RNA-RNA interactions in the spliceosome: Unraveling the ties that bind

Timothy W. Nilsen Department of Molecular Biology and Microbiology Case Western Reserve University School of Medicine Cleveland, Ohio 44106 The well-documented mechanistic similarities between nuclear pre-mRNA splicing and group II self-splicing has suggested that spliceosomal catalysis may be RNA medi- ated (see Weiner, 1993, and Wise, 1993, for recent discus- sion and references). The definitive demonstration that splicing of pre-mRNAs proceeds through successive in line transesterification reactions analogous to those observed in group I autocatalysis (Moore and Sharp, 1993) leaves little doubt that this is the case. Although it has been known for many years that splicing requires the participation of five small nuclear RNAs (Ul, U2, U4, U5, and U6) function- ing as ribonucleoproteins (U snRNPs), a detailed under- standing of their specific role(s) has been elusive. Recently, however, the combined efforts of several laboratories working in a variety of systems have provided significant insight into the inner workings of the spliceosome. While many important gaps remain, a picture is beginning to emerge in which assembly of and catalysis by the spliceo- some is orchestrated through an intricate and ordered se- ries of snRNA-pre-mRNA as well as snRNA-snRNA inter- actions. This minireview summarizes recent advances in the understanding of spliceosomal architecture. It is im- possible to cite here the extensive body of research that has contributed to the current working model of assembly and catalysis; however, excellent comprehensive reviews of the field have recently been published (Guthrie, 1991; Moore et al., 1993; Madhani and Guthrie, 1994b). Further- more, the discussion is perforce RNA-centric, since space constraints make it impossible to integrate the equally im- pressive recent progress toward understanding the role of protein factors in pre-mRNA splicing. RNA-RNA Interactions during Spliceosome Assembly For a productive (i.e., complete) splicing reaction to occur, three critical sequence elements (the 5’ splice site, the 3’ splice site, and the branch point) must be recognized in the pre-mRNA. It is well established that early in spliceosome assembly, Ul snRNP base pairs with conserved intronic sequence at the splice donor site, and U2 snRNP base pairs with the branch point region. In the U2/pre-mRNA helix, mounting evidence indicates that the branch point nucleotide is, as previously suspected, bulged (Query et al., 1994). In contrast with the 5’ splice site and branch point regions, current understanding of 3’ splice site recog- nition is fragmentary. Genetic suppression analysis via compensatory mutagenesis in fission yeast has indicated that Ul, in addition to its role at the 5’ splice site, concur- rently base pairs to conserved nucleotides at the 3’ splice site (Reich et al., 1992). This interaction provides an attrac- tive mechanism for early juxtaposition of the splice junc- tions (reviewed by Steitz, 1992) but it alone does not ap- pear to be sufficient explain 3’ splice site identification (see below). Following the stable binding of Ul and U2 to the pre- mRNA, U4, U5, and U6 snRNPs join the spliceosome as a tri-snRNP complex. In the tri-snRNP, U4 and U6 are linked through extensive intermolecular base pairing; however, the association of U5 with the U4/U6 snRNP is not well understood, and it is not known whether U5 establishes RNA-RNA contact with either U4 or U6 in the tri-snRNP complex. The mechanism whereby the tri- snRNP enters the spliceosome is also unclear. In mamma- lian cells, this step in assembly is mediated, at least part, by a base pairing interaction between the 3’ end of U6 snRNA and the 5’ end of U2 (helix II) (Datta and Weiner, 1991; Wu and Manley, 1991). Curiously, this U2/U6 pairing does not appear to be important in Saccha- romyces cerevisiae, even though the potential to form the interaction is conserved in yeast. Until recently, it was not known whether any of the RNA constituents of tri-snRNP made contact with the mRNA precursor upon entry into the spliceosome. Site-specific cross-linking in mammalian extracts now indicates that an intimate association is formed earlyon between a phyloge- netically invariant loop sequence in U5 snRNA and exon sequence just upstream of the 5’ splice site (Wyatt et al., 1993). Significantly, several lines of evidence indicate that the functional interaction between U5 snRNA and the pre- mRNA is established before Ul disengages from the 5’ splice site (see below). The First Catalytic Step The first step of splicing involves nucleophilic attack at the 5’ splice junction by the P’ -hydroxyl of the branch point adenosine. The resultant products are the lariat (or two- thirds) intermediate and free 5’ exon. Minimally, cleavage at the 5’ splice site requires identification of the splice junction phosphodiester linkage coupled with juxtaposi- tion and activation of the attacking nucleophile. It is now clear that, prior to the first catalytic step, a dramatic reorga- nization of the spliceosome involving all five of the spliceo- somal snRNAs takes place (Figure 1 B). The emerging un- derstanding of this dynamic rearrangement has provided exciting fundamental insight into the mechanism of splic- ing. The following discussion makes the assumption that all aspects of the reorganization take place concurrently, but this is undoubtedly a gross oversimplification. Im- portantly, it is not known what triggers the conformational rearrangements, and kinetic intermediates (which must exist) have not been isolated. Not surprisingly, the 5’ splice site serves as the focal point for much of the action. Cross-linking studies have shown that the conserved loop in U5 appears to shift and thereby gain a firmer grip on the terminus of the 5’ exon (Sontheimer and Steitz, 1993). Concomitantly, the base pairing between Ul and intron sequence is destabilized, and a new interaction is established in which U6 base pairs to a subset of the same intron nucleotides first recog- nized by Ul (Wassarman and Steitz, 1992; Lesser and