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The past year has witnessed refinements in models ofspliceosome assembly pathways and in the understanding ofhow splicing factors of the serine/arginine-rich (SR) proteinfamily function. The role of splicing in human genetic diseaseshas also received a lot of attention recently as exonic splicingenhancers become better understood.

AddressesCold Spring Harbor Laboratory, 1 Bungtown Road, PO Box 100, ColdSpring Harbor, New York 11724-2208, USA *e-mail: krainer@cshl.org

Current Opinion in Cell Biology 2001, 13:302–309

0955-0674/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

Abbreviations3′′ss 3′ splice site 5′′ss 5′ splice site BPS branch point sequence ESE exonic splicing enhancer Py tract polypyrimidine tract RNAi RNA interference RRM RNA-recognition motif RS arginine/serine-rich SR serine/arginine-rich U2AF U2 auxiliary factor

IntroductionThe precise removal of pre-mRNA introns is a criticalaspect of gene expression. Not only must the splicingmachinery recognize and remove introns to make the cor-rect message for protein production but also, for manygenes, alternative splicing mechanisms must be in place togenerate functionally diverse protein isoforms in a spatiallyand temporally regulated manner. The splicing reaction is carried out by the spliceosome, which consists of fivesmall nuclear ribonucleoprotein complexes U1, U2, U4,U5 and U6 snRNPs and a large number of non-snRNP pro-teins. The spliceosome acts through a multitude ofRNA–RNA, RNA–protein and protein–protein interac-tions to precisely excise each intron and join the exons inthe correct order [1].

For efficient splicing, most introns require a conserved 5′splice site (5′ss), and a branch point sequence (BPS) followedby a polypyrimidine tract (Py tract) and a 3′ splice site (3′ss).Assembly of the spliceosome onto pre-mRNA is an orderedprocess with several distinct intermediates. In metazoans,current models hold that commitment of pre-mRNA to thesplicing pathway occurs upon ATP-independent formation ofthe E complex. Assembly of the E complex involves recogni-tion of the 5′ss by U1 snRNA base pairing and association ofnon-snRNP splicing factors, such as serine/arginine-rich (SR)proteins and the U2 auxiliary factor (U2AF), which binds tothe Py tract and 3′ss. Next, U2 snRNA base pairs with theBPS during ATP-dependent formation of the A complex.

The subsequent association of the U4/U6�U5 tri-snRNP withpre-mRNA results in formation of the B complex, and finally,the C complex is formed by remodelling of RNA–RNA and RNA–protein interactions to create the catalytically competent spliceosome.

Spliceosome assembly is facilitated, in part, by SR proteins,which are a family of splicing factors that have one or twocopies of an RNA-recognition motif (RRM) followed by anarginine/serine-rich (RS) domain [2]. The RRMs mediateRNA binding and determine substrate specificity for individ-ual SR proteins, whereas the RS domain appears to berequired for protein–protein interactions. SR proteins havediverse roles in constitutive and alternative splicing. One suchrole is the recognition of exonic splicing enhancers (ESEs),which mediate splicing stimulation [3]. SR proteins act in asubstrate-specific manner by binding to cognate ESEs, whichconsist of degenerate sequence motifs. The degeneracy ofthe consensus recognition motifs for SR proteins probablyallows overlap in binding, and specificity may result fromcombinatorial effects, different SR protein levels, bindingaffinities and specific interactions with other proteins.

The mechanisms by which the splicing machinery recog-nizes pre-mRNA have been the focus of several key studiesin the past year, and these will be highlighted in this review.The discovery of novel spliceosomal intermediates andtheir implications for the mechanism of early splice-siterecognition will be discussed. Several studies that provideinsights into the function of SR proteins in constitutive andalternative splicing will also be reviewed. In addition, wewill describe reports that illustrate the physiological impor-tance of alternative splicing and consequences of aberrantsplicing. Several topics not discussed here are addressed inrecent reviews on alternative splicing [4,5], nuclear local-ization of splicing [6] and catalytic activity of thespliceosome [1]. In addition, systematic analyses ofsequences from the entire Drosophila melanogaster [7] andSchizosaccharomyces pombe [8] genomes allowed useful com-parisons of the human, yeast and fly splicing machinery.

