Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. In some cases, cyclophilins have also been shown to form stable complexes with specific proteins in vivo and may thus also act as chaperone-like molecules. We have characterized the 20kD protein of the spliceosomal 25S [U4/U6.U5] tri-snRNP complex from HeLa cells and show that it is a novel human cyclophilin (denoted SnuCyp-20). Purified [U4/U6.U5] tri-snRNPs, but not U1, U2, or U5 snRNPs, exhibit peptidyl-prolyl cis/trans isomerase activity in vitro, which is cyclosporin A-sensitive, suggesting that SnuCyp-20 is an active isomerase. Consistent with its specific association with tri-snRNPs in vitro, immunofluorescence microscopy studies showed that SnuCyp-20 is predominantly located in the nucleus, where it colocalizes in situ with typical snRNP-containing structures referred to as nuclear speckles. As a first step toward the identification of possible targets of SnuCyp-20, we have investigated the interaction of SnuCyp-20 with other proteins of the tri-snRNP. Fractionation of RNA-free protein complexes dissociated from isolated tri-snRNPs by treatment with high salt revealed that SnuCyp-20 is part of a biochemically stable heteromer containing additionally the U4/U6-specific 60kD and 90kD proteins. By coimmunoprecipitation experiments performed with in vitro-translated proteins, we could further demonstrate a direct interaction between SnuCyp-20 and the 60kD protein, but failed to detect a protein complex containing the 90kD protein. The formation of a stable SnuCyp-20/60kD/90kD heteromer may thus require additional factors not present in our in vitro reconstitution system. We discuss possible roles of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conformational changes occurring during the splicing process.

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, protein-protein 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 US-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.

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, protein-protein 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.

Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. In some cases, cyclophilins have also been shown to form stable complexes with specific proteins in vivo and may thus also act as chaperone-like molecules. We have characterized the 20kD protein of the spliceosomal 25S [U4/U6.U5] tri-snRNP complex from HeLa cells and show that it is a novel human cyclophilin (denoted SnuCyp-20). Purified [U4/U6.U5] tri-snRNPs, but not U1, U2, or U5 snRNPs, exhibit peptidyl-prolyl cis/trans isomerase activity in vitro, which is cyclosporin A-sensitive, suggesting that SnuCyp-20 is an active isomerase. Consistent with its specific association with tri-snRNPs in vitro, immunofluorescence microscopy studies showed that SnuCyp-20 is predominantly located in the nucleus, where it colocalizes in situ with typical snRNP-containing structures referred to as nuclear speckles. As a first step toward the identification of possible targets of SnuCyp-20, we have investigated the interaction of SnuCyp-20 with other proteins of the tri-snRNP. Fractionation of RNA-free protein complexes dissociated from isolated tri-snRNPs by treatment with high salt revealed that SnuCyp-20 is part of a biochemically stable heteromer containing additionally the U4/U6-specific 60kD and 90kD proteins. By coimmunoprecipitation experiments performed with in vitro-translated proteins, we could further demonstrate a direct interaction between SnuCyp-20 and the 60kD protein, but failed to detect a protein complex containing the 90kD protein. The formation of a stable SnuCyp-20/60kD/90kD heteromer may thus require additional factors not present in our in vitro reconstitution system. We discuss possible roles of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conformational changes occurring during the splicing process.

