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During stress, mRNAs were found to cluster (arrows) within P-bodies that resulted in an additional layer of translational regulation for the sequestered mRNAs.











FMI

March 20, 2015

A spotlight on protein translation


Jeff Chao, Junior group leader at the FMI, together with colleagues in New York and Heidelberg, has developed a sophisticated technique that makes it possible to monitor the precise time and place of the first translation event within a cell. This novel method, known as TRICK, is described and initial results are reported in Science today.

For the fate of a cell, it is crucial what proteins are produced, at what time and in what part of a cell. But what is curtly termed transcription and translation, – the copying of genetic information and subsequent protein synthesis – are controlled by thousands of different proteins. After transcription, the resulting messenger RNA (mRNA) has to be processed, exported out of the nucleus and transported to the right location within the cell before it can be translated. To date, however, detailed analysis of these processes has been hampered by the limitations of available methods.

Jeff Chao, Junior group leader at the Friedrich Miescher Institute for Biomedical Research (FMI), together with colleagues from the Albert Einstein College of Medicine and the European Molecular Biology Laboratory, has now developed a technique that makes the translation of individual mRNAs visible and quantifiable.

Chao and his colleagues took advantage of the fact that, immediately after transcription, mRNAs are coated with RNA-binding proteins and that, during translation, the ribosomal machinery displaces these proteins bound within the coding sequence of transcripts. To visualize the timing of translation, the scientists engineered a transcript that is labeled with two RNA-binding proteins fused to two distinct fluorescent tags: one of the coat proteins – PP7 (tagged with green fluorescent protein) – binds to the coding region of the mRNA, while the other – MS2 (with a red tag) – binds to the 3′ untranslated region. Thus labeled, the individual untranslated mRNAs appear yellow. As the ribosome traverses the coding sequence during the first round of translation, the green-tagged PP7 is displaced, leading to a detectable and quantifiable color change.

Chao comments: “Using this technique, which we named TRICK (translating RNA imaging by coat protein knock-off), we have been able to examine the timing and the site of mRNA translation. To our surprise, not all the mRNAs were treated the same way. Most were translated within minutes after leaving the nucleus. But others were sequestered to specific areas within the cytoplasm, undergoing additional regulatory processes.”

TRICK revealed that, during normal growth, mRNAs are not translated in the nucleus, and that translation occurs homogenously throughout the cytoplasm. During cellular stress, however, the situation is slightly different: mRNAs can be sequestered within cytoplasmic compartments called P-bodies, where an additional level of regulation takes place. Finally, precise translational regulation is necessary during Drosophila development, when mRNAs must be transported over a long distance to a particular location within the oocyte and then activated at a specific stage; TRICK also allowed this process to be monitored.

“TRICK allows us to monitor translation ‘live’ and to pinpoint the precise time and place of the first translation event,” says Chao. “This will now enable us to address mechanisms of regulation that have been inaccessible using genetic and biochemical approaches. We no longer have to imagine how this regulation is occurring within cells – we can simply image it.”

Publication
Halstead JM#, Lionnet T#, Wilbertz JH#, Wippich F#, Ephrussi A*, Singer RH*, Chao JA* (2015) An RNA biosensor for imaging the first round of translation from single cells to living animals. Science, 347:1367-1671
# equal contributions, *corresponding authors

Contact
Jeffrey Chao, jeffrey.chao@fmi.ch, Tel. +41 (0)61 697 51 73
Jeffrey Chao is a Junior group leader who joined the FMI in 2013 from the Albert Einstein College of Medicine in New York. He and his 5-strong research group study the temporal and spatial orchestration of mRNA processing, transport, translation and degradation.

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