Science Spotlight

They stumble that run fast

From the Subramaniam lab, Basic Sciences Division

“Wisely and slow; they stumble that run fast.”

William Shakespeare, Romeo and Juliet 

It is unlikely that Shakespeare was thinking of ribosomes when he warned against hastiness. But it turns out, for ribosomes too, “wisely and slow” does not really cut it as they go about their business translating mRNA into protein. In every cell, a single mRNA molecule may be occupied by multiple ribosomes or polysomes. That way mRNA is protected from degradation and at the same time the cells can maximize their protein synthesis efficiency.

At any given time, an mRNA molecule loaded with ribosomes is akin to a highway loaded with vehicles and it is no surprise that ‘ribosome collisions’ are an undesirable destiny. Ribosome collisions may arise from events that lead to ribosome slowing or stalling. So what is it that makes ribosomes stumble?

Research in the Subramaniam lab (Basic Sciences Division) aims to answer such questions by investigating the molecular mechanisms that enable cells to maximize their protein expression without compromising mRNA stability, using a combination of computational modeling and biochemical approaches. “This kind of approach, i.e. combining the mathematical kinetic modeling and well-defined quantitative experiment, is not that common. I think this approach is our strength which makes it possible for us to do some unique researches in the translation field”, said Dr. Heungown Park, a postdoc in the Subramaniam lab. 

Using this combinatorial approach, the Subramaniam lab found in a previous study that protein expression in bacteria followed a kinetic model in which ribosome collisions stimulate abortive termination of translation. The proposed model was therefore called the collision-stimulated abortive termination model. The next question was whether ribosome collisions had a similar regulatory role in eukaryotic cells. Drs. Park and Subramaniam recently published new insights to answer this question in PLoS Biology.

The canonical model of translation in eukaryotic cells postulates that higher translation initiation rates would increase protein expression and mRNA stability. Park and Subramaniam observed however that this model was inconsistent with their findings showing that increasing the initiation rate can in fact decrease both protein expression and stability of certain mRNAs in the budding yeast, a eukaryotic model organism. Therefore, they decided to build a model that was more faithful to their biochemical observations.  

A schematic of different models that aim to explain how ribosome stalling disrupts translation elongation.
A schematic of different models that aim to explain how ribosome stalling disrupts translation elongation. Figure 3 from the publication. Courtesy of the Subramaniam Lab.

Using complementary computational and biochemical approaches, the Subramaniam laboratory modeled ribosome stalling sites in yeast by artificially introducing stall sites into a reporter gene that they integrated into the yeast genome. They also used mutagenesis to manipulate the initiation rate and monitor the dynamics of mRNA and protein production in the cells. 

Their computational approach predicted that the collision-stimulated mRNA decay they observed at high initiation rates was consistent with an abortive termination model in which a stalled ribosome simply falls off the mRNA. To test this model, they depleted known collision-associated quality-control factors and measured the effect thereof on protein expression and mRNA stability. Depletion of quality-control factors such as Hel2 and Asc1 rescued the protein expression defects observed in stall-containing reporters at high initiation rates. Therefore, they concluded that at high initiation rates Hel2 and Asc1 are necessary to reduce protein expression and mRNA stability. 

Computer simulations of ribosome collision models accurately recapitulate experimental observations from yeast cells. i.e CSAT (Collision-Stimulated Abortive Termination) and CSEC (Collision-Stimulated Endonucleolytic Cleavage) predicted computationally were confirmed impirically in vivo.
Computer simulations of ribosome collision models accurately recapitulate experimental observations from yeast cells. i.e CSAT (Collision-Stimulated Abortive Termination) and CSEC (Collision-Stimulated Endonucleolytic Cleavage) predicted computationally were confirmed empirically in vivo. Image provided by Heungown Park

Dr. Park explained the significance of their work: “Model simulations explained well our interesting experimental observations i.e. the decrease of protein expression and mRNA levels when the initiation rate is high and the presence of stall inducing codon repeats. When ribosomes collide on mRNA due to the stall-inducing codon repeats, it is not clear if the front ribosome falls off from mRNA or if the ribosome-collisions induce degradation of mRNA. The new model simulations reproduce well our experimental results.”

Intriguingly, hundreds of yeast mRNAs contain ribosome stall sequences similar to those engineered into the reporter mRNA; do they also shape the translation of endogenous mRNAs? Indeed, the authors observed that such endogenous mRNAs exhibited signatures of inefficient translation. Understanding the biological function of these stall sequences is still a work in progress.

Park H, Subramaniam AR. 2019. “Inverted translational control of eukaryotic gene expression by ribosome collisions.” PLoS Biology.

This work was funded by the National Institute of Health and the National Science Foundation.

Cancer Consortium member Arvind Subramaniam contributed to this research.

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