Introduction to mRNA nucleotides

Messenger RNA (mRNA) is the intermediate between DNA and proteins, transferring the information obtained from a DNA template in the nucleus to the ribosome in the cytoplasm. It acts as a template for translating nucleotide sequences into amino acid chains, which are linked together to form functional proteins. Amino acid synthesis does not occur randomly during translation; instead, the specific mRNA nucleotide sequence informs it. Each amino acid corresponds to a three-nucleotide-long sequence called a codon. The nucleotides in mRNA and their sequences influence the structure and function of proteins that govern essential cellular processes.

Structure and composition of mRNA nucleotides

Composition of mRNA

mRNA is a single-stranded nucleic acid composed of nucleotides with one of the four nitrogenous bases, adenine (A), cytosine (C), guanine (G) and uracil (U). A, G and C are common to both the DNA and the mRNA, while DNA contains thymine (T) instead of U. It possesses a flexible structure able to exit the nucleus and interact with the translational machinery in the ribosome.

Components of each nucleotide

Each mRNA nucleotide contains the following:

1. Nitrogenous bases: One of the four bases (A, C, G or U) that carry genetic information
2. Ribose sugar: The five-carbon sugar in mRNA. A nucleoside combines a nitrogenous base, ribose sugar and the connecting glycosidic link.
3. Phosphate group: The nucleotides of an mRNA are linked together via phosphodiester bonds, forming the mRNA backbone.

Transcription and mRNA synthesis

Transcription is the process of generating a preliminary single-stranded RNA structure from DNA.

Role of RNA polymerase in mRNA synthesis

RNA polymerase is an enzyme that facilitates mRNA synthesis. It recognizes a specific DNA site called the promoter and initiates transcription by separating the strands. It uses one strand as a template to add RNA nucleotides in the 5' to 3' direction. The elongation of the mRNA sequence continues until the RNA polymerase encounters a termination signal. An mRNA sequence is terminated with a poly(A) tail, a set of adenine nucleotides added to the 3' end. The poly(A) tail in mRNA ensures that the mRNA is protected from degradation by exonucleases and exported into the cytoplasm.

Complementary base pairing during transcription

Complementary base pairing rules determine the precise nucleotide sequence in mRNA. Accordingly, the DNA strand-mRNA strand pairs are as follows:

1. A in DNA: U in RNA
2. T in DNA: A in RNA
3. C in DNA: G in RNA
4. G in DNA: C in RNA

These rules are crucial for ensuring that the mRNA contains the precise genetic information from the DNA. More specifically, an mRNA strand is complementary to the DNA template strand, which is complementary to the coding DNA strand not used for transcription. In other words, the final mRNA sequence corresponds to the coding DNA strand, except that it contains U instead of T.

Modified nucleotides and epitranscriptomic changes

Definition of modified nucleotides

Modified nucleotides are RNA bases that are chemically altered after transcription. Although these modifications do not change the essence of genetic information, they refine mRNA capping efficiency, stability, splicing, nuclear export and translational efficiency.

Role of modified nucleotides in mRNA function and stability

Enhanced mRNA molecules are modified to improve stability, facilitate nuclear export, and optimize interactions with RNA-binding proteins and ribosomes. These modifications not only extend mRNA lifespan but also help regulate gene expression to ensure homeostasis.

Epitranscriptomics: chemical modifications of mRNA

The posttranscriptional chemical changes in RNA are similar to the epigenetic changes in DNA in that they alter the final mRNA structure and function without changing the genetic code embedded in it. These changes are the focus of the research field called epitranscriptomics.

Common examples of epitranscriptomic changes include:

1. N6-methyladenosine (m6A) is a modified adenosine with a methyl group added to the nitrogen at position 6
2. Pseudouridine (Ψ) is a uridine isomer, where the uracil is attached to the ribose sugar via a carbon-carbon bond instead of a nitrogen bond
3. 5-methylcytidine is a modified cytidine with a methyl group added to its 5th carbon
4. N1-methyladenosine (m1A) involves adenosine methylation at the N1 position
5. 2'-O-Methylation (Nm) is formed when a methyl group is added to the 2'-hydroxyl group of the sugar

These modifications often improve mRNA stability to protect it from immune response and degradation. They enhance translation efficiency by promoting mRNA interaction with the ribosomal translation machinery. As such, they have significant roles in embryogenesis and organ development.¹ For instance, m6A modification is vital for modulating mRNA translation during early development, which helps control the balance between stem cell self-renewal and differentiation.² In other words, this modification instructs cells to divide and differentiate precisely during tissue development. On the other hand, dysregulated epitranscriptomic changes drive tumor development. The m6A modification that was essential in tissue development is also associated with several cancers, including acute myeloid leukemia (AML), lung cancer and glioblastoma.³ More specifically, the overexpression of the enzyme METTL3, which facilitates m6A-type methylation, can increase the frequency of this modification on oncogenes, leading to their stable and persistent expression.⁴

Applications in mRNA-based medicine

Besides their biological functions, these modifications are applied individually or in combination to in vitro-transcribed (IVT) mRNA to optimize the activity of mRNA-based medicine.

mRNA-based vaccines

Vaccine development is one of the most prominent uses of synthetic mRNA. An mRNA-based vaccine is designed to encode a protein, which the immune system recognizes and develops a specific immune response by producing antibodies. Thus, the vaccine product instructs the immune system to recognize and attack the same protein when the host is infected with an actual pathogen. Modified mRNA nucleotides play a central role in successfully administering the vaccine. They improve mRNA stability so that the vaccine construct stays long enough in the body to induce antigen production without prematurely being recognized as foreign RNA and degraded. Their significance is evident in the development of COVID-19 vaccines. The mRNA construct was improved with a modified nucleotide called N1-methylpseudouridine (m1Ψ), which involved the methylation of pseudouridine at its N1 position.⁵ As a result, the vaccine's durability and translation efficiency increased, while the risk of immunogenicity was minimized.

Conclusion

mRNA nucleotides are the building blocks of gene expression that carry the genetic information necessary for protein synthesis. The mRNA nucleotide sequence ensures error-free protein synthesis by informing the precise amino acid sequence during translation. Furthermore, mRNA nucleotides can possess chemical modifications, either naturally or synthetically, that impact mRNA activity. Such modifications contributed to the clinical success of vaccines during the COVID-19 pandemic. With further research on mRNA nucleotide modifications, more effective mRNA-based vaccines and therapeutics can be developed to treat and prevent infectious diseases, cancer and genetic disorders.

References

1. Seo KW, Kleiner RE. Mechanisms of epitranscriptomic gene regulation. Biopolymers 2021;112(1):e23403.
2. Zhang M, Zhai Y, Zhang S, Dai X, Li Z. Roles of N6-Methyladenosine (m6A) in stem cell fate decisions and early embryonic development in mammals. Front Cell Devel Biol 2020;8:782.
3. Deng X, Qing Y, Horne D, Huang H, Chen J. The roles and implications of RNA m6A modification in cancer. Nat Rev Clin Oncol 2023;20(8):507-526.
4. Wei X, Huo Y, Pi J, Gao Y, Rao S, He M, et al. METTL3 preferentially enhances non-m6A translation of epigenetic factors and promotes tumourigenesis. Nat Cell Biol 2022;24(8):1278-1290.
5. Xia X. Detailed dissection and critical evaluation of the Pfizer/BioNTech and Moderna mRNA vaccines. Vaccines 2021;9(7):734.

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