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.

Nuclear precursor messenger RNA (pre-mRNA) splicing is the removal of introns and joining of exon sequences to form mRNA. The excision of each intron involves two sequential transesterification reactions that occur within a large, dynamic complex termed the spliceosome. Spliceosome assembly requires ATP and multiple trans-acting factors that interact with one another and with conserved cis-elements in the pre-mRNA. The mechanism of the two-step splicing reaction is highly conserved from yeast to mammals (reviewed by Green, 1991; Rymond and Rosbash, 1992; Moore et al., 1993; Sharp, 1994), as are at least some of the splicing factors (reviewed by Guthrie and Patterson, 1988; Hodges and Beggs, 1994; Hodges et al., 1995). First, the scissile phosphate at the 5′end of the intron (5′splice site) is attacked by the 2′OH of an adenosine (the branchpoint) residue in the 3′region of the intron. As the 3′-5′phosphodiester bond at the 5′splice site is cleaved there is concomitant formation of a 2′-5′phosphodiester bond between the phosphate at the 5′end of the intron and the attacking adenosine to form a branched structure, and producing the reaction intermediates: the linear 5′exon and the lariat intron-3′exon. An important question that will be addressed here is -how is the cleaved-off 5′exon retained in the catalytic centre of the spliceosome? In the second transesterification reaction the phosphate at the 3′splice site is attacked by the 3′OH of the 5′exon, resulting in joining of the two exons, and excision of the intron in lariat form.

The major subunits of the spliceosome are five small nuclear ribonucleoprotein particles (snRNPs); UI, U2, U4, U5 and U6. These snRNPs play critical roles in defining introns and folding pre-mRNAs into a conformation suitable for catalysis, and may also have catalytic roles in the splicing reactions. As characterised in metazoans each snRNP, with the exception of U6, is composed of a single small nuclear RNA (snRNA) with a trimethylguanosine cap, a set of eight common or ‘core’ proteins, and a variable number of snRNA-specific proteins (reviewed by Lührmann et al., 1990; Will et al., 1993). Unlike the others, U6 snRNA is transcribed by RNA polymerase III, has a γ-monomethyl guanosine cap structure and does not directly bind the core proteins that are common to the other snRNPs since it lacks the appropriate structural motif, the Sm-site. However, at least in Saccharomyces cerevisiae, U6 RNA associates with proteins that are structurally related to the core proteins (Cooper et al., 1995; Seraphin, 1995).

In vitro, the snRNPs and other protein splicing factors assemble on the substrate pre-mRNA in precisely defined consecutive steps to form the spliceosome, within which a network of RNA interactions develops (summarised below and in Fig. 1; for more details and references see reviews by Madhani and Guthrie, 1994; Newman, 1994; Nilsen, 1994). The U1 snRNP is the first to associate with the pre-mRNA at the 5′splice site, and subsequently the U2 snRNP assembles at the branchpoint sequence of the intron. There is substantial evidence from both biochemical and genetic suppression studies that highly conserved sequences in the U1 and U2 snRNAs interact through Watson-Crick basepairing with conserved sequences at the 5′splice site and branchpoint, respectively, in the substrate RNA. The U4 and U6 snRNAs contain extensive sequence complementarity with each other and are predominantly found base paired within a U4/U6 snRNP complex. The U4/U6 snRNP interacts with the U5 snRNP to form a U4/U6.U5 triple snRNP which then associates with the U1 -U2-pre-mRNA complex to form the spliceosome. Of the spliceosomal snRNAs, U6 is the most highly conserved in primary sequence, and it has been proposed that essential conserved motifs in U6 snRNA might be involved in the catalysis of splicing. Prior to the first transesterification reaction (step 1 of splicing) the U4/U6 basepairing appears to be destabilized, which led to the suggestion that U4 sequesters U6 in an inactive form until the spliceosomal function of U6 is required. Following this conformational change, U6 snRNA anneals with U2 snRNA to form two helices, one of them (helix I) immediately upstream of the branchpoint-binding domain of U2. A conserved sequence, ACAGAG, in U6 immediately adjacent to the helix I region interacts with a conserved intron sequence at the 5′splice site, bringing the branchpoint adenosine into close proximity with the scissile phosphate for the first transesterification reaction. The U5 snRNA primary sequence is not phylogenetically conserved except for a singlestranded loop (loop I) consisting of an invariant 9 nucleotide pyrimidine-rich sequence. Genetic suppression studies and photo-crosslinking experiments have shown that this conserved loop interacts with the last three nucleotides of the 5′exon prior to and following its cleavage from the remainder of the pre-mRNA (step 1), and with the first two nucleotides of the 3′exon prior to the second step, thus maintaining contact with the free upstream exon after the first transesterification reaction and possibly aligning it with the downstream exon for the second transesterification reaction. Since exon sequences at the splice sites are highly variable, the predominance of uridine residues in the U5 loop could be explained by their capacity for promiscuous basepairing.

