The Spliceosome Comes Assembled

Graphic: Courtesy of Scott Stevens ASSEMBLY INSTRUCTIONS: In the penta-snRNP model, all five spliceosomal snRNPs interact with the substrate pre-mRNA as a single, discrete particle. Spliceosome assembly in yeast extracts also could occur by interaction of the U1 snRNP with the pre-mRNA, followed by a tetra-snRNP joining to form a functionally identical particle. (Reprinted with permission from Elsevier Science, Molecular Cell, 9:31-4, 2002.) In multicellular organisms, the earliest products of transcription, called pre-mRNAs, undergo a molecular makeover before shipping out to the cytoplasm, where the modified mRNAs spell out the recipe for protein synthesis. Preparing the pre-mRNA involves modifying the molecule's two ends and splicing out one or more introns in between. This splicing reaction occurs in large complexes known as spliceosomes, which consist of five small nuclear RNAs (snRNAs) called U1, U2, U4, U5, and U6, as well as various proteins. Together, these snRNAs and proteins form small nuclear ribonucleoprotein particles (snRNPs), setting the stage for splicing.1 The traditional view posits that spliceosome formation is a stepwise affair, with one snRNP binding to the pre-mRNA first, followed by the others in a temporal order. This model is widely held as dogma and described in every undergraduate molecular biology and biochemistry text. But now, a discovery by California Institute of Technology investigators challenges this canonical view. These researchers have demonstrated the existence of a preassembled, five-unit complex, or penta-snRNP, which associates with the mRNA transcript all at once. The discovery of a preformed, purifiable, penta-snRNP will shed considerable light on how splicing occurs and will enable structural studies of the dynamic spliceosome, which have been impossible until now. Biologists Scott Stevens and John Abelson have described the purification and isolation of a penta-snRNP, complete with 85% of known splicing factors that demonstrate splicing activity.2 Their discovery is "absolutely gorgeous," says cell biologist and Howard Hughes Medical Institute (HHMI) investigator Joan Steitz, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine. "Finding this physical evidence, I think, is stunning." Numerous researchers agree. From an in vivo point of view, a preassembled complex makes more sense to molecular biologist and HHMI investigator Melissa Moore, Department of Biochemistry, Brandeis University. "Otherwise, rates in vivo would be limited by how fast species can assemble," she says. "By having a preformed complex, it's not limited by the rate of assembly of that complex. [This finding] really accelerates our understanding of the molecular mechanism of splicing." The notion of a preformed snRNP complex is more consistent with increasing evidence of macromolecular assemblages operating in the cell. If individual components of cellular machinery always had to assemble and disassemble every time, Steitz says, "the cell would be a hopeless mess. The entropy involved would be enormous." "Everything points to the fact that the cell likes to work in these huge assemblages of molecules," says Abelson. He believes that the spliceosome will join the ranks of other macromolecular complexes such as the ribosome, replication fork, and the nuclear pore. "What [Abelson's] data strongly suggest is that the model we need to think about is a whole complex that binds at one time," says cell biologist Phillip Sharp, Center for Cancer Research, Massachusetts Institute of Technology (MIT). A DIFFERENT SET OF INTERACTIONS In the stepwise model, the U1 snRNP first recognizes its substrate and binds to the 5' splice site. Following this interaction, the U2 snRNP makes contact with the branch point region of the mRNA. Finally, a complex consisting of U4/U6×U5 joins U2. The splicing reaction then proceeds as the snRNPs undergo rearrangement with the intron removed and the two exons joined. Conventional studies of spliceosome units involve sedimentation and electrophoretic analyses to isolate individual components, by using high salt and heparin concentrations that disrupt all but the hardiest of molecular interactions. However, high salt concentrations are incompatible with splicing inside the cell. In an experimental stroke of luck, the CalTech researchers noticed changes, at varying salt concentrations, in the makeup of the snRNP complexes they were purifying. Using gentle affinity-purification techniques, they isolated tetra- and penta-snRNPs. At first, this was not surprising, since low salt concentrations are known to result in nonspecific aggregates. But their data suggested a new discovery was afoot. The snRNPs were present in stoicheometric amounts, rather than the expected mish-mash of different units. Furthermore, the complex contained not only all five snRNAs, but it also contained 13 additional proteins involved in splicing that had never been found associated with individual snRNPs; they are thought to be involved in the late stages of spliceosome assembly. On its own, the penta-snRNP complex demonstrated no functional activity. However, splicing activity did occur when the researchers added the complex to a soluble