Rethinking spliceosome assembly The current model of spliceosome assembly proposes thatU2 snRNP first associates with the pre-mRNA in an ATP-dependent manner in the A complex. However, U2snRNP has now been identified as a component of a puri-fied, functional E complex [9••]. U2 snRNP associationwith the E complex occurs in the absence of ATP anddoes not require BPS interactions. The most straightfor-ward interpretation of the data is that the U2 snRNP firstbinds loosely to the pre-mRNA in the E complex via theintegral U2 snRNP-associated protein SF3b, and then anATP-dependent process leads to stable binding of U2snRNP to the BPS in the A complex (Figure 1). However,

Pre-mRNA splicing in the new millenniumMichelle L Hastings and Adrian R Krainer*

Pre-mRNA splicing in the new millennium Hastings and Krainer 303

under physiological conditions, in the presence of ATP,this temporal sequence may not be obligatory.

Similar to spliceosome assembly in higher eukaryotes, thefirst ATP-dependent step in Saccharomyces cerevisiaepre-mRNA splicing also involves the stable binding of U2 snRNP. Interestingly, in yeast extracts lacking the U2 suppressor protein, CUS2, a complex containing U2snRNP can form in the absence of ATP [10••]. The authors

propose that U2 snRNP addition to the spliceosome doesnot strictly require ATP but may be under the negativecontrol of CUS2. Another study in yeast showed that therequirement for the SUB2 ATPase could be bypassed bydeletion of the yeast homolog of U2AF65, MUD2 [11••].Both studies describe situations in which ATP-dependentsteps in splicing can be bypassed, suggesting that some ofthe energy-requiring steps in splicing are not essential butmay have evolved as regulatory checkpoints.

Figure 1

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Current Opinion in Cell Biology

Intermediates in early spliceosome assembly. In the E complex, U1snRNP binds at the 5′ss (red GU), and U2 snRNP loosely binds to thepre-mRNA near the 3′ss (red AG) in an ATP-independent manner,perhaps via interactions between SF3b and the U2AF heterodimerand/or U1 snRNP [9•• ]. In the presence of ATP, the U4/U6�U5 tri-snRNP binds to the 5′ss region, in part through interactions betweenPrp8 and the pre-mRNA, and U2 snRNP stably binds to the BPS (singlered A) [12•• ]. These two events can apparently occur independently (notshoen). In this model, subsequent formation of the B complex is not a

consequence of tri-snRNP association with the pre-mRNA but probablyreflects a stabilization that occurs upon rearrangement of RNA–RNAinteractions, including replacement of U1 by U6 at the 5′ss. A definedspliceosomal intermediate containing both the U1 snRNP and tri-snRNP,with or without U2 snRNP, has not been identified for the major splicingpathway, although evidence for an analogous interaction has beenobtained for the U12-dependent pathway [13•• ]. Red vertical linesindicate RNA base-pairing interactions, and filled circles and squaresdepict snRNA 5′ cap structures.

304 Nucleus and gene expression

In contrast to current models for spliceosome assembly,which depict stable binding of U2 snRNP to the BPSbefore association of the U4/U6�U5 tri-snRNP, a recentstudy exploited a cis- and trans-splicing competition sys-tem to reveal a new intermediate in spliceosome assembly[12••]. This study reports that the tri-snRNP interacts withthe 5′ss and the upstream 5′ exon at an earlier step inspliceosome assembly than previously thought (Figure 1).These interactions occur in an ATP-dependent manner inthe absence of stable U2 snRNP binding and appear to beguided, in part, by the U5 snRNP-associated proteinPrp8/220. The authors propose that U1 snRNP and the

U4/U6�U5 tri-snRNP both define the 5′ss before interac-tions with the 3′ss and U2 snRNP occur. Recognition ofeach splicing signal by multiple components is probablyvery important for splicing fidelity.

