Flickering black and white movies show components of the spliceosome coming and going from bits of RNA as they await processing.

Microsope lasersTechnology and perseverance have enabled scientists to create the first movies tracking the assembly of one of the cell’s massive machines: the spliceosome.

Spliceosomes chop out long strings of genetic material and stitch the remaining segments back together to prepare the genetic code to be translated into proteins. By cutting and pasting the genetic sequences in different ways, a process called alternative splicing, spliceosomes enable a single gene to code for multiple proteins.

The flickering black and white movies on display in the lab of Howard Hughes Medical Institute investigator Melissa J. Moore show components of the spliceosome coming and going from bits of RNA as they await processing. This continuous shifting of parts suggests that spliceosome assembly can be shut down or reversed once it has begun—meaning cells do not have to commit to splicing a particular gene as early as once thought.

After labeling the spliceosome component U1 with a fluorescent tag, researchers could watch it come and go as spliceosomes assembled on RNAs awaiting processing. Movie: Aaron Hoskins

The findings were published in the March 11, 2011, issue of the journal Science—the culmination of five years of work by a diverse team of collaborators led by Moore, a professor of biochemistry and molecular pharmacology at the University of Massachusetts Medical School, and Jeff Gelles, a professor of Biochemistry at Brandeis University. Also integral to the project’s success was a group led by Virginia Cornish, a professor of chemistry at Columbia University.

Moore says understanding the fundamentals of spliceosome assembly will help scientists explain conditions in which that process goes awry, which can skew gene processing and cause disease.

Watch as introns are removed from RNA during an animation of the splicing process.Video: HHMI Biointeractive

To track spliceosome assembly molecule by molecule, the Gelles/Moore team devised an imaging strategy that can now be applied to other large cellular machines. "We think that this [method is] going to allow many researchers to do much more detailed, deep mechanistic analysis on their systems," she says.

We think that this [method is] going to allow many researchers to do much more detailed, deep mechanistic analysis on their systems.

Melissa J. Moore

The human genome is relatively small, carrying about 25,000 genes, but those genes hold the blueprints for producing far more than 25,000 proteins. That’s because most human genes can be spliced a variety of ways.

Each gene is made up of segments that directly encode protein—known as exons—and interrupting segments of noncoding DNA called introns. When a gene is transcribed into RNA—the molecule that will relay the gene’s information to the cell’s protein-making machinery—that RNA is a direct copy of the gene: all the exons and introns are included. To make the finished blueprint, the introns must be removed. That job is carried out by the spliceosome.

A complex of many proteins, the spliceosome latches onto RNA as it unspools off the nuclear genome, then it stitches the proper genetic segments together into a protein-making code. "It's a much more complicated machine," Moore says of the spliceosome, comparing it to the better known ribosome, which uses the spliced RNA blueprints to make proteins. "The spliceosome has five major parts, and they're very dynamic in how they move and interact with RNA—how they come and go.

To do its work, a single spliceosome must identify where a gene needs to be snipped—which might change depending on time, place, or conditions—cut out the unnecessary genetic material (the introns), and then stitch the exons back together. To accomplish this, it uses five small pieces of RNA and scores of proteins—all of which much find one another and assemble into a spliceosome every time a newly made RNA needs to be spliced. In most human cells, this happens millions of times a day.

Scientists have tried for years to understand how the parts of spliceosomes assemble. But most of the techniques they have used in the past focused on tracking stable intermediates in the spliceosome assembly pathway. The problem with that approach, Moore says, is that it’s like observing traffic on a highway by looking only at cars that have reached a stoplight: you don't get a sense of the dynamics and interplay

In 2008, the Gelles/Moore group published a study in the journal RNA showing introns being spliced by individual spliceosomes. In that study, they could see only the RNA molecule being spliced. Other labs are also starting to publish single-molecule studies of the spliceosome, but existing technology equally limited those studies to observing just the RNAs.

Several new technologies have now coalesced to let the Gelles/Moore group track the real-time assembly of individual spliceosomes. Key to the new study was a microscope equipped with four different color lasers developed by Gelles' long-time collaborator Larry Friedman, a research scientist at Brandeis University. With the microscope, the team was able to use a technique they have dubbed colocalization single molecule spectroscopy, or CoSMoS, to visualize distinct pieces of the spliceosome as they moved toward and away from the genetic material.

To make the components of their system visible, the team, spearheaded by first author Aaron Hoskins, a postdoctoral researcher in the Moore and Gelles laboratories, genetically engineered baker's yeast, Saccharomyces cerevisiae, so that components of the spliceosome were attached to different fluorescent labels. This is where Virginia Cornish's research group came in – they developed one of the methods for attaching the fluorescent labels to pieces of the spliceosome.

After stripping away the cells' outer membranes, the researchers let the cellular innards come in contact with intron-containing RNA molecules attached to a slide. They had also labeled those RNAs with fluorescent molecules that would be visible under the microscope.

In their initial experiments, the team examined four pieces of the spliceosome complex, watching each of them attach to the intron. They made sure they were looking at working complexes by monitoring the disappearance of the intron that they had fluorescently labeled. Indeed, the spliceosomes were fully functional, and the more pieces that added on to the complex, the more committed the spliceosome became to snipping out the intron.

As expected from previous experiments looking at stable complexes, the group found that the spliceosome parts latched onto its RNA target in a precise order. But surprisingly, their observations revealed that this binding is reversible. "Just based on this one paper we're going to have to rewrite the textbooks," Moore says.

Knowing that spliceosome assembly is reversible is important, Moore says, because it indicates that there are more opportunities for regulating gene expression than previously thought. When scientists have tried to understand the different ways a gene can be spliced, they have usually focused on possible regulatory mechanisms only at the very earliest steps. "What our data show is that since we now know the assembly pathway is reversible, it is possible to regulate it at any step along the pathway," Moore says.

That spliceosome assembly can be controlled at multiple levels gives the cell more opportunities to practice quality control in expressing genes, Moore adds. "If you're totally committed to splicing after the very first step in the assembly pathway, then that step has to be perfect or near perfect. You only have one chance to get it right."

For more than a decade, Moore and Gelles have dreamed of being able to study spliceosome assembly at the single-molecule level. Now that they have achieved that goal, they want to share the strategy with other biologists. This methodology should be particularly accessible and adaptable, Moore says, because there is no need to purify the molecule to be studied, which can often be a tricky and time-consuming task. "The beauty of it is that we don't have to add anything—we can just look at what's there," she says.

Moore points out that data analysis can be one of the most challenging and laborious aspects of studying macromolecular complexes in this way. For the Science paper, this involved tracking thousands of individual molecules, each of which were represented as spots of light on a computer screen. They manually followed each spot over time and documenting its association with particular splicing complexes. That all had to be done by hand, each one by a person — in this case Hoskins, who worked on the project for nearly five years. Gelles and his coworkers are now working on software to automate parts of the data analysis.

In the meantime, the group plans to use the technique to answer many more questions about the spliceosome. "We're very rapidly going down a list," Moore says. For example, they are looking at how some pieces of the spliceosome complex are ejected after the assembly process. They are also interested in finding out whether spliceosome assembly follows the same order for all introns. "Obviously we can't test all introns, but we can take some educated guesses on which introns might be different," she adds.

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