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Microscope technique reveals for first time when and where proteins are made

Microscope technique reveals for first time when and where proteins are made
2015-03-19
(Press-News.org) March 19, 2015--(Bronx, NY)--Scientists at Albert Einstein College of Medicine of Yeshiva University and their international collaborators have developed a novel fluorescence microscopy technique that for the first time shows where and when proteins are produced. The technique allows researchers to directly observe individual messenger RNA molecules (mRNAs) as they are translated into proteins in living cells. The technique, carried out in living human cells and fruit flies, should help reveal how irregularities in protein synthesis contribute to developmental abnormalities and human disease processes including those involved in Alzheimer's disease and other memory-related disorders. The research will be published the March 20 edition of Science.

"We've never been able to pinpoint exactly when and where mRNAs are translated into proteins," said study co-leader Robert H. Singer, Ph.D., professor and co-chair of anatomy and structural biology and co-director of the Gruss Lipper Biophotonics Center at Einstein. "This capability will be critical for studying the molecular basis of disease, for example, how dysregulation of protein synthesis in brain cells can lead to the memory deficits that occur in neurodegeneration." Dr. Singer also holds the Harold and Muriel Block Chair in Anatomy & Structural Biology at Einstein.

The directions for making proteins are encoded in genes in the cell nucleus. Two steps--transcription and translation--must occur so that the gene's protein-making instructions will lead to actual proteins. In the first step, called transcription, the gene's DNA is "read" by molecules of mRNA. These mRNAs then migrate from the nucleus into the cytoplasm and attach to structures called ribosomes. That's where translation, the second step in protein synthesis, occurs: the mRNAs attached to ribosomes function as templates on which proteins are constructed.

To visualize translation, Dr. Singer and his colleagues took advantage of a key occurrence during the first round of translation: the ribosome to which mRNAs attach must displace so-called RNA-binding proteins from the mRNAs. The researchers synthesized identical copies of mRNA molecules containing two fluorescent proteins, one green and one red. This meant that in the nucleus (where mRNAs are made), mRNAs labeled with both red and green proteins appear yellow. After migrating to the cytoplasm, the mRNAs can change color depending on their fate.

For mRNAs landing on ribosomes, the ribosome displaces the mRNAs' green fluorescent protein. As a result, these mRNA molecules--stripped of their green fluorescent proteins, bound to ribosomes, and ready to be translated into a protein--appear red. Meanwhile, all the untranslated mRNA molecules remain yellow. The technique was dubbed TRICK (for Translating RNA Imaging by Coat protein Knock-off).

In a test of TRICK, the collaborators in Germany studied when and where mRNAs for a gene called oskar are expressed in Drosophila eggs, or oocytes. (Drosophila, or fruit flies, are a frequently used model for understanding human disease, and oskar is critical for normal development of fruit fly embryos.) The researchers made oskar mRNAs tagged with red and green fluorescent proteins and inserted the tagged mRNAs into the nuclei of Drosophila oocytes. "Using TRICK, oskar mRNAs were not translated until they reached the posterior pole of the oocyte," said Dr .Singer. "We suspected this, but now we have definitive proof. Going forward, researchers can use this technique to dissect the cascade of regulatory events required for mRNA translation during Drosophila development."

The researchers also found that protein translation doesn't start immediately after mRNAs exit the nucleus but instead gets underway several minutes after mRNAs have entered the cytoplasm. "We never knew that timeframe before," said Dr. Singer. "That's another example of what we can learn using this technique."

INFORMATION:

The paper is titled "An RNA biosensor for imaging the first round of translation from single cells to living animals." The other contributors are: Timothée Lionnet, Ph.D., now at Janelia research Campus of the HHMI; James M. Halstead, Ph.D., and Jeffrey A. Chao, Ph.D., at Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; Johannes H. Wilbertz, Ph.D. student, at Friedrich Miescher Institute for Biomedical Research and University of Basel, Basel, Switzerland; Frank Wippich, Ph.D., and Anne Ephrussi, Ph.D., at European Molecular Biology Laboratory, Heidelberg, Germany.

This study was supported by grants from the Novartis Research Foundation, National Institutes of Health (NS83085, EB13571 and GM57071), Howard Hughes Medical Institute, and European Molecular Biology Laboratory.

The authors report no conflicts of interest.

About Albert Einstein College of Medicine

Albert Einstein College of Medicine of Yeshiva University is one of the nation's premier centers for research, medical education and clinical investigation. During the 2013-2014 academic year, Einstein is home to 743 M.D. students, 275 Ph.D. students, 103 students in the combined M.D./Ph.D. program, and 313 postdoctoral research fellows. The College of Medicine has more than 2,000 full-time faculty members located on the main campus and at its clinical affiliates. In 2013, Einstein received more than $150 million in awards from the National Institutes of Health (NIH). This includes the funding of major research centers at Einstein in aging, intellectual development disorders, diabetes, cancer, clinical and translational research, liver disease, and AIDS. Other areas where the College of Medicine is concentrating its efforts include developmental brain research, neuroscience, cardiac disease, and initiatives to reduce and eliminate ethnic and racial health disparities. Its partnership with Montefiore Medical Center, the University Hospital and academic medical center for Einstein, advances clinical and translational research to accelerate the pace at which new discoveries become the treatments and therapies that benefit patients. Through its extensive affiliation network involving Montefiore, Jacobi Medical Center -- Einstein's founding hospital, and three other hospital systems in the Bronx, Brooklyn and on Long Island, Einstein runs one of the largest residency and fellowship training programs in the medical and dental professions in the United States. For more information, please visit www.einstein.yu.edu, read our blog, follow us on Twitter, like us on Facebook, and view us on YouTube. END


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Microscope technique reveals for first time when and where proteins are made

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[Press-News.org] Microscope technique reveals for first time when and where proteins are made