Fig. 1.

RNA interactions in the spliceosome. This is a highly schematic representation of interactions between the substrate RNA and snRNAs in the spliceosome. Boxes represent exon sequences, the intron is represented by a line between the boxes, A in the intron indicates the branchpoint, and the bold arrows indicate the formation of helix I and helix II between U2 and U6 snRNAs after destabilization of the U4/U6 interaction. For simplicity, loop I of U5 snRNA is discontinuous.

Fig. 1.

RNA interactions in the spliceosome. This is a highly schematic representation of interactions between the substrate RNA and snRNAs in the spliceosome. Boxes represent exon sequences, the intron is represented by a line between the boxes, A in the intron indicates the branchpoint, and the bold arrows indicate the formation of helix I and helix II between U2 and U6 snRNAs after destabilization of the U4/U6 interaction. For simplicity, loop I of U5 snRNA is discontinuous.

ROLES FOR PROTEINS IN MODIFYING RNA INTERACTIONS

Most of these RNA interactions involve rather short conserved motifs and are unlikely to be sufficiently stable by themselves to build up and hold the complex and dynamic spliceosomal structure. The question arises what provides the necessary stability for such fragile, yet crucial interactions. There is mounting evidence that proteins contribute to the stability and specificity of these interactions and regulate conformational changes. For example, in higher eukaryotes, certain members of the SR and hnRNP protein families have RNA annealing activities and function as constitutive or alternative splicing factors, influencing splice site usage by modifying snRNP interactions with the pre-mRNA (reviewed by Dreyfuss et al., 1993; Lamm and Lamond, 1993; Burd and Dreyfuss, 1994; Horowitz and Krainer, 1994; Norton, 1994).

In yeast, there are five protein splicing factors, PRP2, PRP5, PRP16, PRP22 and PRP28, that are members of the DEAD/H-box protein family of putative RNA helicases and it has been proposed that they might influence RNA-RNA interactions in splicing (Wassarman and Steitz, 1991). PRP2 protein is required for the first transesterification reaction and interacts only transiently with spliceosomes at that time. A dominant negative mutant form of PRP2 protein has been isolated that blocks the first step of splicing and remains associated with stalled spliceosomes, directly bound to the substrate pre-mRNA (Plumpton et al., 1994; Teigelkamp et al., 1994). This protein contains a mutation in the conserved SAT motif that is proposed to be important for the RNA unwinding activity of DEAD/H proteins. Thus, the pre-mRNA may be a substrate for the putative RNA unwinding activity of PRP2 protein. PRP16 was identified through suppression of a branchpoint mutation in an intron-containing reporter gene (Couto et al., 1987). It has been proposed that PRP16 influences the accuracy of branchpoint recognition by regulating the use of a discard pathway for aberrant lariat intermediates (Burgess and Guthrie, 1993). Based on genetic experiments, it has been suggested that another DEAD box protein, PRP28, destabilizes the U4/U6 snRNA interaction prior to step 1 of splicing (Strauss and Guthrie, 1991). Thus, protein splicing factors have an essential impact on the formation, fidelity and stability of RNA-RNA interactions in early spliceosome formation.

PRP8 PROTEIN

Biochemical studies revealed that PRP8 of S. cerevisiae is a U5 snRNP-specific protein (Lossky et al., 1987; Whittaker et al., 1990), as is p22O, the human homologue of PRP8 (Anderson et al., 1989; Pinto and Steitz, 1989). Although PRP8 protein shows an extraordinarily high degree of conservation among eukaryotes, it has no obvious homology to other proteins (Hodges et al., 1995), suggesting a critical role in the splicing process. Since the high molecular mass (280 kDa in yeast) is also conserved among homologues (Jackson et al., 1988; Anderson et al., 1989; Paterson et al., 1991; Kulesza et al., 1993), the large size might be essential for the function of PRP8 protein. In vitro, PRP8 protein appears to play a role in the formation of the U4/U6.U5 tri-snRNP complex and its assembly into spliceosomes, while in vivo depletion of PRP8 protein results in degradation of U4, U5 and U6 snRNAs (Brown and Beggs, 1992). An allele of PRP8 has been isolated as a suppressor of a prp28 mutation, and it was proposed that PRP8 may counterbalance the putative U4/U6 destabilizing activity of PRP28 (Strauss and Guthrie, 1991). Following incorporation of the U4/U6.U5 triple snRNP into spliceosomes, PRP8 protein as well as the human homologue p22O interact directly with substrate RNA, as shown by UV-crosslinking using uniformly 32P-labelled pre-mRNA (Garcia-Blanco et al., 1990; Whittaker and Beggs, 1991). The PRP8 interaction 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 (Teigelkamp et al., 1995a). RNase T1 treatment of spliceosomes revealed that fragments of the substrate RNA from the 5′splice site and the branchpoint-3′splice site regions could be coimmunoprecipitated with PRP8-specific antibodies, indicating that these are potential sites of interaction of PRP8 protein with substrate RNA. Protection of the branchpoint-3′splice site region was detected only after step 1 of splicing (Teigelkamp et al., 1995a).

