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 assembly pathways and in the understanding of pre-mRNA results in formation of the B complex, and finally, how splicing factors of the /-rich (SR) 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 , 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 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 . 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 the consensus recognition motifs for SR proteins probably Py 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 is a critical The discovery of novel spliceosomal intermediates and aspect of 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 , 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 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 [7] and tions to precisely excise each and join the in Schizosaccharomyces pombe [8] allowed useful com- the correct order [1]. parisons of the human, 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 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 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 , the requirement for the SUB2 ATPase could be bypassed by first ATP-dependent step in 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

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 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••]. (RS-domain-dependent) Current Opinion in Cell Biology Insights into SR protein function Previous studies showed that the RS domain of the SR RS domain-dependent and -independent activities of SR proteins. (a) The RS domain of SF2/ASF is needed to mediate splicing that protein SF2/ASF is required for constitutive splicing depends on intron-definition interactions. The RS domain of SF2/ASF in vitro and for cell viability in vivo, although it is dispens- may interact with the RS domains of U1-70K and U2AF35 to facilitate able for dose-dependent switching between alternative cross-intron bridging. (b) Splicing of some introns with a weak Py tract 5′ss [2]. However, a report this year demonstrated that the requires U2AF35 binding at the 3′ss, as well as the RS domain of SF2/ASF. (c) The RS domain and U2AF35 are dispensable for splicing RS domain of SF2/ASF is not required for in vitro splicing of some introns with strong splice sites. The RS domain may not be of all pre-mRNAs [17•]. Specifically, the RS domain is dis- required for mediating the activity of certain ESEs. In this case, two pensable for processing of several constitutively spliced possible mechanisms are envisaged: first, other portions of the protein, pre-mRNAs with strong splice sites. In contrast, the RS besides the RS domain, may mediate critical protein–protein interactions; second, binding of SF2/ASF to the pre-mRNA via the domain is required for splicing of an intron with a weak- RRMs may be sufficient to promote splicing by competing with ened Py tract and for some, but not all, ESE-dependent inhibitory factors (I), such as hnRNP proteins, which have antagonistic substrates. The same substrates also require U2AF35, the activities in splice-site selection. U2AF subunit that specifically recognizes the 3′ss AG [2,3,17•]. The RS domain is also required when splicing is dependent on intron-definition interactions. Discrepancies In contrast to current models for spliceosome assembly, with earlier work probably result from the previous use of which depict stable binding of U2 snRNP to the BPS protein tags. Taken together, these results suggest that before association of the U4/U6U5 tri-snRNP, a recent there are two pathways by which SR proteins promote study exploited a cis- and trans-splicing competition sys- splicing (Figure 2). One pathway requires the RS domain tem to reveal a new intermediate in spliceosome assembly and the other does not. Two possible mechanisms for the [12••]. This study reports that the tri-snRNP interacts with latter activity of SR proteins can be envisaged. First, other the 5′ss and the upstream 5′ exon at an earlier step in portions of the protein, besides the RS domains, may spliceosome assembly than previously thought (Figure 1). mediate critical protein–protein interactions. Second, These interactions occur in an ATP-dependent manner in binding of SF2/ASF to the pre-mRNA via the RRMs may the absence of stable U2 snRNP binding and appear to be be sufficient to promote splicing by competing with other guided, in part, by the U5 snRNP-associated protein factors, such as hnRNP proteins, that antagonize SR Prp8/220. The authors propose that U1 snRNP and the proteins in splice-site selection. Pre-mRNA splicing in the new millennium Hastings and Krainer 305

