Epitranscriptomics

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. 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. 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 RNA methylomes of different eukaryotic organisms have two common characteristics. First of all, the mark is usually found in the Rm6AC 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. The terms, “writer”, “eraser” and “reader” have been associated with RNA modification. An 'Eraser' is a category of enzymes that demethylate m6A. Proteins that recognize and bind to m6A are known as 'readers'. However, some of the mRNA modifications prevent the binding of some RNA binding proteins; these are called 'anti-readers'. The 'writers' and 'erasers' of the m6A mark are mostly located in nuclear speckles (subnuclear structures enriched in pre-mRNA splicing factors), where mRNA is processed and stored. The m6A mark is added by a m6A methyltransferase 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 proved that m6A is a reversible mark, and this fact was crucial for the development of 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 methylations. 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, methylated mRNA has 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.