A new tool using genetic code expansion to study circadian rhythms

Controlling the body clock with an expanded genetic code
Controlling the body clock with an expanded genetic code

Circadian rhythms dominate our lives through our daily cycle of sleep and wakefulness. These rhythms are controlled by a master clock in the brain: the suprachiasmatic nucleus (SCN). Studying neuronal cell biology and how the SCN drives behaviour in humans and all animals has been made easier by the development of tools that allow rapid, reversible, and conditional control of these systems. A collaboration between researchers in Michael Hastings’ group in the LMB’s Neurobiology Division and Jason Chin’s group in the LMB’s PNAC Division has resulted in a new technique to manipulate circadian behaviour using genetic code expansion.

Genetic code expansion techniques allow scientists to teach cells to read their genetic code in new ways that mean that non-canonical amino acids – compounds that are not normally used by our bodies – can be incorporated into new proteins. Production of proteins requires transfer RNA (tRNA) molecules to bring individual amino acids to the growing protein chain. Introduction of non-canonical amino acids requires the use of a specific new tRNA. This tRNA has been engineered to recognise a particular sequence of the nucleotide letters that usually functions as a full stop to end production of the protein, but in the presence of the non-canonical amino acid allows protein production to continue. This process is termed translational switching.

Timekeeping in the SCN pivots around the function of Cryptochrome (Cry) proteins, such that loss of these proteins results in the clock not working. Cry1 is one of these proteins required for maintaining circadian rhythms. Liz Maywood, a researcher in Michael Hastings’ group, initially used small isolated slices of mouse brains containing the SCN to show that tissue in which Cry proteins had been lost and circadian rhythms were not functioning, could have the rhythms reinstated with addition of the non-canonical amino acid. This was achieved by also inserting a version of the Cry1 gene that contained a premature full stop, so that a functional protein could not normally be produced, but the presence of the new tRNA and special non-canonical amino acid would allow production of a full-length, functional Cry1 protein.

The researchers then went on to show that the same could be done in live mice; addition of the non-canonical amino acid to the drinking water allowed production of a full-length, functional Cry1 protein and rapidly initiated circadian behaviours in mice that were normally arrhythmic as they produce no Cry proteins. This study showed that producing just Cry1 only in the SCN is sufficient to drive circadian behaviour in these mice. Importantly, because the mice are not able to produce the non-canonical amino acid themselves, this system is reversible and can be completely and simply controlled by addition or removal of the non-canonical amino acid to or from the drinking water. Beyond studying circadian rhythms, these techniques could be useful for the study of many different functions within mammalian brains through the reversible control of production of key proteins that regulate different systems and behaviours.

This work was funded by the MRC.

Further references:

Paper in PNAS
Michael Hastings’ group page
Jason Chin’s group page
PNAS Commentary: Regulating behaviour with the flip of a translational switch