302 Pre-mRNA splicing in the new millennium Michelle L Hastings and Adrian R Krainer* The past year has witnessed refinements in models of The subsequent association of the U4/U6U5 tri-snRNP with spliceosome assembly pathways and in the understanding of pre-mRNA results in formation of the B complex, and finally, how splicing factors of the serine/arginine-rich (SR) protein the C complex is formed by remodelling of RNA–RNA family function. The role of splicing in human genetic diseases and RNA–protein interactions to create the catalytically has also received a lot of attention recently as exonic splicing competent spliceosome. enhancers become better understood. Spliceosome assembly is facilitated, in part, by SR proteins, Addresses which are a family of splicing factors that have one or two Cold Spring Harbor Laboratory, 1 Bungtown Road, PO Box 100, Cold copies of an RNA-recognition motif (RRM) followed by an Spring Harbor, New York 11724-2208, USA arginine/serine-rich (RS) domain [2]. The RRMs mediate *e-mail: [email protected] RNA binding and determine substrate specificity for individ- Current Opinion in Cell Biology 2001, 13:302–309 ual SR proteins, whereas the RS domain appears to be required for protein–protein interactions. SR proteins have 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. diverse roles in constitutive and alternative splicing. One such role is the recognition of exonic splicing enhancers (ESEs), Abbreviations which mediate splicing stimulation [3]. SR proteins act in a 3′ss 3′ splice site 5′ss 5′ splice site substrate-specific manner by binding to cognate ESEs, which BPS branch point sequence consist of degenerate sequence motifs. The degeneracy of ESE exonic splicing enhancer the consensus recognition motifs for SR proteins probably Py tract polypyrimidine tract allows overlap in binding, and specificity may result from RNAi RNA interference combinatorial effects, different SR protein levels, binding RRM RNA-recognition motif RS arginine/serine-rich affinities and specific interactions with other proteins. SR serine/arginine-rich U2AF U2 auxiliary factor The mechanisms by which the splicing machinery recog- nizes pre-mRNA have been the focus of several key studies Introduction in the past year, and these will be highlighted in this review. The precise removal of pre-mRNA introns is a critical The discovery of novel spliceosomal intermediates and aspect of gene expression. Not only must the splicing their implications for the mechanism of early splice-site machinery recognize and remove introns to make the cor- recognition will be discussed. Several studies that provide rect message for protein production but also, for many insights into the function of SR proteins in constitutive and genes, alternative splicing mechanisms must be in place to alternative splicing will also be reviewed. In addition, we generate functionally diverse protein isoforms in a spatially will describe reports that illustrate the physiological impor- and temporally regulated manner. The splicing reaction tance of alternative splicing and consequences of aberrant is carried out by the spliceosome, which consists of five splicing. Several topics not discussed here are addressed in small nuclear ribonucleoprotein complexes U1, U2, U4, recent reviews on alternative splicing [4,5], nuclear local- U5 and U6 snRNPs and a large number of non-snRNP pro- ization of splicing [6] and catalytic activity of the teins. The spliceosome acts through a multitude of spliceosome [1]. In addition, systematic analyses of RNA–RNA, RNA–protein and protein–protein interac- sequences from the entire Drosophila melanogaster [7] and tions to precisely excise each intron and join the exons in Schizosaccharomyces pombe [8] genomes allowed useful com- the correct order [1]. parisons of the human, yeast and fly splicing machinery. For efficient splicing, most introns require a conserved 5′ Rethinking spliceosome assembly splice site (5′ss), and a branch point sequence (BPS) followed The current model of spliceosome assembly proposes that by a polypyrimidine tract (Py tract) and a 3′ splice site (3′ss). U2 snRNP first associates with the pre-mRNA in an ATP- Assembly of the spliceosome onto pre-mRNA is an ordered dependent manner in the A complex. However, U2 process with several distinct intermediates. In metazoans, snRNP has now been identified as a component of a puri- current models hold that commitment of pre-mRNA to the fied, functional E complex [9••]. U2 snRNP association splicing pathway occurs upon ATP-independent formation of with the E complex occurs in the absence of ATP and the E complex. Assembly of the E complex involves recogni- does not require BPS interactions. The most straightfor- tion of the 5′ss by U1 snRNA base pairing and association of ward interpretation of the data is that