A competitive binding mechanism for the RS-domain- follow-up study this year demonstrates that other SR independent SR protein activity is consistent with a report proteins complement the loss of B52 in most tissues [26•]. demonstrating that competition for pre-mRNA binding However, in the brain, where B52 is the predominant SR between SF2/ASF and hnRNP A1 underlies their antago- protein, the levels of other SR proteins are not sufficient to nistic functions in alternative splicing; this competition compensate for loss of B52 in the null mutant. These does not require the RS domain of the SR protein [18]. In results further indicate that the requirement for a particu- terms of a mechanism for alternative splice-site selection, lar SR protein may be due to specific functions in the SF2/ASF enhances U1 snRNP binding at all 5′ss and/or tissue or developmental stage in which a particular SR interferes with hnRNP A1 binding, resulting in a situation protein is predominant. that favors splicing via the 5′ss nearest to the 3′ss. In con- trast, hnRNP A1 binds indiscriminately to the pre-mRNA, Splice-site recognition which interferes with U1 snRNP binding and results in Although many sequences within mammalian transcripts splicing at the 5′ss with the highest affinity for U1 snRNP. match the consensus splice sites, most of them are not used. A recent report provides evidence that these pseudo- The use of alternative and inefficient splice sites may be splice sites have multiple defects and are inhibited by influenced by competitive binding of SR proteins and surrounding splicing silencer sequences [27]. The arrange- hnRNP proteins, and the mechanism by which splice-site ment of positive and negative cis-acting sequence usage is dynamically regulated appears to be determined, elements is probably one solution to the problem of at least in part, by the relative ratio of hnRNP A1 to SR finding authentic splice sites. Positive elements promote proteins in the nucleus. A natural stimulus that influences splicing at appropriate times and at correct splice sites, the ratio of these proteins in the nucleus is genotoxic whereas negative elements bound by trans-acting factors stress, which induces hnRNP A1 by the may block splicing at pseudo-splice sites and partially or p38-MAP pathway and results in accumulation of completely repress splicing at inefficient or regulated hnRNP A1 in the [19•]. The resulting decrease splice sites. It seems increasingly clear that SR proteins in nuclear hnRNP A1 relative to SR proteins alters splice- and hnRNP proteins are at least some of the factors that site selection, thereby linking alternative splicing mediate these regulatory activities. regulation to a specific signal transduction pathway. Recent work has also provided insights into the mechanism SR proteins modulate an unusual mode of alternative of 3′ss selection ([28•]; see also Update) and led to the iden- splicing in interferon regulatory factor-3 (IRF-3) tification of splicing factors involved in the selection of pre-mRNA [20]. In this transcript, the core sequence correct splice sites [29•,30•,31–34] in constitutive or alterna- AG/GUGCGU serves in a mutually exclusive manner as tive splicing, especially when weak or regulated splice sites either a 3′ss by use of the AG or as a 5′ss by use of the first are involved. Progress has also been made in understanding GU. Because A and G are the most conserved the role of polypyrimidine-tract-binding protein in the at the –2 and –1 exonic positions of a 5′ss, this alternative regulation of tissue-specific alternative splicing [35•,36–38]. splicing scenario may well take place in other pre-mRNAs as well. A subset of SR proteins