Through UV-crosslinking experiments, we previously provided evidence suggesting that a U5 snRNP protein with a molecular weight in the 100-kDa range is an ATP-binding protein (Laggerbauer B, Lauber J, Lührmann R, 1996, Nucleic Acid Res 24:868-875). Separation of HeLa U5 snRNP proteins on 2D gels revealed multiple variants with apparent molecular masses of 100 kDa. Subsequent microsequencing of these variants led to the isolation of a cDNA encoding a protein with an N-terminal RS domain and a C-terminal domain that contains all of the conserved motifs characteristic of members of the DEAD-box family of RNA-stimulated ATPases and RNA helicases. Antibodies raised against cDNA-encoded 100-kDa protein specifically recognized native U5-100kD both on immunoblots and in purified HeLa U5 snRNPs or [U4/U6.U5] tri-snRNP complexes, confirming that the bona fide 100-kDa cDNA had been isolated. In vitro phosphorylation studies demonstrated that U5-100kD can serve as a substrate for both Clk/Sty and the U1 snRNP-associated kinase, and further suggested that the multiple U5-100kD variants observed on 2D gels represent differentially phosphorylated forms of the protein. A database homology search revealed a significant degree of homology (60% similarity, 37% identity) between the Saccharomyces cerevisiae splicing factor, Prp28p, which lacks an N-terminal RS domain, and the C-terminal domain of U5-100kD. Consistent with their designation as structural homologues, anti-Prp28 antibodies recognized specifically the human U5-100kD protein on immunoblots. Together with the DEXH-box U5-200kD protein (Lauber J et al., 1996, EMBO J 15:4001-4015), U5-100kD is the second example of a putative RNA helicase that is tightly associated with the U5 snRNP. Given the recent identification of the U5-116kD protein as a homologue of the ribosomal translocase EF-2 (Fabrizio P, Laggerbauer B, Lauber J, Lane WS, Lührmann R, 1997, EMBO J 16:4092-4106), at least three integral U5 snRNP proteins thus potentially facilitate conformational changes in the spliceosome during nuclear pre-mRNA 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.

The removal of introns from precursor messenger RNAs occurs in a large complex, the spliceosome, that contains many proteins and five small nuclear RNAs (snRNAs). The snRNAs interact with the intron-containing substrate RNA and with each other to form a dynamic network of RNA interactions that define the intron and promote splicing. There is evidence that protein splicing factors play important roles in regulating RNA interactions in the spliceosome. PRP8 is a highly conserved protein that is associated in particles with the U5 snRNA and directly binds the substrate RNA in spliceosomes. UV crosslinking has been used to map the binding sites, and shows extensive interaction between PRP8 protein and the 5' exon prior to the first step of splicing and with the 3' splice site region subsequently. It is proposed that PRP8 protein may stabilize fragile interactions between the U5 snRNA and exon sequences at the splice sites, to anchor and align them in the catalytic centre of the spliceosome.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.

Splice site recognition and catalysis of the transesterification reactions in the spliceosome are accompanied by a dynamic series of interactions involving conserved or invariant sequences in the spliceosomal snRNAs. We have used site-specific photoactivated crosslinking in yeast spliceosomes to monitor interactions between snRNAs and exon sequences near the 5' and 3' splice sites. The last nucleotide of the 5' exon can be crosslinked to an invariant loop sequence in U5 SnRNA before and after 5' splice site cleavage. The first nucleotide of the 3' exon can also be crosslinked to the same U5 loop sequence, but this contact is only detectable after the first transesterification. These results are in close agreement with earlier data from mammalian splicing extracts, and they are consistent with a model in which U5 snRNA aligns the 5' and 3' exons for the second transesterification. After the first catalytic step of splicing, the first nucleotide of the 3' exon can also crosslink to nt U23 in U2 snRNA. This is one of a cluster of residues in U2-U6 helix I implicated by mutational analysis in the second catalytic step of splicing. The crosslinking data suggest that these residues in U2-U6 helix I are in close proximity to the scissile phosphodiester bond at the 3' splice site prior to the second transesterification. These results constitute the first biochemical evidence for a direct interaction between the 3' splice site and U2 snRNA.

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 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.

Journal Article Branched poly-labelled oligonucleotides: enhanced specificity of fork-shaped biotinylated oligoribonucleotides for antisense affinity selection Get access Stefan Teigelkamp, Stefan Teigelkamp Search for other works by this author on: Oxford Academic PubMed Google Scholar Susanne Ebel, Susanne Ebel 11 University of Edinburgh, Department of ChemistryWest Mains Road, Edinburgh EH9 3JJ, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar David W. Will, David W. Will 11 University of Edinburgh, Department of ChemistryWest Mains Road, Edinburgh EH9 3JJ, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Tom Brown, Tom Brown 11 University of Edinburgh, Department of ChemistryWest Mains Road, Edinburgh EH9 3JJ, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Jean D. Beggs Jean D. Beggs * *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 21, Issue 19, 25 September 1993, Pages 4651–4652, https://doi.org/10.1093/nar/21.19.4651 Published: 25 September 1993 Article history Received: 14 July 1993 Accepted: 11 August 1993 Published: 25 September 1993