PHOTO-CROSSLINKING STUDIES USING 4-THIOURIDINE

As PRP8 protein interacts with substrate RNA only in assembled spliceosomes, studies of PRP8 interactions with the substrate RNA are complicated by the presence of other RNA-binding proteins in the spliceosome. To enhance the efficiency of crosslinking and to facilitate mapping of the PRP8 binding site(s), UV-crosslinking experiments were carried out using substrate RNAs containing the photoactivatable uridine analogue, 4-thiouridine, incorporated at unique sites (Teigelkamp et al., 1995b). High efficiency crosslinking of 4-thiouridine to contacting amino acids or nucleotides (usually in non-Watson-Crick interactions) can be induced by relatively low energy and brief duration UV-irradiation (Favre, 1990; Sontheimer, 1994). In 4-thiouridine the keto oxygen at position 4 on the pyrimidine ring is replaced by the fractionally larger atom sulphur, a change that is unlikely to cause major perturbations. Thus crosslinking of RNA or proteins to pre-mRNA containing 4-thiouridine is expected to reflect normal molecular interactions, and has been used to detect RNA-RNA and RNA-protein interactions in mammalian spliceosomes (Wyatt et al., 1992; Sontheimer and Steitz, 1993).

Splicing substrate RNAs containing 4-thiouridine were prepared in several stages (Fig. 2). The 3′portion, starting at the test site was produced by T7 RNA polymerase transcription primed with the dinucleotide 4-thioUpG (Sigma), and then 5′end-labelled with 32P using T4 polynucleotide kinase. The 5′portion of the substrate RNA was transcribed in vitro and the two RNAs were directionally ligated by annealing to a complementary bridging oligodeoxynucleotide and joining with T4 DNA ligase (Moore and Sharp, 1992). For each test RNA a corresponding control RNA was generated using UpG instead of 4-thioUpG to prime synthesis of the 3′portion. As these RNAs containing only normal uridine were not stimulated to crosslink by irradiation with long wavelength UV light, they acted as negative controls to confirm the site-specificity of any detected crosslink. Following UV-irradiation of splicing reactions containing the test or control substrate RNAs, samples were treated with RNase Tl, and PRP8 protein was immunoprecipitated from the reaction and analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. If PRP8 protein became 32P-labelled this indicated crosslinking to the 4-thiouridine residue at the test position in the substrate RNA. To investigate the timing of detected crosslinks with respect to the first step of the splicing reaction, results obtained with wild-type yeast splicing extracts were compared with crosslinks detected in heat-inactivated extracts produced from a temperature-sensitive prp2-1 strain. In such extracts, which lack functional PRP2 protein, spliceosomes assemble but do not carry out the first transesterification reaction unless supplemented with purified wild-type PRP2 protein (Kim and Lin, 1993; Teigelkamp et al., 1994).

Fig. 2.

The method of preparation of substrate RNA containing 4-thiouridine at a unique position. Boldface U represents 4-thiouridine, and the neighbouring 5′32P is indicated by an asterisk. In this example 4-thiouridine is incorporated as the last residue of the 5′exon (5’SS-l).

Fig. 2.

The method of preparation of substrate RNA containing 4-thiouridine at a unique position. Boldface U represents 4-thiouridine, and the neighbouring 5′32P is indicated by an asterisk. In this example 4-thiouridine is incorporated as the last residue of the 5′exon (5’SS-l).

The results are summarised in Fig. 3. PRP8 protein became crosslinked to 4-thiouridine at all three positions tested in the 5′exon: the last and the penultimate position of the 5′exon (5′SS-l and 5′SS-2), and 8 residues upstream of the 5′splice site (5′SS-8). In each case the crosslink was detected in spliceosomes lacking functional PRP2 protein, demonstrating that contact with the 5′exon was initiated prior to PRP2 action (that is prior to the first transesterification reaction). Comparable signals were obtained in control extracts supplemented with functional PRP2 protein, indicating that the contact may be maintained following PRP2 action. In contrast, PRP8 protein did not crosslink to 4-thiouridine at the 4th position of the intron (5’SS+4). Because of the sequence constraints at the 5′splice site, this was the closest intron position to the 5′splice site that was testable by this method.

Fig. 3.

