Ribosomes Team Up to Translate Tricky mRNA Segments

During translation, multiple ribosomes travel along the nucleic acid chain to build polypeptides that become functional proteins. Occasionally, these molecular decoders pause on the mRNA, either because they are instructed to do so or they have difficulty traversing the sequence. Previous studies that investigated these events looked at isolated ribosomal proteins as opposed to the multiple ribosomes typically involved in translation, leaving questions about how these pauses affect translation and how they are overcome.¹

To elucidate this process, a team led by Marvin Tanenbaum, a molecular biologist studying single-molecule dynamics at Hubrecht Institute, developed a novel imaging method to study ribosome dynamics. In a study published in Cell, Tanenbaum and his team demonstrated that ribosomes use collisions to move past pause sites and other complicated segments of mRNA to increase translation efficiency.² These findings introduce a new mechanism in translation and polypeptide formation.

First, the researchers generated stopless-ORF circular RNAs (socRNAs) that promoted continuous translation for extended periods of time. The researchers visualized translation, including ribosome pauses, by using the intensity of GFP-tagged antibodies on the polypeptide to measure polypeptide elongation.

As Tanenbaum and his team watched translation unfold, they found that at any given time, each socRNA was bound by one to four ribosomes and that these molecular machines exhibited varied translation speeds. With the help of computational models, the researchers demonstrated how faster incoming ribosomes “bump into” the stalled-out ribosomes. These collisions occurred rapidly even when there were as few as two ribosomes on the socRNA.

Previous studies showed that when the leading protein stalls, the ribosome collisions that occur trigger the removal of the affected ribosomes.^3,4 However, Tanenbaum’s team didn’t see this occurring in their experiments. They estimated that the collisions that they observed lasted between a few milliseconds to seconds, so they set out to determine whether the length of time a collision lasts influences its outcome.

Using a socRNA that included a pause site and another with a mutation that caused a longer pause period, the team determined that the mutant construct caused more collisions that lasted longer. Additionally, compared to the control socRNA, the number of ribosomes on the mutant socRNA decreased over time, suggesting an increase in degradation.

Since these brief collisions did not induce translation termination and ribosome disassembly, the researchers explored other ways that these encounters influenced ribosome activity. They found that when a sequence included a pause site, translation occurred faster if there were two ribosomes translating the socRNA compared to just one. This effect, which they termed ribosome cooperativity, extended to delays in other difficult mRNA regions, such as translation through repetitive sequences and complicated RNA structures.

“This allows ribosomes to endure short collisions on problematic RNA sections, thereby promoting continuous protein production”, Maximilian Madern, a PhD student in Tanenbaum’s group at Hubrecht Institute and study coauthor, said in a statement.

Lastly, the team evaluated the effect of ribosome cooperativity on socRNA sequences that did not include specific pause sequences or problematic patterns. Whereas translation with a single ribosome led to extended pauses in about five percent of the translation runs, sequences translated with two or more ribosomes experienced reduced pausing.

These findings support a new mechanism in maintaining translation efficiency and help elucidate the process of ribosome recycling.