Laser microscope demonstrates gene splicing process in real time

Researchers at Brandeis University (Waltham, MA) have developed a laser microscope to study the splicing of pre-messenger RNA molecules. This essential process creates proteins to sustain advanced organisms, including human life, and the splicing is carried out by a cellular micro-machine called the spliceosome (see http://bit.ly/kokDPM).

With the spliceosome, the single yeast components are tagged with fluorescent dyes, and then the sample is placed into the microscope. The lasers act as a light source that causes individual tagged molecules to light up so one can actually watch, in unprecedented detail, the splicing process through its various stages.

"If we have one component of the spliceosome that has a green dye on it and one that has a red dye on it, then we see a green spot and a red spot coming together on the RNA, we know that we are studying part of that assembly process," says Jeff Gelles, the Aron and Imre Tauber professor of biochemistry and molecular pharmacology, whose lab at Brandeis developed the multi-laser imaging system used in the research. "By looking at individual molecules one at a time, we can actually follow the stages of the assembly process. We can determine whether it happens in the same order on each molecule, or if some spliceosomes assemble differently than others."

There are easily a hundred or so components that comprise the microscope, according to Larry Friedman, a senior scientist in the biochemistry department who was a key contributor in building the elaborate microscope. There are so many parts, in fact, that it is housed on a platform that looks much like a billiard table, with small, tower-like structures and glass lenses scattered throughout.

The molecular process known as the "central dogma of molecular biology" concerns the flow of information from DNA to RNA to proteins. RNA contains the chemical information that tells the cells what proteins to make—for instance, muscle cells use the genes that tell the cell how to make the proteins that are important for muscle, and blood cells use the genes that tell the cell how to make proteins that are important for blood cells.

With the methods to study the splicesome now at their fingertips, the Gelles lab is also researching the process by which the RNA copy is made, called transcription, and processes by which cells change their shape and move.

The researchers' paper, "Ordered and Dynamic Assembly of Single Spliceosomes," reports on a five-year-long collaboration of three research laboratories with diverse expertise to study the splicing process. The paper appears in the March 11 issue of Science. In addition to the paper's first author, Aaron A. Hoskins, a postdoc fellow who is a visiting scientist at Brandeis, authors include Gelles, Friedman, and Melissa J. Moore, a Howard Hughes Medical Institute Investigator and professor of biochemistry and molecular pharmacology at the University of Massachusetts Medical School and members of Virginia Cornish's laboratory in the Department of Chemistry at Columbia University, whose lab synthesized the special dyes that were attached to the spliceosomal proteins so that the proteins could be viewed with the laser microscope.

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Posted by Lee Mather

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