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CELL BIOLOGY Protein Components Identified in RNA Splicer Network Coordinates Steps of Gene Expression If an organism's genome really were a "book of life," then a bacterial genome would be an editor's dream, each letter of DNA translating into a perfectly meaningful protein product. But eukaryotic genomes are far more unwieldy--they ramble on nonsensically for long stretches, have huge leaps and interruptions, and require extensive culling for each RNA product to make sense as a protein. The editorial department overseeing this undertaking is the spliceosome, five RNA molecules surrounded by complexes of proteins that carry out the task of identifying and weeding out the noncoding introns from meaningful exons. With careful biochemical techniques and powerful proteomic tools, Zhaolan Zhou (left) and Robin Reed captured a complete set of proteins involved in RNA splicing. (Photo by Steve Gilbert) A proteomic analysis of this complex published in the Sept. 12 Nature captures for the first time the complete set of proteins in a functional human spliceosome. The Coupling Connection Led by Robin Reed, HMS professor of cell biology, the study reveals an even larger staff of proteins than originally thought. Many of the newfound members also share duties in other aspects of gene expression and might be serving as a link between splicing and other processes. It supports the idea that turning DNA into proteins is not the job of several separate departments but the product of a dynamic gene expression factory in which different steps are linked by common players. Capturing a spliceosome in action has been a long-sought goal for Reed. As such a large complex of proteins, it has been difficult to isolate in its native environment. Instead, what is known about its contents is based on spliceosomes generated in vitro or extracted with salt solutions that can break pieces apart. But Zhaolan Zhou, then a graduate student in Reed's lab and currently a postdoc in HMS professor Michael Greenberg's lab, was able to develop a method of binding spliceosomes using beads that can be disengaged with maltose, a gentle sugar that leaves the complex intact and functional. "In contrast to previous methods, everything can be done under conditions that are optimal for splicing so that we don't disrupt the complex," Reed said. "It still splices efficiently." In a separate paper in the Sept. 17 Proceedings of the National Academy of Sciences (Sept. 5 online), the team outlines their purification method, and offers a first look at the structure of a functional spliceosome through electron microscopy. A complex network of coupled interactions is involved in gene expression. The major steps of gene expression are shown at left, and to the right are the processes within each of these steps. The arrows indicate that evidence has been found for physical or functional coupling between two steps in the form of common coupling proteins. (Illustration by Jeff Cleary) With the precious sample in hand, Reed and Zhou then collaborated with Steven Gygi's mass spectrometry lab to determine the contents. For good measure, Gygi and the facility's assistant director Lawrence Licklider looked at gels from two spliceosomes assembled on separate strands of RNA and only considered those proteins common to both. As for many researchers using mass spectrometry, the ability to see all the proteins was exciting for Reed after years of studying one protein at a time (see sidebar). To the team's surprise, the mass spectrometer pulled out of the two samples a whopping 145 proteins. In 1990, when Reed first presented data on spliceosomes assembled in vitro and reported that she had identified around 50 proteins, "the audience burst out laughing," she said. At that time, no one thought that so many proteins would be necessary for the task of removing introns. Since then, the number 50 has been cited so much it has become standard, and triple that number is another surprise. But Reed points out that early studies were done with yeast, which has very few introns, so it is reasonable to expect that human spliceosomes would have developed a more sophisticated machinery to handle a larger editorial load: reams of junk DNA and many alternate splices. Mass Spec at HMS Amassing Fans Sometimes we forget the middlemen who provide the resources, tools, and expertise that let investigators move their ideas forward. Steven Gygi, HMS assistant professor of cell biology, has become something of an MVP among HMS researchers studying proteins. In addition to directing the Taplin Biological Mass Spectrometry Facility at HMS, Gygi runs his own lab and collaborates with researchers like Robin Reed to carry their research further with the power of proteomic tools. "It's really a wonderful thing to be able to report back to people," he said. "Sometimes people almost start crying." Mass spectrometry has completely changed the way people study proteins. While the sequence of a single protein would have once required painstaking work, a mass spectrometer can, with one ionizing blast, determine the mass and amino acid sequence of each peptide in a sample, whether it is a single protein or a large complex. It can also help characterize a protein by spotting modifications such as phosphorylation among the amino acids. And the availability of the human genome sequence means that only a handful of peptides need to be fully sequenced before the computer spots a match. So it is no surprise that people get emotional sometimes. Gygi said that some researchers are reluctant to hand over their coveted protein samples, which are unfortunately still difficult to isolate. But this is where his laboratory excels: getting the analysis to work in cases when only a single sample can be procured. He added that investigators are only beginning to realize what this still nascent technology can accomplish. "Sometimes you feel like you're still in first gear when you're using it." Familiar Protein Players Among the proteins identified, many are already known to be involved in other aspects of gene expression like transcription, mRNA export, and polyadenylation. "Those proteins being present in the spliceosome could mean that they're involved in coupling splicing to other steps," Reed said. She has been a proponent of the idea that these processes, often looked at as separate events, are bound into a single system by common proteins. Over the past few years, several groups have identified proteins that serve as links between one process and another--sometimes between steps that are not consecutive, such as transcription and export. Such a mechanism would overcome some logistical hurdles, like the need for rapid timing or close proximity of two events. For instance, Reed's team previously identified the protein Aly, which is involved in mRNA export but is recruited during splicing and connects the two steps (see Focus, Sept. 29, 2000). Reed believes these individual examples of coupling represent a large network coordinating the different steps of gene expression. "We can't really focus on each step, but instead we have to look at how all of the steps are interrelated," she said. Stephen Buratowski, HMS professor of biological chemistry and molecular pharmacology, shares this perspective: "In the past, we used to think of these things in a reductive mode." But ideas of coupling and integration are catching on: his lab, for instance, has looked at the transcription complex and how it is linked to RNA capping and polyadenylation. Coupling, he said, is not always a one-way street: there seems to be information traveling in both directions. One question that remains is whether coupling processes are hard-wired or happen dynamically, and whether they are required for the individual steps of gene expression. --Courtney Humphries