New findings from research on the U12-dependent AT-ACsplicing pathway also provide clues to how the spliceosomeforms. An oligonucleotide displacement method allowedidentification of U4atac–U6atac and U12–U6atac interac-tions that form without displacement of U11 by U6atac atthe 5′ss [13••]. However, the U12–U6atac–U4atac presump-tive intermediate is not required for displacement of U11snRNP, suggesting that an alternative pathway for assemblyof the minor spliceosome exists. This work also demon-strates that the establishment of U6atac snRNA interactionswith the 5′ss requires sequences within the 5′ exon and thatU5 and U6atac interact simultaneously with the pre-mRNAnear the 5′ss even in the absence of U12. These results areconsistent with observations made with the U2-dependentsplicing pathway ([12••] and references therein).

In addition to early spliceosome assembly, insights into thesecond catalytic step of splicing have been reportedrecently, including the function of the adenine at the 3′ss[14] and the critical role of RNA helicase activity in yeastpre-mRNA splicing [15•]. In addition, the finding thatmetal ion coordination by yeast U6 snRNA is required forsplicing, provides strong evidence that U6 snRNA partici-pates in splicing catalysis [16••].

Insights into SR protein function Previous studies showed that the RS domain of the SRprotein SF2/ASF is required for constitutive splicingin vitro and for cell viability in vivo, although it is dispens-able for dose-dependent switching between alternative5′ss [2]. However, a report this year demonstrated that theRS domain of SF2/ASF is not required for in vitro splicingof all pre-mRNAs [17•]. Specifically, the RS domain is dis-pensable for processing of several constitutively splicedpre-mRNAs with strong splice sites. In contrast, the RSdomain is required for splicing of an intron with a weak-ened Py tract and for some, but not all, ESE-dependentsubstrates. The same substrates also require U2AF35, theU2AF subunit that specifically recognizes the 3′ss AG[2,3,17•]. The RS domain is also required when splicing isdependent on intron-definition interactions. Discrepancieswith earlier work probably result from the previous use ofprotein tags. Taken together, these results suggest thatthere are two pathways by which SR proteins promotesplicing (Figure 2). One pathway requires the RS domainand the other does not. Two possible mechanisms for thelatter activity of SR proteins can be envisaged. First, otherportions of the protein, besides the RS domains, maymediate critical protein–protein interactions. Second,binding of SF2/ASF to the pre-mRNA via the RRMs maybe sufficient to promote splicing by competing with otherfactors, such as hnRNP proteins, that antagonize SR proteins in splice-site selection.

Figure 2

RS domain-dependent and -independent activities of SR proteins.(a) The RS domain of SF2/ASF is needed to mediate splicing thatdepends on intron-definition interactions. The RS domain of SF2/ASFmay interact with the RS domains of U1-70K and U2AF35 to facilitatecross-intron bridging. (b) Splicing of some introns with a weak Py tractrequires U2AF35 binding at the 3′ss, as well as the RS domain ofSF2/ASF. (c) The RS domain and U2AF35 are dispensable for splicingof some introns with strong splice sites. The RS domain may not berequired for mediating the activity of certain ESEs. In this case, twopossible mechanisms are envisaged: first, other portions of the protein,besides the RS domain, may mediate critical protein–proteininteractions; second, binding of SF2/ASF to the pre-mRNA via theRRMs may be sufficient to promote splicing by competing withinhibitory factors (I), such as hnRNP proteins, which have antagonisticactivities in splice-site selection.

GU A YYYYYYYY AG

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Pre-mRNA splicing in the new millennium Hastings and Krainer 305

A competitive binding mechanism for the RS-domain-independent SR protein activity is consistent with a reportdemonstrating that competition for pre-mRNA bindingbetween SF2/ASF and hnRNP A1 underlies their antago-nistic functions in alternative splicing; this competitiondoes not require the RS domain of the SR protein [18]. Interms of a mechanism for alternative splice-site selection,SF2/ASF enhances U1 snRNP binding at all 5′ss and/orinterferes with hnRNP A1 binding, resulting in a situationthat favors splicing via the 5′ss nearest to the 3′ss. In con-trast, hnRNP A1 binds indiscriminately to the pre-mRNA,which interferes with U1 snRNP binding and results insplicing at the 5′ss with the highest affinity for U1 snRNP.