appears to promote the use Applying splicing models to human disease of the site as a 5′ss by interacting with a region down- and genetic diversity stream. In this case, although the putative enhancer is An important mechanism for regulation of gene expression is within an alternative exon, it functions from an intronic alternative splicing, which can expand the coding capacity of position, as it enhances the use of the upstream 5′ss rather a single gene to allow production of different protein isoforms, than the adjacent upstream 3′ss. which often have very different functions [4]. An analysis of the prevalence of alternative splicing, on the basis of align- SR proteins are functionally redundant in splicing of some ment of available EST sequences to the , estimates introns but exhibit unique functions in removal of others, that at least 35% of human genes are alternatively spliced consistent with their multiple functions in splicing. The ([39,40]; see also Update). The Drosophila Dscam gene, which notion of partial redundancy is supported by results from codes for a cell surface protein involved in neuronal connec- RNA interference (RNAi) knockouts of SR proteins in tivity, provides a striking example of alternative splicing . An SF2/ASF knockout resulted in [41••]. Dscam has 24 exons, with 12 alternative versions of defects in early embryonic development and lack of viability exon 4, 48 versions of exon 6, 33 versions of exon 9 and 2 [21,22•]. However, when all the other SR proteins were also versions of exon 17. The protein sequences coded by each set targeted by RNAi, no obvious was observed. of alternative exons are highly related. Each version of a Instead, lethality was only seen when combinations of these particular exon is used to the exclusion of all the others. Thus, SR proteins were targeted simultaneously [22•,23]. the combinatorial use of alternative exons in the Dscam pre-mRNA can potentially generate 38,016 different protein In Drosophila, the B52 gene, a homolog of human SRp55, isoforms. In addition to the remarkable diversity of potential is essential for development, although several genes show proteins produced from this single pre-mRNA, the splicing of proper pre-mRNA splicing in the arrested larvae [24,25]. A Dscam exemplifies the rigid regulation of alternative splicing 306 Nucleus and gene expression

Figure 3

Exonic point that cause altered RS splicing. (a) Nonsense mutations (red circles) can result in both degradation of spliced RRM mRNAs (nonsense-mediated decay) and abnormal splicing, often manifested as wt ESE skipping of the mutant exon. The skipping of exons containing nonsense mutations may Nonsense Nonsense or result from presumptive nuclear missense mutation scanning or disruption of secondary structure. (b) can also result from the (a) (b) disruption of a splicing enhancer sequence within the skipped exon. In the case of ESE mut ESE mut X abrogation, aberrant exon skipping may be associated with any type of base substitution Exon scanning ESE disruption in the exon (black circle). or secondary structure disruption?

Nonsense-mediated Exon skipping Exon skipping decay Current Opinion in Cell Biology

that must be in place to somehow enforce not only the choice Progress has also been made in understanding the role of of each version of a particular exon but also to exclude all aberrant splicing in other human diseases [42,46]. A single other homologous versions of the exon once one version has difference between the two non-allelic copies of been selected. the survival of motor neurons gene, SMN1 and SMN2, affects the resulting mRNA species [47]. SMN1 mRNA On one hand, alternative splicing contributes to genetic codes for full-length SMN, whereas the SMN2 gene mostly diversity by generating multiple protein isoforms; on the produces an aberrantly spliced message lacking exon 7. Loss other hand, mutations in either alternatively or constitu- of the SMN1 gene, and the corresponding decrease in full- tively spliced genes can trigger aberrant splicing, which length SMN protein, correlates with the development of can lead to human disease [3,42]. A recent report finds that (SMA). An ESE within exon 7 that mutations affecting mRNA splicing are the most common is critical for exon inclusion has been identified [48]. This cause of neurofibromatosis type 1 (NF1), one of the most ESE is 13 nucleotides downstream of the single nucleotide prevalent inherited disorders in humans [43•]. Similar difference between SMN2 and SMN1, which is critical for findings were reported for the ATM gene, which is respon- the splicing patterns in human cells. However, this down- sible for ataxia telangiectasia [44]. Interestingly, in both stream ESE does appear to be relevant to SMN splicing, cases the identification of the mutations at the DNA level because activation of the identified ESE by overexpression predicted a lower level of splicing mutations, whereas the of the splicing factor Htra2-β1 can compensate for the splic- proportion was significantly increased when the effect of ing defect associated with the natural SMN2 substitution each mutation was examined at the mRNA level. Part of [49]. Whether the natural substitution in the SMN2 gene this discrepancy results from the fact that some nonsense disrupts another ESE, thereby accounting for the mutations cause skipping of exons, a phenomenon known prevalence of exon 7 skipping, remains to be determined. as nonsense-associated altered splicing (NAS; Figure 3). Targeting of the splicing pathway as a means for human A recent study has examined the role of ESEs in exon skip- disease therapy is an attractive alternative to . ping associated with nonsense mutations [45••]. A nonsense The feasibility of such approaches has been demonstrated mutation in the breast susceptibility gene BRCA1 that by a report of successful treatment of erythroid progenitor results in abnormal exon skipping actually disrupts an ESE cells from thalassemic patients carrying mutations in the consisting of a high-score recognition motif for the SR pro- HBB (β-globin) gene [50•]. The mutations activate cryptic tein SF2/ASF (Figure 3). The exon-skipping phenotype splice sites in β-globin pre-mRNA, resulting in a deficiency could be reproduced invitro and was shown not to result from of adult hemoglobin A. Using antisense oligonucleotides disruption of the translational reading frame. These results targeted to the cryptic splice sites, correct splicing was appear to be applicable to many other mutations, since other restored, and an increase in hemoglobin production was genes with nonsense, missense or silent substitutions known observed. The oligonucleotides were able to enter the cell, to cause exon skipping reduced or eliminated high-score migrate to the nucleus, hybridize to the β-globin pre- motifs for SR proteins in over half of the cases. mRNA and block splicing at the targeted site, resulting in Pre-mRNA splicing in the new millennium Hastings and Krainer 307

increased splicing at the authentic splice site and correction SC35 is important for T cell development, as well as for of the splicing defect. Because aberrant splicing is a com- alternative splicing of the receptor tyrosine phosphatase mon cause of genetic disorders, this approach to correcting CD45. This study establishes a role for an SR protein in a splicing defects may be widely applicable in human disease physiological process and in alternative processing in vivo. therapy, although targeting less accessible cell types may be In the future, it should be possible to use similar methods more challenging than in the case of peripheral blood cells. to analyze the involvement of additional SR proteins in tissue- and developmentally-specific processes. Conclusions In summary, the year 2000 has brought insights into Acknowledgements spliceosome assembly, indicating that the recognition of We thank Jim Duffy for artwork, and Mikko Frilander, Joan Steitz and ′ ′ Christine Guthrie for communicating results prior to publication. We the 5 ss and 3 ss by several components of the spliceosome acknowledge support from a post-doctoral fellowship from the American takes place earlier than previously thought. In addition, Cancer Society, grants GM42699 from the National Institutes of Health and the role of SR proteins in