There exists a cellular machine in our bodies, called the spliceosome, which just like a film director cuts out extraneous footage to create a blockbuster, snips out unwanted stretches of genetic material and joins the remaining pieces to fashion a template for protein production.
However, if the spliceosome makes a careless cut, it could result in a disease.
AdvertisementUsing a new approach to studying the spliceosome, a team led by University of Michigan chemistry and biophysics professor Nils Walter, spied on the splicing process in single molecules.
The new study, which utilizes a technique called fluorescence resonance energy transfer (FRET) and a sophisticated microscope that watches single molecules in action, allows researchers to observe in real time the contortions involved in spliceosome assembly and operation.
If molecular-scale standards are considered, the spliceosome is made up of five RNA and 100 or more protein subunits that agilely assemble, step-by-step, into the giant complex when it's time to carry out its work.
True to the movie director analogy, the spliceosome not only wields the scissors, it's also "the brain that decides where to cut," said Walter.
The "footage" it works on is the genetic material contained in RNA molecules.
RNA carries coded instructions for producing the proteins our body needs for building and repairing tissues, regulating body processes and many other sections called introns. The spliceosome's task is to recognize and excise introns.
Once the introns are removed, the spliceosome can stitch together exons in various combinations.
And owing to this mixing and matching of exons, a relatively small number of genes (a little over 20,000 in humans) can serve as blueprints for a great variety of proteins.
Walter and colleagues spied on the splicing process by attaching fluorescent tags to exons on either side of an intron in a short section of RNA they designed specifically for such studies.
"Conventional wisdom has been that the spliceosome directs the whole splicing process, that the RNA itself has little influence on it. But we saw the RNA molecule flexing on its own, with the intron folding and unfolding in a way that brings the exons closer together, suggesting a more active role for introns," Nature quoted Walter as saying.
When the team added an extract containing spliceosome components, along with ATP---the energy currency that fuels spliceosome assembly---the distance between exons first increased, then decreased even more, and splicing occurred.
Interestingly, the series of contortions that RNA went through during splicing was not a one-way path; the steps were reversible.
"Imagine the movie director having doubts about what scenes to cut and continuously going back and forth in holding different pieces of footage together before actually making a decision and splicing the film. That's what we saw happening at the molecular level. To our knowledge, our data provide the first direct glimpse of such reversible conformational changes during the splicing process," said Walter.
The research is published in Nature Structural and Molecular Biology.
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