Epitranscriptome

Within the field of molecular biology, the epitranscriptome includes all the biochemical modifications of the RNA (the transcriptome) within a cell. In analogy to epigenetics that describes "functionally relevant changes to the genome that do not involve a change in the nucleotide sequence", epitranscriptomics involves all functionally relevant changes to the transcriptome that do not involve a change in the ribonucleotide sequence. Thus, the epitranscriptome can be defined as the ensemble of such functionally relevant changes.

There are several types of RNA modifications that impact gene expression. These modifications happen to many types of cellular RNA including, but not limited to, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and small nuclear RNA (snRNA). The most common and well-understood mRNA modification at present is N⁶-Methyladenosine (m⁶A), which has been observed to occur an average of three times in every mRNA molecule.

Currently, work is focused on determining the types of and location of RNA modifications, determining if these modification have function, and if so, what is their mechanism of action. Similar to the epigenome, the epitranscriptome has "writers" and "erasers" that mark RNA and "readers" that translate those marks into function. One function that has been elucidated involves the enzyme adenosine deaminase (ADAR), which acts on RNA. ADAR affects a series of cellular processes, including alternative splicing, microRNAs, the innate immune system, and leads to protein recoding especially for important receptors in the central nervous system.

Chemical Modifications of RNA

N⁶-Methyladenosine (m⁶A)

m⁶A describes the methylation of the nitrogen at position 6 in the adenosine base within mRNA. Discovered in 1974, m⁶A is the most abundant eukaryotic mRNA modification; most mRNAs contain approximately three m⁶A residues. However, some mRNA transcripts do not contain any m⁶A at all, while others may have 10 or more. The term "epitranscriptome" was coined following transcriptome-wide mappings of m⁶A sites, but does not necessarily exclude other post-transcriptional mRNA modifications. How, and in response to what stimulus, the cell endogeneously regulates the level of m⁶A methylation remains unclear at present. However, it is known that the levels of this epitranscriptional mark are dynamically altered during embryonic development. Moreover, environmental stimuli such as stress can also alter the levels of m⁶A.

The m⁶A mRNA methylomes of different eukaryotic organisms have two common characteristics. First of all, the mark is usually found in the R > A⁶AC >A>C>or RRm⁶ACH sequence. Secondly, this mark is enriched in specific regions of the transcriptome; it is mostly found close to stop codons, in 3’-UTRs and in long internal exons. Nevertheless, m⁶A levels vary between different RNAs within a cell and between different cell types of the same organism. The mechanisms controlling the addition of m⁶A to some types of RNA have been described, but others remain unknown.

Writers, Readers, and Erasers

In eukaryotes, the use of m6A on mRNA involves a methyltransferase complex, commonly termed the 'Writer', that installs the methyl group. This m6A modification is recognized by special proteins known as 'Readers'. The number of readers varies across different organisms. Notably, in vertebrates, the presence of proteins categorized as 'Erasers' is suggested to facilitate the removal of m6A, which enables a dynamic regulation of m6A deposition on mRNAs.

The m⁶A mark is added by a m⁶A methyltransferase writer complex post-transcriptionally. This writer complex is composed of METTL3, METTL14, Wilms tumor 1-associated protein (WTAP), KIAA1429 and RBM15. METTL3 is the catalytic subunit, whereas METTL14 is involved in the stability of the complex and RNA recruitment. WTAP is also needed in aiding the recruitment of mRNA, whereas RBM15 and its paralog RBM15B are only involved in the recruitment of lncRNAs. The role RBM15 and RBM15B may have in recruiting other types of RNA to the methyltransferase complex remains unknown. The specific recognition sites of the writers are not known, but the minimal sequence required is 5’-Rm⁶AC-3’. METTL3 has been proposed to also be a "reader" of the m⁶A mark. This function is localized in the cytoplasm, where it promotes the recruitment of eIF3. Discovery of the METTL3 complex indicated that m6A installation might be a regulated process, which was pivotal for the advancement and interest in the field of epitranscriptomics.