The use of alternative and inefficient splice sites may beinfluenced by competitive binding of SR proteins andhnRNP proteins, and the mechanism by which splice-siteusage is dynamically regulated appears to be determined,at least in part, by the relative ratio of hnRNP A1 to SRproteins in the nucleus. A natural stimulus that influencesthe ratio of these proteins in the nucleus is genotoxicstress, which induces hnRNP A1 phosphorylation by thep38-MAP kinase pathway and results in accumulation ofhnRNP A1 in the cytoplasm [19•]. The resulting decreasein nuclear hnRNP A1 relative to SR proteins alters splice-site selection, thereby linking alternative splicingregulation to a specific signal transduction pathway.

SR proteins modulate an unusual mode of alternativesplicing in interferon regulatory factor-3 (IRF-3) pre-mRNA [20]. In this transcript, the core sequenceAG/GUGCGU serves in a mutually exclusive manner aseither a 3′ss by use of the AG or as a 5′ss by use of the firstGU. Because A and G are the most conserved nucleotidesat the –2 and –1 exonic positions of a 5′ss, this alternativesplicing scenario may well take place in other pre-mRNAsas well. A subset of SR proteins appears to promote the useof the site as a 5′ss by interacting with a region down-stream. In this case, although the putative enhancer iswithin an alternative exon, it functions from an intronicposition, as it enhances the use of the upstream 5′ss ratherthan the adjacent upstream 3′ss.

SR proteins are functionally redundant in splicing of someintrons but exhibit unique functions in removal of others,consistent with their multiple functions in splicing. Thenotion of partial redundancy is supported by results fromRNA interference (RNAi) knockouts of SR proteins inCaenorhabditis elegans. An SF2/ASF knockout resulted indefects in early embryonic development and lack of viability[21,22•]. However, when all the other SR proteins were alsotargeted by RNAi, no obvious phenotype was observed.Instead, lethality was only seen when combinations of theseSR proteins were targeted simultaneously [22•,23].

In Drosophila, the B52 gene, a homolog of human SRp55,is essential for development, although several genes showproper pre-mRNA splicing in the arrested larvae [24,25]. A

follow-up study this year demonstrates that other SR proteins complement the loss of B52 in most tissues [26•].However, in the brain, where B52 is the predominant SRprotein, the levels of other SR proteins are not sufficient tocompensate for loss of B52 in the null mutant. Theseresults further indicate that the requirement for a particu-lar SR protein may be due to specific functions in thetissue or developmental stage in which a particular SR protein is predominant.

Splice-site recognitionAlthough many sequences within mammalian transcriptsmatch the consensus splice sites, most of them are notused. A recent report provides evidence that these pseudo-splice sites have multiple defects and are inhibited bysurrounding splicing silencer sequences [27]. The arrange-ment of positive and negative cis-acting sequenceelements is probably one solution to the problem of finding authentic splice sites. Positive elements promotesplicing at appropriate times and at correct splice sites,whereas negative elements bound by trans-acting factorsmay block splicing at pseudo-splice sites and partially orcompletely repress splicing at inefficient or regulatedsplice sites. It seems increasingly clear that SR proteinsand hnRNP proteins are at least some of the factors thatmediate these regulatory activities.

Recent work has also provided insights into the mechanismof 3′ss selection ([28•]; see also Update) and led to the iden-tification of splicing factors involved in the selection ofcorrect splice sites [29•,30•,31–34] in constitutive or alterna-tive splicing, especially when weak or regulated splice sitesare involved. Progress has also been made in understandingthe role of polypyrimidine-tract-binding protein in the regulation of tissue-specific alternative splicing [35•,36–38].