the U2 snRNP first non-snRNP splicing factors, such as serine/arginine-rich (SR) binds loosely to the pre-mRNA in the E complex via the proteins and the U2 auxiliary factor (U2AF), which binds to integral U2 snRNP-associated protein SF3b, and then an the Py tract and 3′ss. Next, U2 snRNA base pairs with the ATP-dependent process leads to stable binding of U2 BPS during ATP-dependent formation of the A complex. snRNP to the BPS in the A complex (Figure 1). However, Pre-mRNA splicing in the new millennium Hastings and Krainer 303 Figure 1 mRNA Exon 1 GU A YYYYYYYYYY AG Exon 2 U2 U2snRNP snRNP SF3a SF3a SF3b SF3b U1 U1 snRNP snRNP ATP U2AF65 35 U2AF65 35 Exon 1 GU A YYYYYYYYYY AG Exon 2 Exon 1 GU A YYYYYYYYYY AG Exon 2 E complex Prp8 U4/U6•U5 U5 snRNP ATP U6 U4 U1 U2 U1 U2 snRNP snRNP snRNP snRNP U2AF65 35 U2AF65 35 Exon 1 GU A YYYYYYYYYY AG Exon 2 Exon 1 GU A YYYYYYYYYY AG Exon 2 A complex Prp8 U4/U6•U5 U5 snRNP U6 U4 Current Opinion in Cell Biology Intermediates in early spliceosome assembly. In the E complex, U1 consequence of tri-snRNP association with the pre-mRNA but probably snRNP binds at the 5′ss (red GU), and U2 snRNP loosely binds to the reflects a stabilization that occurs upon rearrangement of RNA–RNA pre-mRNA near the 3′ss (red AG) in an ATP-independent manner, interactions, including replacement of U1 by U6 at the 5′ss. A defined perhaps via interactions between SF3b and the U2AF heterodimer spliceosomal intermediate containing both the U1 snRNP and tri-snRNP, and/or U1 snRNP [9••]. In the presence of ATP, the U4/U6U5 tri- with or without U2 snRNP, has not been identified for the major splicing snRNP binds to the 5′ss region, in part through interactions between pathway, although evidence for an analogous interaction has been Prp8 and the pre-mRNA, and U2 snRNP stably binds to the BPS (single obtained for the U12-dependent pathway [13••]. Red vertical lines red A) [12••]. These two events can apparently occur independently (not indicate RNA base-pairing interactions, and filled circles and squares shoen). In this model, subsequent formation of the B complex is not a depict snRNA 5′ cap structures. under physiological conditions, in the presence of ATP, propose that U2 snRNP addition to the spliceosome does this temporal sequence may not be obligatory. not strictly require ATP but may be under the negative control of CUS2. Another study in yeast showed that the Similar to spliceosome assembly in higher eukaryotes, the requirement for the SUB2 ATPase could be bypassed by first ATP-dependent step in Saccharomyces cerevisiae deletion of the yeast homolog of U2AF65, MUD2 [11••]. pre-mRNA splicing also involves the stable binding of Both studies describe situations in which ATP-dependent U2 snRNP. Interestingly, in yeast extracts lacking the U2 steps in splicing can be bypassed, suggesting that some of suppressor protein, CUS2, a complex containing U2 the energy-requiring steps in splicing are not essential but snRNP can form in the absence of ATP [10••]. The authors may have evolved as regulatory checkpoints. 304 Nucleus and gene expression Figure 2 U4/U6U5 tri-snRNP both define the 5′ss before interac- tions with the 3′ss and U2 snRNP occur. Recognition of each splicing signal by multiple components is probably (a) RS very important for splicing fidelity. RRM U1 New findings from research on the U12-dependent AT-AC snRNP splicing pathway also provide clues to how the spliceosome 70K forms. An oligonucleotide displacement method allowed U2AF65 35 identification of U4atac–U6atac and U12–U6atac interac- tions that form without displacement of U11 by U6atac at GU A YYYYYYYY AG the 5′ss [13••]. However, the U12–U6atac–U4atac presump- Intron definition tive intermediate is not required for displacement of U11 (RS-domain-dependent) snRNP, suggesting that an alternative pathway for assembly of the minor spliceosome exists. This work also demon- (b) RS strates that the establishment of U6atac snRNA interactions with the 5′ss requires sequences within the 5′ exon and that RRM U2AF65 35 U5 and U6atac interact simultaneously with the pre-mRNA ′ GU A YRYRYRYY AG ESE near the 5 ss even in the absence of U12. These results are consistent with observations made with the U2-dependent Weak 3′ss/U2AF35-dependent splicing pathway ([12••] and references therein). (RS-domain-dependent) In addition to early spliceosome assembly, insights into the (c) second catalytic step of splicing have been reported recently, including the function of the adenine at the 3′ss ? [14] and the critical role of RNA helicase activity in yeast RRM I U2AF65 pre-mRNA splicing [15•]. In addition, the finding that GU A YYYYYYYYY AG ESE metal ion coordination by yeast U6 snRNA is required for splicing, provides strong evidence that U6 snRNA partici- Strong 3′ss/U2AF35-independent pates in splicing catalysis [16••].