Members of the YTH domain protein family act as "readers" of m⁶A. The study of these proteins has been key in understanding the functions and effects of mRNA methylation. It has been shown that three members of the human YTH domain family of proteins have higher binding affinities to methylated mRNA. The YTH protein YTHDF2 affects mRNA by directing methylated mRNA from the translational pool to mRNA decay sites. As a result, presence of m6A on mRNA is correlated with a shorter half-life than unmethylated mRNA.

So far, two "erasers" of the m⁶A mark have been identified. ALKBH5 is a demethylase found in mammals that removes the methyl group of m⁶A. The second one is the fat mass and obesity associated protein ( FTO), a demethylase that converts m⁶A back to adenosine. FTO preferentially demethylates the m⁶A found closer to the mRNA cap. This oxidative process has three steps and two intermediates: N⁶-hydroxymethyladenosine (hm⁶A) and N⁶-formyladenosine (f⁶A). FTO is most commonly found in nuclear speckles; however, in some species low levels of FTO can also be found in the cytoplasm. Dysfunctional FTO correlates with alterations in body weight and disease, while Alkbh5 knockout mice have impaired fertility. These two facts reflect how important the proper regulation of the m⁶A modification is for normal body function. Moreover, mutations in FTO can lead to developmental failures, brain atrophy and physiological disorders in adulthood.

Role in the life-cycle of mRNA

mRNA methylation is important throughout the entire life-cycle of the mRNA, starting with the alternative polyadenylation (APA) of some transcripts. m⁶A sites are often located in the last exon, mostly in the 3’ untranslated region (3'-UTR). The presence of m⁶A in the 3’-UTR promotes the use of the proximal APA site, resulting in a shorter 3’-UTR. Splicing of the pre-mRNA transcripts may be influenced by m⁶A, although this effect can vary across different biological systems. Furthermore, nuclear export of mature mRNAs depends on m⁶A; when the m⁶A "writers" are inhibited, there is a delay in the export of the mature mRNAs. However, normal nuclear export does not solely depend on m⁶A, other mRNA marks such as 5'-methylcytosine (m⁵C) are also involved.

The m⁶A mark has a notable effect on translational dynamics. There are various ways in which m⁶A is involved in translational efficiency. For instance, this modification modulates multiple steps in the process of tRNA incorporation. On the one hand, it slows down GTP hydrolysis by EF-Tu by 12-fold and the peptidyl transfer reaction by 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. Moreover, 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 a m⁶A-modified codon interacts with its cognate tRNA (the tRNA with the anticodon that is complementary to a particular codon), it acts more like a near-cognate codon interaction instead of the cognate codon interaction. This can be seen in 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. Overall, this m⁶A modification leads to a kinetic loss of a factor of 18. 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.

However, this mark can also increase translational efficiency. The m⁶A "reader" YTHDF1 induces the association of the modified mRNA with the ribosome. Furthermore, it also recruits the translation initiation factor eIF3 to the mRNA independently of METTL3. Additionally, eIF3 also acts as a "reader" of a m⁶A located in the 5’-UTR of the mRNA, which results in recruitment of the 40S translational preinitiation complex. This interaction is involved in cap-independent translation, which happens during the cellular response to heat shock stress.

m⁶A methylation also modulates mRNA stability. The "reader" YTHDF2 binds to m⁶A-containing mRNAs and decreases their stability by recruiting them to P-bodies, in a process called methylation-dependent mRNA decay. This process is needed to rapidly degrade pluripotency transcription factor transcripts, to enable the commitment of a pluripotent stem cell to a specific cell lineage. Reduced levels of m⁶A in mice embryos lead to embryonic lethality during the early stages of development.

