Researchers from the Institute for Molecules and Materials (IMM) of Radboud University have reported significant findings about the role of poly(A) tail length in messenger RNAs (mRNAs) and its impact on regulating the amplitude and frequency of protein fluctuations. Understanding how specific mRNA sequences, such as the poly(A) tail, can influence these fluctuations is crucial for comprehending diseases that involve alterations in poly(A) tails, but also for improving mRNA-based therapies and vaccinations. The findings have been published in the journal Cell Systems.
mRNA
mRNAs are vital to cellular functions, translating genetic information from DNA into proteins. To properly functions, cells have developed ways to tightly regulate for how long mRNAs can be present in the cells before being degraded, but also to ensure the right amount of protein is produced from each mRNA molecule, in a process called translation. A crucial feature of mRNAs that is able to regulate both their degradation and translation is the poly(A) tail, a sequence of repeated adenosine molecules at the end of the mRNA trail, which varies in length. However, until now the relationship between poly(A)-tail length and their degradation and translation was unclear. This is because on endogenous mRNAs there are many other regulatory sequences that similarly to the poly(A)-tail can regulate their function. Therefore, trying to study the effect of poly(A)-tail length on mRNA regulation on endogenous genes is complicated.
Poly(A) tail length
The study aimed to understand how different lengths of the poly(A) tail influence mRNA stability and protein production. The research team synthesized identical mRNAs with varying poly(A) tail lengths and transfected them into cells. Once in the cells, these mRNAs produce fluorescent markers that allow to track protein production, revealing that intermediate poly(A) tail lengths produced the most proteins, while mRNA degradation rate correlated directly with tail length. These results suggest that mRNA translation and degradation are decoupled for mRNAs that differ only in their poly(A)-tail length. This means that changes in protein production due to changes in the poly(A)-tail length do not directly influence mRNA degradation. “We were very surprised to discover that intermediate tail lengths are associated with the highest protein production, because until now research has focused on trying to find a correlation between the two, expecting the longest or shortest tails to have the highest translation rates. Sometimes we just need to look at things from a different perspective, researcher Carmen Grandi says.
These findings could revolutionize how we understand mRNA behavior in cells. The researchers also described how identical mRNAs are often not present in cells with a single poly(A)-tail length, but with a distribution of lengths that can be more or less variable. With their synthetic mRNAs they were able to reproduce this variability and describe its effect on protein production. How this variability in poly(A) tail length affects protein production kinetics has never been taken into account by other studies, and it therefore complicates studies on endogenous mRNAs, suggesting a need for more advanced techniques to study these variations.
Challenges
Recreating endogenous behaviors using synthetic molecules is not easy. The research faced initial challenges in synthesizing mRNAs with specific poly(A) tail lengths, but overcame the initial problems when the team found a simpler, more efficient method to synthesize the molecules. Additional challenges raised when it was time to transfect these molecules into cells. Cells easily die when imaged for many hours, and therefore maintaining cell viability during live cell imaging also caused difficulties, but the team successfully optimized the process to obtain reliable results.
Advancements in understanding mRNA regulation
The fundamental results provide a better understanding of how poly(A) tail regulates mRNAs, and could potentially advance mRNA-based therapies and vaccinations. For example, optimizing the poly(A) tail length in mRNA vaccines could ensure better protein production once the mRNA is introduced into the body, improving vaccine efficacy. The current study has laid groundwork for future research in mRNA behavior and its applications in medical science.
Biophysical Chemistry
The studies were conducted in the team of dr. Maike Hansen, Carmen Grandi is PhD Candidate in this group. The group is part of the Biophysical Chemistry department, which is embedded in IMM. The Biophysical Chemistry group focuses on the study of gene expression dynamics by employing techniques at the interface of computational modeling, cell-free biochemistry, and quantitative single-cell biology. They aim to identify design principles that allow for robust outcomes in noisy crowded systems.
