Crystal Structure of the Spliceosomal 15.5kD Protein Bound to a U4 snRNA Fragment

Abstract

We have determined the crystal structure of a spliceosomal RNP complex comprising the 15.5kD protein of the human U4/U6.U5 tri-snRNP and the 5' stem-loop of U4 snRNA. The protein interacts almost exclusively with a purine-rich (5+2) internal loop within the 5' stem-loop, giving an unusual RNA fold characterized by two tandem sheared G-A base pairs, a high degree of purine stacking, and the accommodation of a single RNA base, rotated out of the RNA chain, in a pocket of the protein. Apart from yielding the structure of an important entity in the pre-mRNA splicing apparatus, this work also implies a model for the complex of the 15.5kD protein with box C/D snoRNAs. It additionally suggests a general recognition principle in a novel family of RNA binding proteins.

The role of specific 2′-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA

“…Cold Spring Harbor Laboratory Press on March 22, 2019 -Published by rnajournal.cshlp.org Downloaded from at 2.7 Å resolution, file 1RLG.pdb (Moore et al 2004); human U4 snRNA (bound to 15.5-kDa protein) at 2.9 Å resolution, file 1E7K.pdb (Vidovic et al 2000); SAM-binding riboswitch of Thermoanaerobacter tengcongensis at 2.9 Å resolution, file 2GIS.pdb (Montange and Batey 2006). Molecular graphics images were generated using Insight II and annotated in Photoshop.…”

“…The K-turn was first identified as a novel motif occurring multiple times in the ribosome. Further examples have been found in mRNA (Mao et al 1999;Winkler et al 2001;Allmang et al 2002;Montange and Batey 2006), guide RNAs (Kuhn et al 2002;Watkins et al 2002;Bortolin et al 2003;MarmierGourrier et al 2003;Rozhdestvensky et al 2003), and spliceosomal RNA (Vidovic et al 2000;Watkins et al 2000).…”

“…In this study we have examined the importance of selected 29-hydroxyl groups in the stabilization of the kinked structure by systematic chemical substitution experiments. Examination of crystal structures of the eight K-turns found in the ribosome (Ban et al 2000;Wimberly et al 2000;Brodersen et al 2002) and those of the box C/D snoRNA (Moore et al 2004), U4 snRNA (Vidovic et al 2000), and the SAM riboswitch (Montange and Batey 2006), reveals a number of common direct hydrogen bonding interactions involving 29-OH groups (potential solvent-mediated contacts have not been considered here). These are tabulated for three representative K-turns, those of Kt-7 of the 50S ribosomal subunit (the sequence of which is closest to the consensus), box C/D, and U4 in Table 1.…”

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

“…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...…”

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

“…These proteins bind to a common RNA structural motif consisting of an internal purine-rich asymmetric loop with an unusual fold. The fold is generally characterized by two tandem G⅐A base pairs, a high degree of purine stacking, and a single base rotated out of the RNA loop that inserts into a pocket of the protein (20). The motif has been described either as the ''kink-turn,'' because of the sharp bend present in the phosphodiester backbone of the RNA axis, or as the ''GA motif,'' based on the presence of the highly conserved GA dinucleotide in the asymmetric internal loop (21,22).…”