Journal of Cell Science, Supplement 19,101 -105 (1995) 101 Printed in Great Britain © The Company of Biologists Limited 1995 The role of PRP8 protein in nuclear pre-mRNA splicing in yeast Jean D. Beggs1, Stefan Teigelkamp1’* and Andrew J. Newman2 institute of Cell and Molecular Biology, University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK 2MRC Laboratory for Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 'Present address: Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany SUMMARY The removal of introns from precursor messenger RNAs has been used to map the binding sites, and shows extensive occurs in a large complex, the spliceosome, that contains interaction between PRP8 protein and the 5' exon prior to many proteins and five small nuclear RNAs (snRNAs). The the first step of splicing and with the 3' splice site region snRNAs interact with the intron-containing substrate RNA subsequently. It is proposed that PRP8 protein may and with each other to form a dynamic network of RNA stabilize fragile interactions between the U5 snRNA and interactions that define the intron and promote splicing. exon sequences at the splice sites, to anchor and align them There is evidence that protein splicing factors play in the catalytic centre of the spliceosome. 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 Key words: protein-RNA interaction, photo-crosslinking, RNA binds the substrate RNA in spliceosomes. UV crosslinking splicing, snRNP protein INTRODUCTION pre-mRNAs into a conformation suitable for catalysis, and may also have catalytic roles in the splicing reactions. As charac­ Nuclear precursor messenger RNA (pre-mRNA) splicing is the terised in metazoans each snRNP, with the exception of U6, is removal of introns and joining of exon sequences to form composed of a single small nuclear RNA (snRNA) with a tri- mRNA. The excision of each intron involves two sequential methylguanosine cap, a set of eight common or ‘core’ proteins, transestérification reactions that occur within a large, dynamic and a variable number of snRNA-specific proteins (reviewed complex termed the spliceosome. Spliceosome assembly by Lührmann et al., 1990; Will et al., 1993). Unlike the others, requires ATP and multiple trans-acting factors that interact U6 snRNA is transcribed by RNA polymerase III, has a y- with one another and with conserved d.s-elements in the pre- monomethyl guanosine cap structure and does not directly bind mRNA. The mechanism of the two-step splicing reaction is the core proteins that are common to the other snRNPs since highly conserved from yeast to mammals (reviewed by Green, it lacks the appropriate structural motif, the Sm-site. However, 1991; Rymond and Rosbash, 1992; Moore et al., 1993; Sharp, at least in Saccharomyces cerevisiae, U6 RNA associates with 1994), as are at least some of the splicing factors (reviewed by proteins that are structurally related to the core proteins Guthrie and Patterson, 1988; Hodges and Beggs, 1994; Hodges (Cooper et al., 1995; Séraphin, 1995). et al., 1995). First, the scissile phosphate at the 5' end of the In vitro, the snRNPs and other protein splicing factors intron (5' splice site) is attacked by the 2' OH of an adenosine assemble on the substrate pre-mRNA in precisely defined con­ (the branchpoint) residue in the 3' region of the intron. As the secutive steps to form the spliceosome, within which a network 3'-5' phosphodiester bond at the 5' splice site is cleaved there of RNA interactions develops (summarised below and in Fig. is concomitant formation of a 2'-5' phosphodiester bond 1 ; for more details and references see reviews by Madhani and between the phosphate at the 5' end of the intron and the Guthrie, 1994; Newman, 1994; Nilsen, 1994). The U l snRNP attacking adenosine to form a branched structure, and is the first to associate with the pre-mRNA at the 5' splice site, producing the reaction intermediates: the linear 5' exon and the and subsequently the U2 snRNP assembles at the branchpoint lariat intron-3' exon. An important question that will be sequence of the intron. There is substantial evidence from both addressed here is - how is the cleaved-off 5' exon retained in biochemical and genetic suppression studies that highly the catalytic centre of the spliceosome? In the second transes­ conserved sequences in the Ul and U2 snRNAs interact térification reaction the phosphate at the 3' splice site is through Watson-Crick basepairing with conserved sequences attacked by the 3' OH of the 5' exon, resulting in joining of the at the 5' splice site and branchpoint, respectively, in