mediating splice-site recognition CA13106 from the National Cancer Institute. is complex and involves RS-domain-dependent and -inde- pendent activities that may be determined by the strength References and recommended reading of the splice sites, as well as the presence of competing Papers of particular interest, published within the annual period of review, have been highlighted as: factors that negatively affect splicing. Finally, there has • of special interest been a realization that the incidence of disease-associated •• of outstanding interest mutations that affect splicing is probably much higher than 1. Collins CA, Guthrie C: The question remains: is the spliceosome a previously thought, highlighting the importance of eluci- ribozyme? Nat Struct Biol 2000, 7:850-854. dating splicing mechanisms in order to develop methods to 2. Graveley BR: Sorting out the complexity of SR protein functions. therapeutically correct aberrant splicing. RNA 2000, 6:1197-1211. 3. Blencowe BJ: Exonic splicing enhancers: mechanism of action, Update diversity and role in human genetic diseases. Trends Biochem Sci The recent completion of a draft of the 2000, 25:106-110. and initial analysis of the sequence has led to important 4. Black DL: Protein diversity from alternative splicing: A challenge for bioinformatics and post-genome biology. Cell 2000, insights into the process of pre-mRNA splicing. Of partic- 103:367-370. ular interest is the conclusion that the human genome 5. Smith CWJ, Valcárcel J: Alternative pre-mRNA splicing: the appears to have only 30,000–40,000 protein-coding genes, logic of combinatorial control. Trends Biochem Sci 2000, which is only two to three fold more than the number of 25:381-388. genes in invertebrates like C. elegans and D. melanogaster 6. Lewis JD, Tollervey D: Like attracts like: Getting things together in the nucleus. Science 2000, 288:1385-1389. [51,52]. In view of this finding, it seems likely that post- 7. Mount SM, Salz HK: Pre-messenger RNA processing factors in the transcriptional mechanisms, especially alternative splicing, Drosophila genome. J Cell Biol 2000, 150:F37-F43. play a crucial role in achieving the biological complexity of 8. Kaufer NF, Potashkin J: Analysis of the splicing machinery in fission vertebrates, compared with simpler . Indeed, yeast: a comparison with budding yeast and mammals. Nucleic analysis of potential alternatively spliced transcripts from Acids Res 2000, 28:3003-3010. chromosomes 22 and 19 has led to the extrapolation that at 9. Das R, Zhou Z, Reed R: Functional association of U2 snRNP with least 59% of human genes are alternatively spliced [51]. •• the ATP-independent spliceosomal complex E. Mol Cell 2000, 5:779-787. Because this analysis only considered known transcripts, U2 snRNP is found to associate with the E complex in an ATP-independent alternative splicing is likely to be even more prevalent manner that does not require the BPS. than the current estimates. 10. Perriman R, Ares M Jr: ATP can be dispensable for •• prespliceosome formation in yeast. Genes Dev 2000, 14:97-107. Continuing efforts to understand 3′ss selection have A pre-spliceosomal complex containing U2 snRNP can form in the absence uncovered a complex mechanism for recognition of the of ATP, in yeast extracts lacking the U2 suppressor protein CUS2. correct 3′ss AG [53•]. This mechanism does not conform to 11. Kistler AL, Guthrie C: Deletion of MUD2, the yeast •• homologue of U2AF65, can bypass the requirement for Sub2, an previous scanning or measuring models. Instead, when two essential spliceosomal ATPase. Genes Dev 2001, 15:42-49. AG dinucleotides are located downstream of the BPS, the In yeast lacking Mud2 (the homolog of human U2AF65), the ATPase Sub2 (the homolog of human UAP56) is dispensable for wild-type growth. The AG closest to the BPS is initially recognized, and then data suggest that Sub2 may function to promote Mud2 association with, and dictates the location of the AG that is finally utilized in the removal from, the pre-mRNA. splicing reaction. Thus, if the positioning AG is too close 12. Maroney PA, Romfo CM, Nilsen TW: Functional recognition of the to the BPS, it