Translation-Coupled mRNA Decay: The Role of m6A Modifications in Fine-Tuning Gene Expression

Significance 

The complexity of cellular regulation depends heavily on the delicate control of messenger RNA (mRNA) stability, a process central to determining protein levels and ensuring precise gene expression. Among the various layers of post-transcriptional regulation, chemical modifications on RNA molecules, collectively referred to as the epitranscriptome, play pivotal roles. One such modification, N6-methyladenosine (m6A), has emerged as the most prevalent internal modification in eukaryotic mRNAs. This dynamic mark influences nearly every aspect of RNA metabolism, including splicing, export, translation, and decay. Despite substantial progress in understanding m6A’s functional significance, many questions remain unanswered, particularly regarding how its specific placement within an mRNA molecule dictates its regulatory impact. Until recently, most research has focused on m6A modifications located in untranslated regions (UTRs), especially the 3′ UTR, where they are known to recruit decay factors and destabilize mRNAs. However, coding sequence (CDS)-localized m6A modifications have received comparatively little attention. This knowledge gap limits our understanding of how these modifications contribute to transcript stability and the broader landscape of RNA regulation. The challenges in deciphering m6A’s functional complexity are numerous. The m6A methylation machinery, led by methyltransferase complexes like METTL3 and METTL14, exhibits sequence specificity but can also be modulated by RNA structure and interacting proteins, complicating predictions of m6A sites. Furthermore, the consequences of m6A modifications can vary significantly depending on their location within the transcript. For example, while UTR m6A marks are strongly associated with translation-independent decay pathways, the role of m6A in coding regions and its potential connection to translation remains largely unexplored. Recognizing these challenges, new research paper published in Molecular Cell Journal and led by Drs. Kathi Zarnack & Julian König from the University of Wuerzburg in Germany addressed a critical gap in understanding: how m6A sites within the coding sequence influence mRNA stability. The team hypothesized that these sites might engage unique regulatory mechanisms distinct from those active in untranslated regions. This hypothesis was driven by emerging evidence suggesting that m6A in the CDS may intersect with translation machinery, potentially linking ribosome dynamics to transcript turnover.

Their study aimed to clarify the mechanistic basis of CDS-specific m6A effects, using a combination of high-resolution mapping, translation profiling, and targeted molecular assays. The researchers also sought to determine whether CDS-m6A modifications might regulate specific classes of genes, such as developmental regulators and retrogenes, which are critical for cellular function and organismal development.

To uncover the role of m6A modifications in CDS, the researchers began with mapping m6A modifications across mRNAs in human cells using a technique called miCLIP, which provides nucleotide-resolution data on where m6A modifications occur. The researchers discovered that m6A sites within the CDS were highly enriched and particularly associated with sequences where ribosome dynamics are critical during translation. This mapping hinted at a functional role for CDS m6A modifications distinct from those in untranslated regions. To explore how these modifications influence RNA stability, the team used RNA sequencing in cells treated with a METTL3 inhibitor to suppress m6A deposition. They observed that transcripts with m6A in the CDS were rapidly stabilized when m6A was depleted, much faster than those with m6A in untranslated regions. This finding suggested that m6A modifications in coding regions act as powerful signals for transcript destabilization. Interestingly, the effect was specific to transcripts actively engaged in translation, implying that ribosomes might play a central role in this degradation process. To probe this connection further, the researchers performed ribosome profiling, a technique that captures the positions of ribosomes on mRNAs. They found that ribosomes tended to pause at codons containing m6A modifications, particularly when the modified nucleotide was located in the ribosomal A site. This pausing was shown to trigger a decay pathway, now termed CDS-m6A decay (CMD). Using ribosome footprint data, they also demonstrated that this effect was strongest when m6A was present on specific positions within the codon, emphasizing a direct interaction between the ribosome and the m6A modification. The authors next investigated how CDS-m6A modifications were linked to cellular RNA decay machinery. They showed that transcripts with CDS m6A sites were translocated to processing bodies (P-bodies), cytoplasmic structures associated with RNA degradation. By using immunofluorescence microscopy, the researchers visualized this translocation and demonstrated that it was diminished in cells where m6A deposition was inhibited. These findings revealed that P-bodies are essential mediators of CMD, offering a new dimension to how cells regulate transcript turnover. Central to this process was the m6A-binding protein YTHDF2, which the authors identified as a key player in recognizing m6A sites in the CDS. Using knockdown experiments, they demonstrated that reducing YTHDF2 levels significantly stabilized transcripts with CDS m6A modifications. Moreover, they used proximity proteomics to show that YTHDF2 interacts with RNA decay machinery localized to P-bodies, further cementing its role in facilitating CMD. To validate these observations, the team designed reporter constructs that mimicked natural mRNAs with CDS m6A sites. These constructs carried a fluorescent protein-coding sequence, either with or without m6A-modifiable sites. When these reporters were introduced into cells, those with m6A-modified codons exhibited rapid decay compared to their unmodified counterparts. This effect was lost when translation was inhibited, confirming the critical role of ribosome activity in the CMD pathway. One of the most compelling findings came from investigating the specific genes targeted by CMD. By analyzing global transcriptomic changes, the researchers found that CDS m6A modifications primarily regulate transcripts encoding developmental regulators and retrogenes. These are genes that need to be tightly controlled during critical stages of development or in response to cellular stress. This specificity highlights CMD as a mechanism tailored for fine-tuning the expression of genes with high physiological importance.

