N6-methyladenosine, known as m6A, represents the most abundant chemical modification found on messenger RNA (mRNA) within higher organisms. This modification acts like a form of punctuation on a genetic message, guiding how the cell interprets or handles the RNA molecule. The study of these RNA modifications falls under the field of “epitranscriptomics,” which parallels the more widely recognized “epigenetics” but focuses on RNA rather than DNA. Just as epigenetic changes alter gene expression without changing the DNA sequence, epitranscriptomic modifications like m6A dynamically influence gene expression without altering the RNA sequence itself.
The Key Players in m6A Modification
The m6A modification is a dynamic and reversible process, governed by a sophisticated interplay of three main classes of proteins commonly referred to as “writers,” “erasers,” and “readers.”
Writers
The “writers” are enzymes responsible for adding the methyl group to specific adenosine bases on RNA. The primary m6A methyltransferase complex in humans consists of METTL3, METTL14, and Wilms tumor 1-associated protein (WTAP). METTL3 serves as the catalytic subunit, directly adding the methyl group, while METTL14 contributes to the complex’s stability and helps in recruiting RNA targets. This complex recognizes a specific RNA sequence motif, 5′-RRACH-3′ (where R is a purine, and H is A, C, or U), to deposit the m6A mark.
Erasers
Counteracting the writers are the “erasers,” which are enzymes that remove the m6A mark, making the modification reversible. Two well-characterized m6A demethylases are FTO (fat mass and obesity-associated protein) and ALKBH5 (alkylation repair homolog protein 5). FTO removes the methyl group through an oxidative process, converting it back to adenosine. In contrast, ALKBH5 directly removes the methyl group from m6A without detectable intermediates, quickly releasing adenosine and formaldehyde.
Readers
Once the m6A mark is present, “reader” proteins recognize and bind to it, translating the modification into specific cellular actions. The YTH-domain family of proteins, including YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2, are prominent m6A readers in mammals. Each reader protein can elicit different downstream effects depending on its cellular localization and interactions with other cellular machinery. For instance, YTHDC1 primarily functions in the nucleus, influencing processes like RNA splicing, while YTHDF proteins are mainly cytoplasmic and impact mRNA stability and translation.
How m6A Controls Gene Expression
After a “reader” protein identifies an m6A mark on an RNA molecule, a cascade of events can occur, profoundly influencing the life cycle of messenger RNA and, consequently, gene expression.
RNA Stability
One significant way m6A controls gene expression is by affecting RNA stability. The m6A mark can act as a signal that targets mRNA for degradation, effectively reducing the amount of protein produced from that gene. For example, the reader protein YTHDF2 often directs m6A-modified mRNA from the active translational pool to decay sites, leading to a shorter half-life for the modified mRNA. Alternatively, m6A can also contribute to mRNA stabilization, particularly through interactions with non-canonical readers like IGF2BP proteins, which can protect transcripts from degradation.
RNA Splicing
The m6A modification also influences RNA splicing, the process where non-coding introns are removed and coding exons are joined to form the final mRNA message. M6A can be found in introns and exons, impacting how the pre-mRNA is pieced together. For example, the nuclear reader protein YTHDC1 can bind to m6A on pre-mRNAs and influence alternative splicing patterns by recruiting specific splicing factors like SRSF3, promoting the inclusion of certain exons while restricting others.
Translation Efficiency
Beyond stability and splicing, m6A can significantly impact translation efficiency, which is the rate at which mRNA is converted into protein. The modification can enhance the process of translating mRNA into a protein. Reader proteins like YTHDF1 and YTHDF3 can promote the association of m6A-modified mRNA with ribosomes, thereby increasing protein production. Additionally, m6A in the 5′-untranslated region (5′-UTR) can directly recruit the eukaryotic initiation factor 3 (eIF3), facilitating cap-independent translation, especially during cellular stress responses.
Connection to Human Disease
When the sophisticated regulation of m6A modification goes awry, it can contribute to the development and progression of various human diseases. Dysregulation of the “writers,” “erasers,” or “readers” of m6A can lead to abnormal gene expression, impacting cellular functions and contributing to pathological states.
Cancer
In the context of cancer, aberrant levels or activities of m6A regulatory proteins are frequently observed. For instance, an imbalance, such as reduced m6A levels due to decreased METTL3 or METTL14 expression, can lead to the activation of oncogenes or downregulation of tumor suppressors, promoting uncontrolled cell growth. Conversely, increased expression of demethylases like FTO or ALKBH5 can also contribute to cancer progression by removing m6A from specific transcripts, altering their stability or translation and favoring malignant phenotypes.
Neurological Disorders
M6A modification also plays a role in neurological disorders, given its involvement in proper brain development and function. Alterations in m6A levels or the function of its regulatory proteins have been linked to developmental and neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. For example, reduced m6A modification in hippocampal neurons, often due to METTL3 knockdown, has been associated with memory impairments, synaptic loss, and neuronal cell death in Alzheimer’s disease models.
Immune Response
Beyond cancer and neurological conditions, m6A modification is also recognized as a regulator of the immune response. It plays a role in regulating immune cell function and the body’s response to viral infections. M6A can influence the recognition of viral RNA by host immune sensors, modulate cytokine production, and affect the maturation and activation of immune cells like dendritic cells. Dysregulation of m6A in immune cells can impair their ability to respond effectively to pathogens, highlighting the broad importance of this modification in maintaining health.