cannot be used for the second step of •• 5′ splice site by U4/U6.U5 tri-snRNP defines a novel ATP- dependent step in early spliceosome assembly. Mol Cell 2000, splicing; the downstream AG can then function as the 6:317-328. transesterification AG, but only if it is less than six An early interaction between U4/U6U5 tri-snRNP and the 5′ss is identified that occurs independently of U2 snRNP binding at the BPS. nucleotides downstream of the positioning AG. 13. Frilander MJ, Steitz JA: Dynamic exchanges of RNA interactions •• leading to catalytic core formation in the U12-dependent A new report documents the in vivo requirement for the spliceosome. Mol Cell 2001, 7:217-226. SR protein SC35 [54••]. A mouse model based on the con- A novel ternary snRNA interaction of U12–U6atac–U4atac occurring prior to U11 displacement from the 5′ss, is identified using an oligonucleotide ditional deletion of SC35 in T cells was used to show that block and displacement method. 308 Nucleus and gene expression

14. Gaur RK, Beigelman L, Haeberli P, Maniatis T: Role of adenine linear search for a 3′ss when the AG is more than 35 nucleotides from the functional groups in the recognition of the 3′-splice-site AG BPS, the selection of a 3′ss AG is random when the AG is less than 13–22 during the second step of pre-mRNA splicing. Proc Natl Acad Sci nucleotides from the BPS. USA 2000, 97:115-120. 29. Förch P, Puig O, Kedersha N, Martínez C, Granneman S, Séraphin B, 15. Schwer B, Meszaros T: RNA helicase dynamics in pre-mRNA • Anderson P, Valcárcel J: The -promoting factor TIA-1 is a • splicing. EMBO J 2000, 19:6582-6591. regulator of alternative pre-mRNA splicing. Mol Cell 2000, Mutations in the yeast DExH-box protein Prp22p uncouple the NTPase and 6:1089-1098. helicase activities of the protein and formally demonstrate the requirement TIA-1, a protein previously implicated in apoptosis and homologous to the for helicase activity in pre-mRNA splicing. yeast Nam8 splicing factor, is shown to bind to -rich stretches down- stream of certain 5′ss. The levels of the protein influence Drosophila msl-2 16. Yean S-L, Wuenschell G, Termini J, Lin R-J: Metal-ion •• pre-mRNA alternative splicing in vitro, as well as alternative splicing of the coordination by U6 small nuclear RNA contributes to catalysis in mammalian apoptotic gene Fas in vivo. the spliceosome. Nature 2000, 408:881-884. Yeast U6 snRNA coordination of a divalent metal ion is required for the 30. Spingola M, Ares M Jr: A yeast intronic splicing enhancer and catalytic steps of splicing. This report provides compelling evidence that the • Nam8p are required for Mer1p-activated splicing. Mol Cell 2000, splicing reaction is, at least in part, catalyzed by RNA. 6:329-338. Mer1p-dependent splicing enhancers are identified in three yeast introns 17. Zhu J, Krainer AR: Pre-mRNA splicing in the absence of an SR • whose splicing is activated during meiosis. The U1 snRNP protein Nam8p, protein RS domain. Genes Dev 2000, 14:3166-3178. which is normally not required for general splicing, is indispensable for The SR protein SF2/ASF is found to promote pre-mRNA splicing of several Mer1p/enhancer-dependent splicing of these meiosis-specific introns. constitutively spliced substrates even in the absence of its RS domain. Some ESEs act by recruitment of U2AF35 via interactions with an SR 31. Barnard DC, Patton JG: Identification and characterization of a protein RS domain, whereas other ESEs require neither the RS domain nor novel serine-arginine-rich splicing regulatory protein. Mol Cell Biol U2AF35 to promote splicing. 2000, 20:3049-3057. 18. Eperon IC, Makarova OV, Mayeda A, Munroe SH, Cáceres JF, 32. Del Gatto-Konczak F, Bourgeois CF, Le Guiner C, Kister L, Hayward DG, Krainer AR: Selection of alternative 5′ splice sites: Gesnel M-C, Stévenin J, Breathnach R: The RNA-binding protein role of U1 snRNP and models for the antagonistic effects of TIA-1 is a novel mammalian splicing regulator acting through SF2/ASF and hnRNPA1. Mol Cell Biol 2000, 20:8303-8318. intron sequences adjacent to a 5′ splice site. Mol Cell Biol 2000, 20:6287-6299. 