Precursor RNAs containing 4‐thiouridine at specific sites were used with UV‐crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5′ splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3′ splice site region. Crosslinking of PRP8 to mutant and duplicated 3′ splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5′ exon was established before step 1 and to the 3′ splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.

The human small nuclear ribonucleoprotein (snRNP) U5 is biochemically the most complex of the snRNP particles, containing not only the Sm core proteins but also 10 particle-specific proteins. Several of these proteins have sequence motifs which suggest that they participate in conformational changes of RNA and protein. Together, the specific proteins comprise 85% of the mass of the U5 snRNP particle. Therefore, proteinprotein interactions should be highly important for both the architecture and the function of this particle. We investigated protein-protein interactions using both native and recombinant U5-specific proteins. Native U5 proteins were obtained by dissociation of U5 snRNP particles with the chaotropic salt sodium thiocyanate. A stable, RNA-free complex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unwindase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysis of the dissociated proteins. By cDNA cloning, we show that the 40kD protein is a novel WD-40 repeat protein and is thus likely to mediate regulated protein-protein interactions. Additional biochemical analyses demonstrated that the 220kD protein binds simultaneously to the 40-and the 116kD proteins and probably also to the 200kD protein. Since the 220kD protein is also known to contact both the pre-mRNA and the U5 snRNA, it is in a position to relay the functional state of the spliceosome to the other proteins in the complex and thus modulate their activity.Nuclear pre-mRNA splicing is a dynamic process in which intervening sequences (introns) are excised from pre-mRNAs in a two-step mechanism that is catalyzed by the spliceosome (for review, see references 15 and 28). The spliceosome assembles on the pre-mRNA by the stepwise integration of the small nuclear ribonucleoproteins (snRNP), U1, U2, U4/U6, and U5, and an as-yet-undefined number of non-snRNP proteins. In the early phase of spliceosome formation, U1 snRNA base pairs with the 5Ј splice site, and U2 snRNA interacts with the branch site to form the prespliceosomal E and A complexes, respectively. Spliceosomal assembly is completed by addition of the 25S U4/U6.U5 tri-snRNP complex, which is formed under splicing conditions from the U4/U6 and U5 snRNP particles (reviewed in reference 28). After assembly, the spliceosome undergoes several RNA and protein conformational rearrangements. For example, the U4/U6 duplex, which is present in the tri-snRNP, is unwound, and the U6 snRNA interacts instead with the 5Ј end of the U2 snRNA (7,26,47,58) and with intron sequences at the 5Ј splice site (14,21,40,41,44,56). The rearranged U2/U6 snRNA network is thought to be involved in the catalytic step of splicing (25), and U5 snRNA is involved in aligning the two exons for ligation (32).After the splicing reaction, the spliceosome is dissolved, and the tri-snRNP is generally believed to be assembled on the U5 snRNP again. The U5 snRNP therefore plays an important role in both spliceosome assembly and splicing....

The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5‐200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5‐200kD is distinct from U5‐220kD we demonstrate by protein sequencing that the human U5‐specific 220 kDa protein is homologous to the yeast U5‐specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5–200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH‐box protein family of putative RNA helicases and RNA‐stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5–200kD protein on Western blots and co‐immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co‐immunoprecipitated from yeast splicing extracts containing an HA‐tagged derivative of Snu246p with HA‐tag specific antibodies. U5–200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti‐U5–200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH‐box protein is involved in mammalian splicing. We propose that U5–200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA‐RNA interactions in the spliceosome.

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The RNA helicase‐like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre‐mRNAs, as demonstrated by UV‐crosslinking experiments. The use of various mutant and truncated pre‐mRNAs revealed that this interaction requires a spliceable pre‐mRNA and an assembled spliceosome; a 3′ splice site is not required. To extend these observations to the wild‐type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre‐mRNA in these spliceosomes in an ATP‐independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S‐ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre‐mRNA. As PRP2 is an RNA‐stimulated ATPase, these experiments strongly suggest that the pre‐mRNA is the native co‐factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre‐mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.