The epitranscriptome is the collection of biochemical modifications of the RNA transcripts present in a cell. These modifications allow functional changes to the transcript but do not involve a change in the ribonucleotide sequence. There are various types of modifications and studying the epitranscriptome has been important to drug discovery as well as disease identification.
Image Credit: https://www.nature.com/articles/nmeth.4110
Type of modifications
RNA modifications are carried out by a group of enzymes known as “writer”, “eraser”, and “reader”. “Writers” are a group of methylases that add methyl groups to the RNA; “erasers” demethylate RNAs and “readers” are proteins that recognize and bind to methylated RNAs.
There are different types of RNA modifications that impact gene expression. These include methylation of adenosines and cytosines (N6-Methyladenosine (m6A), N1-Methyladenosine (m1A), and 5-methylcytosine (m5C)), conversion of adenosine to inosine, modification of guanosine to queuine, and pseudouridylation (an isomer of the nucleotide uridine).
Methylation of RNA is the most common type of modification. Adding a methyl group at different positions on the nucleobase has distinct implications. m6A describes the methylation of the nitrogen at position 6 in the adenosine base; it destabilizes pairing with uracil through steric hindrance. m6A is commonly found close to stop codons, in 3’-UTRs, and in long exons.
This suggests a role in alternative splicing and slowing down translation. In m1A, the methyl group protrudes from the Watson–Crick hydrogen-bonding face of adenine, making the nucleotide unpaired. m5C is usually found downstream of translation initiation sites, and thus, it is proposed to play a role in the control of translation.
On the other hand, the conversion of RNA nucleotides to non-Watson-Crick nucleotides can cause changes in gene expression by changing the RNA structure.
How to detect the epitranscriptome
Primary detection of the epitranscriptome can be carried out with dot-blot and high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). This allows quantification of various RNA modifications but does not provide information for widespread identification.
With high throughput sequencing combined with antibodies against a specific type of methylation, the location and abundance of RNA modification can be deciphered accurately.
Since methylases and demethylases are enzymes, they can be inhibited and identified as a drug target. To discover drugs, the correlation between specific epitranscriptome and onset of diseases has to be identified.
For example, it was discovered that an enzyme called methyltransferase-like 3 (METTL3) methylates RNA, changing adenosines into m6A. It was then found that mouse and human embryos missing the METTL3 gene died before birth because their embryonic stem cells never went into differentiation without METTL3 methylating adenosines in the epitranscriptome.
Furthermore, METTL3 also plays a dual role in cancer, it can either inhibit or promote cancer proliferation depending on the specific type of cancer. For example, in leukemia, increased METTL3 levels enhanced the production of proteins linked to cancer. This makes it a good drug target for cancer therapy.
Moreover, tissue samples taken from acute myeloid leukemia patients revealed high levels of the enzyme fat mass and obesity-associated protein (FTO), which is an m6A eraser. This proposes a new treatment method by discovering drugs that inhibit FTO.
Apart from m6A writers and erasers, m6A readers are important for drug discovery as well. An m6A reader called YTHDF1 controls the immune system and inhibiting it may improve the efficacy of existing cancer immunotherapies such as checkpoint inhibitors. This provides an opportunity to discover m6A reader drugs that work in synergy with immunotherapies.
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