the two exons, and excision of the intron in lariat form. substrate RNA. The U4 and U6 snRNAs contain extensive The major subunits of the spliceosome are five small nuclear sequence complementarity with each other and are predomi­ ribonucleoprotein particles (snRNPs); U l, U2, U4, U5 and U6. nantly found base paired within a U4/U6 snRNP complex. The These snRNPs play critical roles in defining introns and folding U4/U6 snRNP interacts with the U5 snRNP to form a 102 J. D. Beggs, S. Teigelkamp and A. J. Newman 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. U4/U6.U5 triple snRNP which then associates with the U1-U2- changes. For example, in higher eukaryotes, certain members pre-mRNA complex to form the spliceosome. Of the spliceo- of the SR and hnRNP protein families have RNA annealing somal snRNAs, U6 is the most highly conserved in primary activities and function as constitutive or alternative splicing sequence, and it has been proposed that essential conserved factors, influencing splice site usage by modifying snRNP motifs in U6 snRNA might be involved in the catalysis of interactions with the pre-mRNA (reviewed by Dreyfuss et al., splicing. Prior to the first transestérification reaction (step 1 of 1993; Lamm and Lamond, 1993; Burd and Dreyfuss, 1994; splicing) the U4/U6 basepairing appears to be destabilized, Horowitz and Krainer, 1994; Norton, 1994). which led to the suggestion that U4 sequesters U6 in an In yeast, there are five protein splicing factors, PRP2, PRP5, inactive form until the spliceosomal function of U6 is required. PRP16, PRP22 and PRP28, that are members of the DEAD/H- Following this conformational change, U6 snRNA anneals box protein family of putative RNA helicases and it has been with U2 snRNA to form two helices, one of them (helix I) proposed that they might influence RNA-RNA interactions in immediately upstream of the branchpoint-binding domain of splicing (Wassarman and Steitz, 1991). PRP2 protein is U2. A conserved sequence, ACAGAG, in U6 immediately required for the first transesterification reaction and interacts adjacent to the helix I region interacts with a conserved intron only transiently with spliceosomes at that time. A dominant sequence at the 5' splice site, bringing the branchpoint negative mutant form of PRP2 protein has been isolated that adenosine into close proximity with the scissile phosphate for blocks the first step of splicing and remains associated with the first transestérification reaction. The U5 snRNA primary stalled spliceosomes, directly bound to the substrate pre- sequence is not phylogenetically conserved except for a single­ mRNA (Plumpton et al.. 1994; Teigelkamp et al., 1994). This stranded loop (loop I) consisting of an invariant 9 nucleotide protein contains a mutation in the conserved SAT motif that is pyrimidine-rich sequence. Genetic suppression studies and proposed to be important for the RNA unwinding activity of photo-crosslinking experiments have shown that this DEAD/H proteins. Thus, the pre-mRNA may be a substrate for conserved loop interacts with the last three nucleotides of the the putative RNA unwinding activity of PRP2 protein. PRP16 5' exon prior to and following its cleavage from the remainder was identified through suppression of a branchpoint mutation of the pre-mRNA (step 1), and with the first two nucleotides in an intron-containing reporter gene (Couto et al., 1987). It of the 3' exon prior to the second step, thus maintaining contact has been proposed that PRP16 influences the accuracy of with the free upstream exon after the first transestérification branchpoint recognition by regulating the use of a discard reaction and possibly aligning it with the downstream exon for pathway for aberrant lariat intermediates (Burgess and Guthrie, the second transestérification reaction. Since exon sequences 1993). Based on genetic experiments, it has been suggested at the splice sites are highly variable, the predominance of that another DEAD box protein, PRP28, destabilizes the uridine residues in the U5 loop could be explained by their U4/U6 snRNA interaction prior to step 1 of splicing (Strauss capacity for promiscuous basepairing. 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. ROLES FOR PROTEINS IN MODIFYING RNA INTERACTIONS PRP8 PROTEIN Most of these RNA interactions involve rather short conserved motifs and are unlikely to be sufficiently stable by themselves Biochemical studies revealed that PRP8 of S. cerevisiae is a to build up and hold the complex and dynamic spliceosomal U5 snRNP-specific protein (Lossky et al., 1987; Whittaker et structure.