Stop does not always mean stop

The genetic code is believed to be strongly conserved through evolution – from the earliest bacteria until today. Now Estienne Swart et al. from the group of Mariusz Nowacki, Institute of Cell Biology of the University of Bern, have found two ciliate species where nature seems to be in the midst of experimenting with the meaning of the stop codon.

We all know language is ambiguous. One word can have several meanings, and different words can sound the same. Still communication between humans is not a perfect mess – we have found ways to determine the meaning of ambiguous words reliably, by interpreting context. What is more, language is a fluent, ever changing system – words can shift their meaning, human languages are always evolving.

For the language of biology this does not seem to be the case - experts usually refer to it as 'frozen'. The genetic code is exceptionally robust: since it has been developed some maybe 4 billion years ago, it does not seem to have undergone any evolutionary changes. Each codon stands for a specific amino acid (or for ‘stop’) - the biological ‘words’ have very clear meanings. There are no ambiguities.

At least that is how it is written in the biological textbooks. But it is a common fate of textbook dogmas: sooner or later they are proven wrong. Now researchers from the Institute of Cell Biology of the University of Bern have for the first time found codons with multiple meanings.

Ciliates, complex protozoans with two nuclei, are known to translate RNA transcripts in unorthodox ways, not always following the classical codon protocol. Now Nowacki and his team have discovered that two ciliate species found in the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP), Condylostoma magnum and an unclassified Parduczia species, have gone even further, reassigning the traditional ‘stop’ codons (UGA, UAA, and UAG) to amino acids. On a first impression it looked like there was ‘stop’ codons spread all over the code, so the researchers quickly considered the option that the codons were used in other ways. But where are the stops, then? "It didn't make sense in the beginning," says Nowacki. "Nobody would expect that there would be a stopless genetic code."

To their big surprise the researchers found that sometimes these same codons have indeed conserved their 'stop' meaning, transmitting very efficiently and reliably the signal to the ribosomes to finish their job. This was found studying the transcripts of the ciliates' histone proteins as their sequences are highly conserved across all eukaryotes. Using protein mass spectrometry and ribosome profiling, the group determined that in the Parduczia species UAA and UAG had no ‘stop’ meaning whatsoever and are always interpreted as glutamine codons, but UGA can be read as a tryptophan codon in some cases and as a stop codon in other cases. Even stranger, the analysis showed that in C. magnum all three traditional stop codons function as either a stop or an amino acid signal.

Nowacki: “That led us to the conclusion that these codons are read in a context-dependent manner.” Further research showed that structural features at the end of the coding sequence have an influence on how the ribosome “understands” the codon, but the exact mechanism of how it reads the context remains a bit of a mystery. What the researchers could determine is that the appearance of the ambiguous codons declined dramatically near the end of transcripts. Additionally, C. magnum and the Parduczia species had a remarkably short length of untranslated mRNA between the translated part of the transcript and the 3' poly(A) tail, compared with other eukaryotes (only around 21-23 nucleotides between a genuine stop codon and the polyadenylation poly(A) sequences, compared with more than 100 nucleotides in most other species). The team suggests that proteins coating or interacting with the poly(A) tail may act as roadblocks to translation when the ribosome bumps up against them. In yeast, poly(A)-binding proteins have been shown to play a role in translation termination.

“We have been fighting about the term 'ambiguous', actually”, says Nowacki. Because the context gives clear hints how the interpret the codon, the biological language remains very clear – the cell machinery does not seem to get confused by these codons with multiple meanings.

The findings are not only interesting because they show that simple truths almost certainly prove wrong when it comes to cellular mechanisms. Nowacki thinks that they may have accidentally found a transitional stage in the evolution of this special cell mechanism, highlighting evolution “as it happens”. Studying this anomaly could help biologists understand how in some species the genetic code might gradually change. Maybe the biological language is not as frozen as we thought.

Read the publication in Cell (Open Access)

Read the publication in Nature Communications (Open Access)

By Roland Fischer