Applying splicing models to human diseaseand genetic diversityAn important mechanism for regulation of gene expression isalternative splicing, which can expand the coding capacity ofa single gene to allow production of different protein isoforms,which often have very different functions [4]. An analysis ofthe prevalence of alternative splicing, on the basis of align-ment of available EST sequences to the genome, estimatesthat at least 35% of human genes are alternatively spliced([39,40]; see also Update). The Drosophila Dscam gene, whichcodes for a cell surface protein involved in neuronal connec-tivity, provides a striking example of alternative splicing[41••]. Dscam has 24 exons, with 12 alternative versions ofexon 4, 48 versions of exon 6, 33 versions of exon 9 and 2 versions of exon 17. The protein sequences coded by each setof alternative exons are highly related. Each version of a particular exon is used to the exclusion of all the others. Thus,the combinatorial use of alternative exons in the Dscampre-mRNA can potentially generate 38,016 different proteinisoforms. In addition to the remarkable diversity of potentialproteins produced from this single pre-mRNA, the splicing ofDscam exemplifies the rigid regulation of alternative splicing

306 Nucleus and gene expression

that must be in place to somehow enforce not only the choiceof each version of a particular exon but also to exclude allother homologous versions of the exon once one version hasbeen selected.

On one hand, alternative splicing contributes to geneticdiversity by generating multiple protein isoforms; on theother hand, mutations in either alternatively or constitu-tively spliced genes can trigger aberrant splicing, whichcan lead to human disease [3,42]. A recent report finds thatmutations affecting mRNA splicing are the most commoncause of neurofibromatosis type 1 (NF1), one of the mostprevalent inherited disorders in humans [43•]. Similarfindings were reported for the ATM gene, which is respon-sible for ataxia telangiectasia [44]. Interestingly, in bothcases the identification of the mutations at the DNA levelpredicted a lower level of splicing mutations, whereas theproportion was significantly increased when the effect ofeach mutation was examined at the mRNA level. Part ofthis discrepancy results from the fact that some nonsensemutations cause skipping of exons, a phenomenon knownas nonsense-associated altered splicing (NAS; Figure 3).

A recent study has examined the role of ESEs in exon skip-ping associated with nonsense mutations [45••]. A nonsensemutation in the breast cancer susceptibility gene BRCA1 thatresults in abnormal exon skipping actually disrupts an ESEconsisting of a high-score recognition motif for the SR pro-tein SF2/ASF (Figure 3). The exon-skipping phenotypecould be reproduced invitro and was shown not to result fromdisruption of the translational reading frame. These resultsappear to be applicable to many other mutations, since othergenes with nonsense, missense or silent substitutions knownto cause exon skipping reduced or eliminated high-scoremotifs for SR proteins in over half of the cases.

Progress has also been made in understanding the role ofaberrant splicing in other human diseases [42,46]. A singlenucleotide difference between the two non-allelic copies ofthe survival of motor neurons gene, SMN1 and SMN2,affects the resulting mRNA species [47]. SMN1 mRNAcodes for full-length SMN, whereas the SMN2 gene mostlyproduces an aberrantly spliced message lacking exon 7. Lossof the SMN1 gene, and the corresponding decrease in full-length SMN protein, correlates with the development ofspinal muscular atrophy (SMA). An ESE within exon 7 thatis critical for exon inclusion has been identified [48]. ThisESE is 13 nucleotides downstream of the single nucleotidedifference between SMN2 and SMN1, which is critical forthe splicing patterns in human cells. However, this down-stream ESE does appear to be relevant to SMN splicing,because activation of the identified ESE by overexpressionof the splicing factor Htra2-β1 can compensate for the splic-ing defect associated with the natural SMN2 substitution[49]. Whether the natural substitution in the SMN2 genedisrupts another ESE, thereby accounting for the prevalence of exon 7 skipping, remains to be determined.

Targeting of the splicing pathway as a means for human disease therapy is an attractive alternative to gene therapy.The feasibility of such approaches has been demonstratedby a report of successful treatment of erythroid progenitorcells from thalassemic patients carrying mutations in theHBB (β-globin) gene [50•]. The mutations activate crypticsplice sites in β-globin pre-mRNA, resulting in a deficiencyof adult hemoglobin A. Using antisense oligonucleotidestargeted to the cryptic splice sites, correct splicing wasrestored, and an increase in hemoglobin production wasobserved. The oligonucleotides were able to enter the cell,migrate to the nucleus, hybridize to the β-globin pre-mRNA and block splicing at the targeted site, resulting in

Figure 3

Exonic point mutations that cause alteredsplicing. (a) Nonsense mutations (red circles)can result in both degradation of splicedmRNAs (nonsense-mediated decay) andabnormal splicing, often manifested asskipping of the mutant exon. The skipping ofexons containing nonsense mutations mayresult from presumptive nuclear reading framescanning or disruption of secondary structure.(b) Exon skipping can also result from thedisruption of a splicing enhancer sequencewithin the skipped exon. In the case of ESEabrogation, aberrant exon skipping may beassociated with any type of base substitutionin the exon (black circle).