Summary of the results for crosslinking of PRP8 protein to CYH2 substrate RNAs containing 4-thiouridine (Teigelkamp et al., 1995). CYH2 RNA is represented by boxes (exons) and a line (intron), and the conserved sequences at both splice sites and at the branchpoint (BP). The positions investigated for crosslinking of PRP8 protein by incorporation of 4-thiouridine are indicated below. Discrimination of crosslinks detected before and after PRP2 action is indicated. + indicates a crosslink of PRP8 protein, -indicates no detectable crosslink, (+) refers to crosslinks that may be maintained after PRP2 action, and [+] represents a crosslink the timing of which was not examined.

Fig. 3.

Summary of the results for crosslinking of PRP8 protein to CYH2 substrate RNAs containing 4-thiouridine (Teigelkamp et al., 1995). CYH2 RNA is represented by boxes (exons) and a line (intron), and the conserved sequences at both splice sites and at the branchpoint (BP). The positions investigated for crosslinking of PRP8 protein by incorporation of 4-thiouridine are indicated below. Discrimination of crosslinks detected before and after PRP2 action is indicated. + indicates a crosslink of PRP8 protein, -indicates no detectable crosslink, (+) refers to crosslinks that may be maintained after PRP2 action, and [+] represents a crosslink the timing of which was not examined.

PRP8 protein also crosslinked to 4-thiouridine at the first position of the downstream exon (3’SS+l) and two residues 3′to the branchpoint (BP+2), however, these contacts were detected only in extracts capable of undergoing the first transesterification reaction (containing functional PRP2 protein). Crosslinking of PRP8 to 4-thiouridine next to a mutant 3′splice site (CAG>CUC) indicated that the primary sequence of the substrate RNA in this region did not direct the PRP8 interaction. Also, as the 3′splice mutant RNA was unable to undergo the second transesterification reaction, contact with the 3′region was initiated after PRP2 function (required for the first step) but prior to the second step of splicing. Further experiments with substrate RNAs containing duplicate 3′splice sites or duplicated 3′exon sequences, suggested that PRP8 protein interacted with at least 13 residues of the 3′exon.

Thus PRP8 protein has at least two distinct interactions with the substrate RNA that have different kinetics with respect to the progress of the splicing reaction. PRP8 protein interacts with at least eight exon residues at the 5′splice site during step 1 and with the 3′splice site region (defined as extending from the branchpoint to the 3′splice site and at least 13 nucleotides into the downstream exon) during step 2 of splicing, indicating its close proximity to the catalytic centre(s) of the spliceosome during both steps of splicing. These contacts between PRP8 protein and the substrate RNA resemble the interactions detected between the U5 snRNA and the substrate (Newman and Norman, 1991, 1992; Cortes et al., 1993; Sontheimer and Steitz, 1993; A.J.N. unpublished). Considering that PRP8 protein is a specific component of U5 snRNPs, we propose a model (Fig. 4) in which PRP8 cooperates with the U5 snRNA, stabilizing the extremely fragile interactions between U5 snRNA and the non-conserved sequences at the ends of both exons, to anchor and align them in the active sites of the spliceosome. In HeLa spliceosomes a protein of approximately 220 kDa that is believed to be the human homologue of PRP8 has been crosslinked to position-2 relative to the 5′splice site (Wyatt et al., 1992; MacMillan et al., 1994) and to the branchpoint nucleotide with similar kinetics to those observed for PRP8 (MacMillan et al., 1994). The strong conservation of the PRP8 protein sequence may therefore reflect the importance of its function in spliceosomes. It will be interesting to determine whether the proposed stabilizing properties of PRP8 protein also affect other RNA interactions in the spliceosome as previously suggested (Strauss and Guthrie, 1991; Brown and Beggs, 1992). As such, PRP8 protein might be a pivotal component of the spliceosome, responsible for the stability of multiple RNA interactions that are subject to conformational change.

Fig. 4.

A model showing PRP8 protein and the U5 snRNA interacting with the pre-mRNA in spliceosomes before and after step 1 of splicing. Exons are shown as boxes, the intron as a curved line. Stem-loop I of U5 snRNA is represented by a thick line, and the interaction of loop I with exon residues adjacent to the splice sites is shown by thin lines. PRP8 protein is drawn as an oval covering the regions of interaction with the substrate RNA.

Fig. 4.

A model showing PRP8 protein and the U5 snRNA interacting with the pre-mRNA in spliceosomes before and after step 1 of splicing. Exons are shown as boxes, the intron as a curved line. Stem-loop I of U5 snRNA is represented by a thick line, and the interaction of loop I with exon residues adjacent to the splice sites is shown by thin lines. PRP8 protein is drawn as an oval covering the regions of interaction with the substrate RNA.

J.D.B. is supported by a Royal Society Cephalosporin Fund Senior Research Fellowship. The work described was funded by a grant from the Cancer Research Campaign. We thank Mary Plumpton for Fig. 1.

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