The role of 5-methylcytosine in mammalian mRNA

The translation of genetic information stored in the DNA into proteins follows a complex series of events starting with the transcription of the DNA into messenger RNA, the correct processing of the messenger RNA (mRNA), the transport of the mRNA to the protein factories in the cell, the ribosomes, and finally, ending in the production of the protein according to the mRNA template. A multitude of regulatory mechanisms is required to ensure the perfect interplay between these events at any given time in the cell or in the life of an organism. One of these regulatory processes is the postsynthetic modification of the mRNA molecule. Thereby, the modification adds information regarding the subsequent fate of the RNA molecule, which is why it is frequently referred to as epitranscriptomic modification. The study of the epitranscriptome is an emerging field of research and although great progress has been made in recent years, there are many open questions. One of the aspects that remains underinvestigated is the functional significance of the observed methylation of cytosine bases in mRNA. With this research grant we will study the generation and function of cytosine methylation in mRNA. We will combine experiments in mammalian cells with in vitro analyses to examine the effect of m5C on several aspects of mRNA metabolism. With the experiments proposed here, we should be able to take a big step towards the elucidation of the molecular machineries involved in epitranscriptomic control. Such an understanding will not only contribute to expand our knowledge of fundamental cellular regulatory mechanisms at the level of RNA, it will have profound ramifications beyond basic research into a variety of research areas including human diseases whose etiology and pathology may involve as yet undiscovered epitranscriptomic aspects.

Our project set out to test how a small chemical mark on mRNA-methylation of the base cytosine, often called m5C-affects the journey from gene to protein. This journey involves making an mRNA copy from DNA, processing and packaging it, sending it to ribosomes, and translating it into protein. Because many control steps must align for this to work, cells add "postscript" signals to mRNA after it is made. These epitranscriptomic marks, such as m5C, are thought to guide the fate of each message, but their true impact has remained unclear. We focused on Nsun2, a main candidate enzyme that can place m5C on mRNA, and asked how it influences mRNA life cycles. One of the key findings of our studies was that Nsun2 controls mRNA turnover during differentiation. When we removed Nsun2, many mRNAs changed how quickly they were made and how long they lasted. Strikingly, the total, amounts of these mRNAs in the cell often changed very little. This shows robust buffering: when production went up, decay often sped up too, and vice versa, keeping overall levels near constant. Unexpectedly, the effects of Nsun2 do not require widespread m5C marks on mRNA. Using an improved method for methylation detection, we found very few m5C sites in the specific mRNAs whose turnover shifted in Nsun2's absence. This argues against a simple model in which Nsun2 marks those messages and directly dictates their fate through m5C. Instead, our data point to Nsun2 shaping mRNA turnover through other routes, likely involving translation, which is the process of protein production. Our study further uncovered that cell differentiation itself reprograms mRNA dynamics. Even without genetic changes, early steps of stem cell differentiation toward the neuronal lineage brought a broad slowdown in mRNA production and a broad increase in mRNA stability. Early in this process, changes in production appeared first; later, changes in stability dominated; eventually, both shifted together. Yet, again, steady-state mRNA levels stayed comparatively stable, reflecting widespread buffering across the transcriptome. Together, these results deliver a clear message: Nsun2 is a major regulator of mRNA turnover during a key cell fate transition, but its influence is largely independent of direct cytosine methylation on the affected mRNAs. Instead, Nsun2 likely acts through links to protein production, indirectly steering how messages are made and removed. The results advance the field's understanding of epitranscriptomic control and opens new paths to study human diseases where NSUN2 or related pathways are misregulated.