19. van der Houven van Oordt W, Diaz-Meco MT, Lozano J, Krainer AR, • Moscat J, Cáceres JF: The MKK3/6-p38-signaling cascade alters 33. McCullough AJ, Berget SM: An intronic splicing enhancer binds U1 the subcellular distribution of hnRNP A1 and modulates snRNPs to enhance splicing and select 5′ splice sites. Mol Cell alternative splicing regulation. J Cell Biol 2000, 149:307-316. Biol 2000, 20:9225-9235. Stress stimuli result in the MKK3/6-p38-mediated cytoplasmic accumulation of hnRNP A1. A concomitant decrease in the nuclear hnRNP A1 to SR 34. Rutz B, Séraphin B: A dual role for BBP/ScSF1 in nuclear pre- protein ratio correlates with a change in the alternative splicing pattern of a mRNA retention and splicing. EMBO J 2000, 19:1873-1886. reporter mRNA, suggesting that signaling mechanisms regulate pre-mRNA Multisite splicing by altering the subcellular distribution of antagonistic splicing factors. 35. Chou M-Y, Underwood JG, Nikolic J, Luu MHT, Black DL: • RNA binding and release of polypyrimidine tract binding protein 20. Karpova AY, Howley PM, Ronco LV: Dual utilization of an during the regulation of c-src neural-specific splicing. Mol Cell acceptor/donor site governs the alternative splicing of the IRF-3 2000, 5:949-957. gene. Genes Dev 2000, 14:2813-2818. Neural-specific splicing of c-src is derepressed in neuronal cell extracts by the ATP-dependent dissociation of polypyrimidine tract-binding protein 21. Kuroyanagi H, Kimura T, Wada K, Hisamoto N, Matsumoto K, (PTB) from intronic elements. Hagiwara M: SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline 36. Carstens RP, Wagner EJ, Garcia-Blanco MA: An intronic splicing development. Mech Dev 2000, 99:51-64. silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract 22. Longman D, Johnstone IL, Cáceres JF: Functional characterization binding protein. Mol Cell Biol 2000, 20:7388-7400. • of SR and SR-related genes in Caenorhabditis elegans. EMBO J 2000, 19:1625-1637. 37. Markovtsov V, Nikolic JM, Goldman JA, Turck CW, Chou M-Y, RNAi with C. elegans SR protein genes reveals that loss of SF2/ASF, but not Black DL: Cooperative assembly of an hnRNP complex induced other SR proteins, causes lethality. Simultaneous RNAi with multiple SR pro- by a tissue-specific homolog of polypyrimidine tract binding teins causes developmental defects or lethality. The results are consistent with protein. Mol Cell Biol 2000, 20:7463-7479. partial SR protein redundancy in splicing. Several other splicing factor protein homologs are analyzed by RNAi, some of which show distinct . 38. Polydorides AD, Okano HJ, Yang YYL, Stefani G, Darnell RB: A brain- enriched polypyrimidine tract-binding protein antagonizes the 23. Kawano T, Fujita M, Sakamoto H: Unique and redundant ability of Nova to regulate neuron-specific alternative splicing. functions of SR proteins, a conserved family of splicing factors, Proc Natl Acad Sci USA 2000, 97:6350-6355. in Caenorhabditis elegans development. Mech Dev 2000, 95:67-76. 39. Croft L, Schandorff S, Clark F, Burrage K, Arctander P, Mattick JS: ISIS, the intron information system, reveals the high of 24. Ring HZ, Lis JT: The SR protein B52/SRp55 is essential for alternative splicing in the human genome. Nat Genet 2000, Drosophila development. Mol Cell Biol 1994, 14:7499-7506. 24:340-341. 25. Peng X, Mount SM: Genetic enhancement of RNA-processing 40. Mironov AA, Fickett JW, Gelfand MS: Frequent alternative splicing defects by a dominant mutation in B52, the Drosophila gene of human genes. Genome Res 2000, 9:1288-1293. for an SR factor. Mol Cell Biol 1995, 15:6273-6282. 41. Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, •• Dixon JE, Zipursky SL: Drosophila Dscam is an axon guidance 26. Hoffman BE, Lis JT: Pre-mRNA splicing by the essential Drosophila receptor exhibiting extraordinary molecular diversity. Cell 2000, • protein B52: tissue and target specificity. Mol Cell Biol 2000, 101:671-684. 