New Research from the Santos Lab: RNA-Level Control of Developmental Pausing

In times of stress, we all need a pause. Whether it's a last-minute report, a transit delay, or restless kids, taking a break from life's pressures is important. Just like us, developing cells also need breaks in times of stress – researchers have now uncovered how cells control such a paused state, and the implications are huge.

Researchers from the Santos Lab at the Lunenfeld–Tanenbaum Research Institute and the Department of Molecular Genetics at the University of Toronto have revealed new insights into the process of developmental pausing – a phenomenon seen in thousands of animal species.

The research was published in Nature Cell Biology this September (read here). Using mouse embryos and stem cells, they showed this dormant state is controlled at the RNA level by a highly interconnected process that restrains key molecules that would otherwise propel growth. Not only does this research increase our understanding of developmental biology but has broader implications for understanding cancer biology and treatment.

Developmental pausing, or diapause, is a stress-induced dormant state where embryonic growth is stopped until stressful conditions pass. You can think of hibernation, but for developing embryos. Despite its recognition, little is known about how cells regulate this state.

“There is a beautiful architecture to how cell put in place this dormancy,” said Dr. Miguel Ramalho-Santos, the principal investigator of the Santos Lab.

His team, led by Dr. Evelyne Collignon, aimed to explore RNA-level cell control of diapause.

“[Dr. Collignon] came into the lab with an interest in RNA biology… and our lab contributed our interest in developmental biology – the combination of those two interests became very fruitful,” said Dr. Ramalho-Santos describing the collaboration.

We often think that the DNA code controls all aspects of a cell’s activity, however that’s not always the case. RNA molecules are produced from DNA and are often precursors to functioning proteins, representing an interesting secondary point of management for the cell. They are regulated by the addition of tags, or chemical modifications, that tell them where to go, how to function, or when to be destroyed.

Here, researchers observed that an RNA modification, called m⁶A, was increased in paused cells. This modification is added by a protein called Mettl3.

After seeing that cells without Mettl3 continued to grow and divide while ‘paused’, the researchers concluded that the Mettl3-m⁶A tagging system was required to enter and maintain diapause.

Although an incredible finding on its own, the researchers decided to turn up the heat and take their study further. Specifically, the reversible nature of diapause intrigued them – what was being silenced to allow diapause and what could bring a cell back from diapause.

“The thought was, let’s look for molecules that need to be repressed in this dormant state but are important for the reawakening” explained Dr. Ramalho-Santos.

They coined these as ‘anti-pausing factors’ – molecules that are involved in growth and therefore need to be inactive during diapause.

This was a tricky line of investigation as their data projected over 465 potential ‘anti-pausing’ molecules. However, there was nothing that conclusively stood out. This meant it was time to bring in new data.

Bringing in outside data from mouse embryos, the team looked for molecules with an inverse relationship to the Mettl3-m⁶A tagging system. This revealed N-Myc as the anti-pausing factor.

Now, this was a significant finding. N-Myc is highly important for embryo development, amplifying the production of numerous molecules related to cell division and growth.

Researchers further found that N-Myc gets tagged by the Mettl3-m⁶A tagging system in diapause, marking it for decay. This disrupts the balance of molecules that N-Myc normally amplifies, leading to cell-wide reduction in active RNA molecules. They also found that if they forced removal of the m⁶A tag from N-Myc RNA, cells resumed growth even in paused conditions. This highlights that N-Myc is a notable ‘anti-pausing factor’ heavily involved in the process of developmental pausing.

Clearly, the molecular interactions that underlie diapause are complex to say the least, but what does this mean in a human context?

“What we learn in diapause, can apply to cancer research, to an extent, in the context of dormancy”, explained Dr. Collignon. Like diapause, cancer cells can enter dormant states to survive treatment. The similarities allow researchers to translate the molecular interactions of diapause to better understand cancer dormancy.

The research discussed here is a prime example.  Exposing the control mechanisms underlying diapause enables exploration into targeted cancer treatments.

Despite the remarkable findings outlined by the Santos Lab, there is much more complexity to uncover. Several questions remain regarding how the environment triggers diapause, how a cell may exit diapause, the control of other anti-pausing factors, and what is applicable to human medicine – all important questions begging to be answered.

“As usual when you peel back layers you start to find other things to unearth” said Dr. Ramalho-Santos and we cannot wait to see what comes next.

Collignon, E., Cho, B., Furlan, G. et al. m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency. Nat Cell Biol 25, 1279–1289 (2023). https://doi.org/10.1038/s41556-023-01212-x