yeast cell extract, pretreated with nuclease to remove any endogenous RNAs. But the most compelling data stemmed from mixing experiments that were designed to test whether the complex dissociates and reassembles, or remains an intact particle. In these tests, Stevens and colleagues tagged snRNAs and studied whether they dissociated from the added penta-snRNPs, mixed with endogenous snRNAs contained in the extract, and then reassembled into complexes consisting of tagged and untagged snRNAs. "The mixing experiment was crucial," says Abelson. "We were looking for evidence of reassortment." But they did not find it: Instead, they learned that tagged U2 remained associated with the original complex and did not exchange with untagged endogenous U2, although U1 could exchange to some extent. These data led them to conclude that the stepwise assembly model is a result of destabilizing experimental methods that disrupt native tetra- and penta-snRNP particles. They proposed that in yeast, splicing occurs through the binding of pre-mRNA to a preformed particle. "The previous model wasn't wrong, it was just based on techniques that picked out strong interactions," says Abelson. "With each step of assembly, you make a different set of interactions." Steitz believes that a preformed multi-snRNP is the real deal. "What they've very beautifully shown argues very strongly that this is a distinct entity and not something that they've just captured." Sharp agrees: "This data is pretty clear that the penta-snRNP is an active component as a unit in splicing. That fact has lots of implications." One researcher was not necessarily surprised with components of the discovery; another cautioned against dismissing the step model out of hand. Michael Rosbash, Department of Biology, Brandeis University, and an HHMI investigator, says that it is not astonishing that the snRNPs copurify when the salt concentration is lowered, considering the affinity the snRNPs have for each other. "Abelson's paper and other data suggest some of [the data found in vitro indicating stepwise assembly] may be oversimplified," Rosbash says. "In vivo the [spliceosome components] may be all together. He's quite likely to be right but for now we're not sure." Timothy Nilsen, Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, who wrote an accompanying commentary in the same issue,3 agrees with the finding's importance, but he does not think the previous model should be discounted. The data, he says, should be compared and contrasted. He argues that the traditional model may reflect stepwise stabilization of interactions rather than the stepwise recruitment of components. FUTURE FINDINGS Increasing evidence suggests that transcription and splicing are coupled. "Having the spliceosome as a unit allows you to understand how there could be a mechanistic couple of splicing as the RNA is being transcribed," says MIT's Sharp. "The idea of the spliceosome as a unit associated onto newly transcribing RNA couples splicing in one step to transcription and gives you a picture of how to do that." Proving that the spliceosome is a preformed entity will require studying the spliceosome in vivo, which until now, has not been possible. One way around this, says Rosbash, is to use chromatin immunoprecipitation (ChIP) analysis on native RNA being transcribed. This can reveal simultaneous deposition of all the snRNPs or temporal separation between association of the snRNPs and the pre-mRNA. "The prediction is that they will all get on at the same time," says Rosbash. "The question is: Could it fall apart and reassemble to be active?" The CalTech team's discovery of a purifiable multi-snRNP also opens the door to structural studies. "It ... provides an indication that you can probe the structure of the spliceosome by isolating this penta-snRNP. Looking at the structure of the penta-snRNP is going to tell you something about the spliceosome," says Sharp. For now, scientists will be busy making sense of earlier data and reconciling it with this latest discovery. "First we have to think, maybe it's not the assay that's wrong but our interpretation," says Magda Konarska, chair of the Laboratory of Molecular Biology and Biochemistry, Rockefeller University, who first described evidence of a preformed complex in mammalian extracts more than a decade ago.4 "We get what we ask for," says Konarska. "We set up conditions and the snRNPs play along. In general, it's a danger in science to accept things as dogma and allow ourselves to be trapped in a fixed mode of thinking." Nicole Johnston (johnstnj@mcmaster.ca) is a freelance writer in Hamilton, Ontario, Canada. References 1. J.P. Staley, C. Guthrie, "Mechanical devices of the spliceosome: motors, clocks, springs, and things," Cell, 92:315-26, 1998. 2. S.W. Stevens et al., "Composition and functional characterization of the yeast spliceosomal penta-snRNP," Molecular Cell, 9:31-44, 2002. 3. T.W. Nilsen, "The spliceosome: no assembly required?" Molecular Cell, 9:8-9, 2002. 4. M. Konarska, P.A. Sharp, "Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA," Proceedings of the National Academy of Sciences, 85:5459-62, 1998.

The Spliceosome Comes Assembled

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