Role of N⁶-Methyladenosine (m⁶A) in alternative splicing

Stem loop structures can sometimes be found in introns. m⁶A residues located in these stem-loops weaken base-pairing interactions within the stem, thus altering the structure of the mRNA. This phenomenon is known as m⁶A-Switch. The m⁶A mark has an important role in alternative splicing, since it increases the accessibility of hnRNPC to its binding site. The heterogeneous nuclear ribonucleoprotein C (hnRNPC) is a RNA-binding protein that complexes with both heterogeneous nuclear RNA (hnRNA) and pre-mRNA to participate in pre-mRNA processing. hnRNPC binds to a uridine-rich region in introns that can usually form stem-loops. The destabilization of the stem-loop exposes the hnRNPC binding site, which increases the accessibility of the protein to the region. Because hnRNPC must be bound to pre-mRNA in order to fulfill its function, increased accessibility means higher activity of hnRNPC. Therefore, m⁶A residues located in stem-loops of introns enhance the activity of hnRNPC, which results in increased alternative splicing. Evidence supporting this claim identified that decreased m⁶A levels in the transcriptome lead to significantly reduced hnRNPC binding.

m⁶A also has additional roles in alternative splicing by acting as the binding site for YTHDC1 (YTHDC1 binds to m⁶A residues located in alternative exons). YTHDC1 has a double role in alternative splicing. First of all, it recruits the serine and arginine-rich splicing factor 3 (SRSF3), which promotes exon inclusion. In addition, YTHDC1 blocks binding of SRSF10, a protein involved in exon-skipping.

Due to the role of m⁶A in alternative splicing, pre-mRNAs have higher levels of m⁶A than mature mRNAs. Moreover, m⁶A is more abundant in mRNAs that undergo alternative splicing compared to genes that code a single isoform. This is because alternatively spliced mRNAs are enriched in METTL3 binding sites. Splicing is affected in Mettl3 knock-out mice, resulting in increased frequency of exon skipping and intron retention. However, m⁶A is not a general unspecific splicing factor, it only participates in the alternative splicing of certain mRNAs and lncRNAs.

Other roles of m⁶A

m⁶A is not only found on mRNAs, various non-coding RNAs also contain this mark. For instance, XIST, the lncRNA that initiates X-inactivation, is enriched in m⁶A. These m⁶A are recognized and bound by the YTH domain protein YTHDC1. XIST mediated silencing of the X chromosome is negatively affected when XIST is not modified with m⁶A.

RNA molecules containing m⁶A are involved in UV-induced DNA damage repair mechanisms. When DNA is damaged, poly(A)+ transcripts containing numerous m⁶A residues accumulate in the region. This facilitates the accessibility of DNA-repairing proteins, such as DNA polymerase K, so that they can fulfil their function.

Disease

Alterations in the pathways leading to the addition of the removal of the m⁶A mark result in impaired gene expression and cellular function, which can lead to disease.

Normal m⁶A levels are altered in a number of cancers. Reduced m⁶A levels due to down regulation of METTL3 and/or METTL14 lead to the activation of a number of oncogenes, such as the gene encoding ADAM metallopeptidase domain 19 (ADAM19). Moreover, loss of m⁶A also results in the down regulation of tumor suppressors like cyclin-dependent kinase inhibitor 2A (CDKN2A) and breast cancer 2 (BRCA2). On the other hand, increased m⁶A levels inhibit tumor progression in certain types of cancer. In addition, single nucleotide polymorphisms (SNPs) on the gene encoding FTO have been associated with increased risk of breast and pancreatic cancer. Altered m⁶A levels also contribute to hypoxia-induced enrichment of breast cancer stem cells phenotype. All things considered, "writers" and "erasers" of the m⁶A mark may be good potential drug targets in cancer therapy.

Metabolic disorders are also affected by the m⁶A mark due to the role of FTO. Overexpression of FTO results in increased body and fat mass, whereas loss of FTO leads to a reduction in lean body mass. However, the mechanisms by which changes in FTO expression affect body and fat mass are not understood.

Current research of the m6a epitranscriptome is continuing to uncover the implications of m6a and its post-physiological effects on ischemic stroke incidents. Microglial-mediated responses and contributing demethylases, including FTO and ALKBH5, appear to be a contributing factor for alterations of the cerebral m6a epitranscriptome. Mood disorders, such as major depressive disorder, have also been identified as disease processes associated with m6a epitranscriptome changes.

N1-methyladenosine (m¹A)

N1-methyladenosine is a modified nucleoside in which a methyl group is added to N1 of the adenosine