20:181-186. Alternative splicing of Drosophila Dscam pre-mRNA can potentially In vitro splicing using extracts from tissues of Drosophila null mutants for generate 38,016 Dscam protein isoforms. This report exemplifies the B52, the human homolog of SRp55, suggests that in brain tissue, where complex regulatory mechanisms involved in alternative splicing. B52 is the predominant SR protein, splicing is compromised. However, in other tissues, sufficient levels of other SR proteins apparently complement 42. Phillips AV, Cooper TA: RNA processing and human disease. Cell the B52 depletion and splicing is not affected. Mol Sci 2000, 57:235-249. 27. Sun H, Chasin LA: Multiple splicing defects in an intronic false 43. Ars E, Serra E, Garcia J, Kruyer H, Gaona A, Lazaro C, Estivill X: • exon. Mol Cell Biol 2000, 20:6414-6425. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum 28. Chen S, Anderson K, Moore MJ: Evidence for a linear search in Mol Genet 2000, 9:237-247. • bimolecular 3′ splice site AG selection. Proc Natl Acad Sci USA A systematic analysis of cDNAs from patients with NF1 reveals that out of 2000, 97:593-598. 44 different mutations, 19 (43%) result in aberrantly spliced transcripts, The authors present evidence that 3′ss selection occurs by a 5′ to 3′ whereas only 13 (30%) were predicted to affect mRNA splicing when the directional search for splice sites. They also propose that although there is a mutations were analyzed at the DNA level. This study highlights the Pre-mRNA splicing in the new millennium Hastings and Krainer 309

prevalence of splicing mutations in human disease and the importance of 50. Lacerra G, Sierakowska H, Carestia C, Fucharoen S, Summerton J, studying mutations at both the genomic and RNA levels. • Weller D, Kole R: Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients. Proc 44. Teraoka SN, Telatar M, Becker-Catania S, Liang T, Önengüt S, Tolun A, Natl Acad Sci USA 2000, 97:9591-9596. Chessa L, Sanal O, Bernatowska E, Gatti RA, Concannon P: Splicing Cells from thalassemic patients with defective alleles of β-globin were treated defects in the ataxia-telangiectasia gene, ATM: Underlying mutations β and consequences. Am J Hum Genet 1999, 64:1617-1631. with antisense modified oligonucleotides complementary to aberrant -globin splice sites, resulting in the restoration of correct splicing and protein 45. Liu H-X, Cartegni L, Zhang MQ, Krainer AR: A mechanism for exon production in a cell type that could eventually be targeted for clinical treatment. •• skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet 2001, 27:55-58. 51. International Human Genome Sequencing Consortium: Initial A nonsense mutation in BRCA1 that causes inappropriate exon skipping sequencing and analysis of the human genome. Nature 2001, does so by disrupting an ESE, rather than by altering the translational 409:860-921. reading frame. Many nonsense or missense mutations probably disrupt splicing by a similar mechanism. 52. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA et al.: The sequence of the 46. Jiang Z, Côté J, Kwon JM, Goate AM, Wu JY: Aberrant splicing of human genome. Science 2001, 291:1304-1351. tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism 53. Chua K, Reed R: An upstream AG determines whether a linked to chromosome 17. Mol Cell Biol 2000, 20:4036-4048. • downstream AG is selected during catalytic step II of splicing. Mol Cell Biol 2001, 21:1509-1514. 47. Monani UR, Coovert DD, Burghes AH: Animal models of spinal A model for 3′ss selection, based on studies of splicing of pre-mRNAs muscular atrophy. Hum Mol Genet 2000, 9:2451-2457. containing two AG 3′ss dinucleotides spaced at varying distances from the 48. Lorson CL, Androphy EJ: An exonic enhancer is required for BPS, proposes a multistep mechanism for AG selection. inclusion of an essential exon in the SMA-determining gene SMN. 54. Wang H-Y, Xu X, Ding J-H, Bermingham JR Jr, Fu X-D: SC35 plays a Hum Mol Genet 2000, 9:259-265. •• role in T cell development and alternative splicing of CD45. Mol 49. Hofman Y, Lorson CL, Stamm S, Androphy EJ, Wirth B: Htra2-β 1 Cell 2001, 7:331-342. stimulates an exonic splicing enhancer and can restore full-length The conditional deletion of the SR protein SC35 in mouse T cells indicates SMN expression to 2 (SMN2). Proc Natl that SC35 is important for T cell maturation and also influences the Acad Sci USA 2000, 97:9618-9623. regulation of a T cell-specific alternative splicing pathway.