Current Opinion in Cell Biology

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increased splicing at the authentic splice site and correctionof the splicing defect. Because aberrant splicing is a com-mon cause of genetic disorders, this approach to correctingsplicing defects may be widely applicable in human diseasetherapy, although targeting less accessible cell types may bemore challenging than in the case of peripheral blood cells.

ConclusionsIn summary, the year 2000 has brought insights intospliceosome assembly, indicating that the recognition ofthe 5′ss and 3′ss by several components of the spliceosometakes place earlier than previously thought. In addition,the role of SR proteins in mediating splice-site recognitionis complex and involves RS-domain-dependent and -inde-pendent activities that may be determined by the strengthof the splice sites, as well as the presence of competing factors that negatively affect splicing. Finally, there hasbeen a realization that the incidence of disease-associated mutations that affect splicing is probably much higher thanpreviously thought, highlighting the importance of eluci-dating splicing mechanisms in order to develop methods totherapeutically correct aberrant splicing.

UpdateThe recent completion of a draft of the human genomeand initial analysis of the sequence has led to importantinsights into the process of pre-mRNA splicing. Of partic-ular interest is the conclusion that the human genomeappears to have only 30,000–40,000 protein-coding genes,which is only two to three fold more than the number ofgenes in invertebrates like C. elegans and D. melanogaster[51,52]. In view of this finding, it seems likely that post-transcriptional mechanisms, especially alternative splicing,play a crucial role in achieving the biological complexity ofvertebrates, compared with simpler organisms. Indeed,analysis of potential alternatively spliced transcripts fromchromosomes 22 and 19 has led to the extrapolation that atleast 59% of human genes are alternatively spliced [51].Because this analysis only considered known transcripts,alternative splicing is likely to be even more prevalentthan the current estimates.

Continuing efforts to understand 3′ss selection haveuncovered a complex mechanism for recognition of thecorrect 3′ss AG [53•]. This mechanism does not conform toprevious scanning or measuring models. Instead, when twoAG dinucleotides are located downstream of the BPS, theAG closest to the BPS is initially recognized, and then dictates the location of the AG that is finally utilized in thesplicing reaction. Thus, if the positioning AG is too closeto the BPS, it cannot be used for the second step of splicing; the downstream AG can then function as thetransesterification AG, but only if it is less than sixnucleotides downstream of the positioning AG.

A new report documents the in vivo requirement for theSR protein SC35 [54••]. A mouse model based on the con-ditional deletion of SC35 in T cells was used to show that

SC35 is important for T cell development, as well as foralternative splicing of the receptor tyrosine phosphataseCD45. This study establishes a role for an SR protein in aphysiological process and in alternative processing in vivo.In the future, it should be possible to use similar methodsto analyze the involvement of additional SR proteins in tissue- and developmentally-specific processes.

AcknowledgementsWe thank Jim Duffy for artwork, and Mikko Frilander, Joan Steitz andChristine Guthrie for communicating results prior to publication. Weacknowledge support from a post-doctoral fellowship from the AmericanCancer Society, grants GM42699 from the National Institutes of Health andCA13106 from the National Cancer Institute.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Collins CA, Guthrie C: The question remains: is the spliceosome aribozyme? Nat Struct Biol 2000, 7:850-854.

2. Graveley BR: Sorting out the complexity of SR protein functions.RNA 2000, 6:1197-1211.

3. Blencowe BJ: Exonic splicing enhancers: mechanism of action,diversity and role in human genetic diseases. Trends Biochem Sci2000, 25:106-110.

4. Black DL: Protein diversity from alternative splicing: A challengefor bioinformatics and post-genome biology. Cell 2000,103:367-370.

