  11. ^ Carlile, Thomas M.; Rojas-Duran, Maria F.; Gilbert, Wendy V. (2015-01-01). He, Chuan (ed.). Methods in Enzymology. RNA Modification. Vol. 560. Academic Press. pp. 219–245. doi:10.1016/bs.mie.2015.03.011.
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