 In conclusion, the research work by Drs. Kathi Zarnack & Julian König and their team represents a paradigm shift in understanding the intricate mechanisms that govern mRNA stability and gene regulation. By identifying the CDS-m6A decay (CMD) pathway, the research highlights an entirely novel function for m6A modifications in coding sequences, separate from previously known roles in untranslated regions. The findings emphasize that the precise location of m6A modifications within mRNAs is not merely a structural feature but a critical determinant of transcript fate. This has profound implications for the study of RNA biology and its intersection with translational control. The study’s discovery that ribosomes pause at m6A-modified codons provides a new perspective on how translation and RNA decay are interconnected. It reveals a mechanism by which cells can tightly regulate protein production in real time, ensuring a swift response to environmental or developmental cues. This mechanism enables a more efficient way of controlling mRNA levels, particularly for transcripts that encode key developmental regulators or genes involved in stress responses. Such precise modulation minimizes energy expenditure while maximizing regulatory potential, a principle fundamental to cellular homeostasis. Another significant implication lies in the CMD pathway’s preference for targeting retrogenes and developmental regulators. These findings suggest that CMD is not merely a passive decay process but a tailored mechanism for refining gene expression. By rapidly degrading specific transcripts, CMD safeguards against aberrant protein accumulation and ensures the fine-tuning of developmental processes, which are often sensitive to even small perturbations in gene expression. The study also opens doors to understanding the role of m6A dysregulation in diseases such as cancer, neurodegenerative disorders, and developmental abnormalities. Aberrant m6A modification or misregulation of CMD could lead to the stabilization of harmful transcripts or the inappropriate degradation of essential ones, contributing to disease pathology. These insights provide a new framework for exploring m6A-targeted therapies that aim to restore proper RNA turnover, especially in conditions where translation-dependent decay is disrupted. From a broader perspective, the research underscores the importance of integrating epitranscriptomic studies with translation and RNA stability analyses. It highlights the dynamic interplay between transcriptional output, translational machinery, and RNA decay, suggesting that gene expression control is far more adaptable and complex than previously appreciated. This understanding could inform the development of novel biotechnological tools and precision medicines, particularly in areas like regenerative medicine and cancer therapy, where fine-tuning gene expression is critical.

Translation-Coupled mRNA Decay: The Role of m6A Modifications in Fine-Tuning Gene Expression - Medicine Innovates
Image credit: Mol Cell. 2024 Dec 5;84(23):4576-4593.e12. doi: 10.1016/j.molcel.2024.10.033.

About the author

Dr. Julian König

Group Leader, Institute of Molecular Biology (IMB), Mainz

Our research programme aims to decipher the molecular mechanisms behind RNA modifications and regulation, as well as their link to ageing and age-related diseases. Our interdisciplinary team employs functional genomics techniques to decode regulatory elements in RNA sequences and has developed high-throughput approaches such as iCLIP and miCLIP2 to map protein-RNA interactions and detect m6A RNA modifications. We seek to unravel the fundamental biological processes that control RNA regulation and the perturbations that lead to age-related diseases such as neurodegeneration and cancer.

Our past achievements include discovering that RNA regulation is extensively modulated by the interactions between RNA-binding proteins. We generated the first maps of the RNA regulatory elements that control specific splicing decisions and decoded their actions in CART-19 therapy resistance in leukaemia. We also found that the RNA-binding protein Makorin acts as a sensor for faulty RNAs, which could be a mechanism contributing to robustness during ageing and in age-related diseases. Recently, we discovered that m6A RNA modifications mediate X-to-autosome dosage compensation in mammals, a mechanism which could balance sex-specific differences during ageing and in disease.

Currently, we are focused on understanding the mechanisms of splicing regulation in ageing and disease, dissecting the roles of m6A RNA modifications in gene expression and dosage compensation, and investigating mechanisms of RNA quality control. Our ultimate goal is to contribute significantly to decoding the molecular principles that govern RNA regulation in human physiology and age-related diseases and guide the development of specific treatments.

About the author

Dr. Kathi Zarnack

Group Leader

Buchmann Institute for Molecular Life Sciences (BMLS) & Institute of Molecular Biosciences, Goethe University Frankfurt, Germany

RNA-binding proteins (RBPs) are critical players in the posttranscriptional control of gene expression. Their functional impact ranges from the alternative splicing and polyadenylation of pre-mRNAs in the nucleus to the subcellular localisation and translation of mRNAs in the cytoplasm. Numerous diseases are characterised by dysregulated RBPs and massively altered transcriptome patterns, particularly prominent in neurodegeneration and cancer. In the era of functional genomics and high-throughput sequencing, we are now able to address these processes at an unprecedented resolution and scale.

Bioinformatics and machine learning open new routes to dissect the complexity of RBP function and posttranscriptional regulation in human physiology and disease. A main pillar of our work is the computational integration of multi-omics data to resolve the regulatory principles of RBP function in alternative splicing, translation and RNA localisation, with a particular interest in clinically relevant scenarios. To achieve this, we constantly expand our toolbox that can be applied to a wide range of questions in RNA biology. For details on projects, collaborators and funding, please refer to the following Projects.

Reference 

Zhou Y, Ćorović M, Hoch-Kraft P, Meiser N, Mesitov M, Körtel N, Back H, Naarmann-de Vries IS, Katti K, Obrdlík A, Busch A, Dieterich C, Vaňáčová Š, Hengesbach M, Zarnack K, König J. m6A sites in the coding region trigger translation-dependent mRNA decay. Mol Cell. 2024 Dec 5;84(23):4576-4593.e12. doi: 10.1016/j.molcel.2024.10.033.

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