30 March 2010

The spliceosome mounts the film "Matrix RNA"

Real-time RNA splicing processNanonewsnet based on ScienceDaily: Spying on a Cellular Director in the Cutting Room
Creating a blockbuster, the film director cuts out extra frames from it, and a cellular machine called a spliceosome cuts out unwanted sections of genetic material and connects the remaining sections, creating a template for protein synthesis.

But if the spliceosome does it carelessly, the disease may develop.

(A spliceosome is a structure consisting of RNA and protein molecules and performing the removal of non-coding sequences (introns) from the precursor molecule of matrix RNA. This process is called splicing, from the English. splicing – splicing. The spliceosome consists of five small nuclear ribonucleoproteins – U1, U2, U4, U5 and U6 – and several additional protein factors. Usually, the spliceosome is reassembled for each pre-mRNA VM).

Using a new approach for the study of spliceosomes, a group of scientists led by Professor of Chemistry and biophysics from the University of Michigan, Nils Walter, in close cooperation with a group led by the world-recognized scientific community experts in the field of splicing, John Abelson and Christine Guthrie from the University California – San Francisco (University of California, San Francisco), investigate the process of splicing in a single molecule.

Article by John Abelson et al. Conformational dynamics of single pre-mRNA molecules during in vitro splicing will be published in the March issue of the journal Nature Structural and Molecular Biology.

Since the Nobel Prize was awarded for the study of the splicing process in 1977, gene splicing has been studied on a number of organisms, including yeast and human cells, using both genetic and biochemical approaches. Although using these methods it is possible to get snapshots, they did not make it possible to observe the process in real time. A new study using a technology called fluorescence resonance energy transfer (fluorescence resonance energy transfer – FRET) and a sophisticated microscope, which make it possible to see movement in a single molecule, allowed scientists to observe changes occurring during the assembly and functioning of spliceosomes in real time.

By analogy with the filmmaker, the spliceosome not only performs the function of scissors. She is also the "brain" that decides which frames to cut, says Walter. The material with which such a "director" works is genetic material enclosed in RNA molecules. RNA contains encoded instructions for the production of proteins that our body needs for the construction and repair of tissues, the regulation of metabolic processes, as well as a large number of sites called introns. The task of the spliceosome is to recognize and cut out introns. When introns are removed, spliceosomes can glue exons in various combinations. Thanks to this mixing and gluing of exons, a relatively small number of genes (slightly more than 20,000 in the human body) creates a huge variety of proteins.

(An example of alternative splicing in humans.
The gene of the structural protein tropomyosin gives rise to different variants of this protein,
which are synthesized in different tissues of the body – VM.)

Walter and his colleagues followed the splicing process using fluorescent tags attached to exons on both sides of the intron, on short sections of RNA specially created for such studies. By illuminating the fluorescent labels with laser light, FRET can detect how close or how far apart the exons are located. The observations repeated over time eventually make up a molecular-scale "movie", from which it is seen how parts of the RNA molecule change before and during splicing.

At first, the researchers studied RNA in the absence of spliceosomes. Common sense suggested that the spliceosome itself directs the entire splicing process, and the RNA molecule has little effect on this process. But they saw changes in the RNA molecule itself: due to compression and stretching of introns, exons became closer to each other. This suggests a more active role of introns.

When the scientists added an extract containing the components of the spliceosomes and ATP – the energy source for their assembly – the distance between the exons first increased, and then became even smaller than before the process began. After such changes, the actual splicing took place. Interestingly, the sequence of events that occurred with the RNA molecule during this process turned out to be reversible.

Imagine that the director has doubts about which scenes from the film need to be cut, and he repeatedly glues various pieces of film before coming to a final decision. We observe all this, only at the molecular level," says Walter. – As far as we know, our data is the first direct observation of reversible conformational changes in the splicing process.

Next, the researchers plan to attach fluorescent labels to various parts of the system in order to see their spatial and temporal relationships during the splicing process. The ultimate goal is to build a comprehensive model showing how RNA and spliceosomes interact with each other so precisely that they avoid the occurrence of the disease.

Portal "Eternal youth" http://vechnayamolodost.ru30.03.2010

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