“…In eukaryotes, the removal of introns from nascent transcripts is mediated by a highly dynamic, macromolecular machine called the spliceosome+ In size and complexity, spliceosomes are comparable to ribosomes+ Like ribosomes, spliceosomes are composed of distinct subunits containing stable RNA molecules in tight association with multiple proteins+ Five of these U-rich small nuclear RNPs (U1, U2, U4, U5, and U6 snRNPs) function together with a plethora of non-snRNP protein factors to accurately identify the termini of each intron, followed by assembly of an active spliceosome to catalyze intron excision (Staley & Guthrie, 1998;Burge et al+, 1999)+ Although the complete set of spliceosomal components is not yet known, it can be estimated that .70 polypeptides contribute to the functional complex (Bennett et al+, 1992a;Gozani et al+, 1994;Neubauer et al+, 1998)+ Recent determination of high-resolution structures of ribosomes has significantly increased our understanding of how these amazing machines operate (Moore, 1998;Wimberly et al+, 2000;Yusupov et al+, 2001)+ For spliceosomes, however, the only available threedimensional information consists of a few single components and subassemblies+ For example, X-ray crystallography has yielded structures of U1A protein bound to its cognate stem loop in U1 snRNA (Oubridge et al+, 1994), U2A9 and U2B0 proteins bound to a portion of U2 snRNA (Price et al+, 1998) and tri-snRNP 15+5 kDa protein bound to a U4 snRNA fragment (Vidovic et al+, 2000)+ X-ray structures of Sm D1/D2 complexes and Sm D3/B have suggested that the core proteins shared by U1, U2, U4, and U5 snRNPs exist as a seven-membered ring (Kambach et al+, 1999), an ultrastructure also visible by electron microscopy (Raker et al+, 1999)+ Finally, a cryo-electron microscopy structure of the entire U1 snRNP, which contains U1 snRNA, the Sm core, and three U1-specific proteins, was recently determined to 15 Å resolution (Stark et al+, 2001)+ To date, however, there has been no report of any three-dimensional structure for an intact spliceosome+ One of the challenges inherent to structural studies of spliceosomes is isolating this remarkably dynamic machine in a single state+ Recognition and removal of each new intron in vitro appears to require full reassembly of a spliceosome from its component parts+ This assembly occurs in a series of distinct steps, primarily defined by the addition and release of the U snRNPs (Fig+ 1A)+ Spliceosome assembly initiates with 59 splice site recognition by U1 snRNP and subsequent recruitment of U2 snRNP to the branch site to form a species termed CC or E (commitment or early) complex+ The U2 snRNP:branch site interaction becomes stabilized with the formation of A complex, and addition of U4, U5, and U6 as a preassembled tri-snRNP results in B complex+ Subsequent formation of the catalytically competent C complex involves recruitment of additional protein factors along with significant structural rearrangements that desta...…”