5. Smith CWJ, Valcárcel J: Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci 2000,25:381-388.

6. Lewis JD, Tollervey D: Like attracts like: Getting things together inthe nucleus. Science 2000, 288:1385-1389.

7. Mount SM, Salz HK: Pre-messenger RNA processing factors in theDrosophila genome. J Cell Biol 2000, 150:F37-F43.

8. Kaufer NF, Potashkin J: Analysis of the splicing machinery in fissionyeast: a comparison with budding yeast and mammals. NucleicAcids Res 2000, 28:3003-3010.

9. Das R, Zhou Z, Reed R: Functional association of U2 snRNP with •• the ATP-independent spliceosomal complex E. Mol Cell 2000,

5:779-787.U2 snRNP is found to associate with the E complex in an ATP-independentmanner that does not require the BPS.

10. Perriman R, Ares M Jr: ATP can be dispensable for •• prespliceosome formation in yeast. Genes Dev 2000,

14:97-107.A pre-spliceosomal complex containing U2 snRNP can form in the absenceof ATP, in yeast extracts lacking the U2 suppressor protein CUS2.

11. Kistler AL, Guthrie C: Deletion of MUD2, the yeast •• homologue of U2AF65, can bypass the requirement for Sub2, an

essential spliceosomal ATPase. Genes Dev 2001, 15:42-49. In yeast lacking Mud2 (the homolog of human U2AF65), the ATPase Sub2(the homolog of human UAP56) is dispensable for wild-type growth. Thedata suggest that Sub2 may function to promote Mud2 association with, andremoval from, the pre-mRNA.

12. Maroney PA, Romfo CM, Nilsen TW: Functional recognition of the •• 5′′ splice site by U4/U6.U5 tri-snRNP defines a novel ATP-

dependent step in early spliceosome assembly. Mol Cell 2000,6:317-328.

An early interaction between U4/U6�U5 tri-snRNP and the 5′ss is identifiedthat occurs independently of U2 snRNP binding at the BPS.

13. Frilander MJ, Steitz JA: Dynamic exchanges of RNA interactions •• leading to catalytic core formation in the U12-dependent

spliceosome. Mol Cell 2001, 7:217-226. A novel ternary snRNA interaction of U12–U6atac–U4atac occurring priorto U11 displacement from the 5′ss, is identified using an oligonucleotideblock and displacement method.

Pre-mRNA splicing in the new millennium Hastings and Krainer 307

14. Gaur RK, Beigelman L, Haeberli P, Maniatis T: Role of adeninefunctional groups in the recognition of the 3′′-splice-site AGduring the second step of pre-mRNA splicing. Proc Natl Acad SciUSA 2000, 97:115-120.

15. Schwer B, Meszaros T: RNA helicase dynamics in pre-mRNA • splicing. EMBO J 2000, 19:6582-6591.Mutations in the yeast DExH-box protein Prp22p uncouple the NTPase andhelicase activities of the protein and formally demonstrate the requirementfor helicase activity in pre-mRNA splicing.

16. Yean S-L, Wuenschell G, Termini J, Lin R-J: Metal-ion •• coordination by U6 small nuclear RNA contributes to catalysis in

the spliceosome. Nature 2000, 408:881-884.Yeast U6 snRNA coordination of a divalent metal ion is required for the catalytic steps of splicing. This report provides compelling evidence that thesplicing reaction is, at least in part, catalyzed by RNA.

17. Zhu J, Krainer AR: Pre-mRNA splicing in the absence of an SR • protein RS domain. Genes Dev 2000, 14:3166-3178. The SR protein SF2/ASF is found to promote pre-mRNA splicing of severalconstitutively spliced substrates even in the absence of its RS domain.Some ESEs act by recruitment of U2AF35 via interactions with an SR protein RS domain, whereas other ESEs require neither the RS domain norU2AF35 to promote splicing.

18. Eperon IC, Makarova OV, Mayeda A, Munroe SH, Cáceres JF,Hayward DG, Krainer AR: Selection of alternative 5′′ splice sites:role of U1 snRNP and models for the antagonistic effects ofSF2/ASF and hnRNPA1. Mol Cell Biol 2000, 20:8303-8318.

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

the subcellular distribution of hnRNP A1 and modulatesalternative splicing regulation. J Cell Biol 2000, 149:307-316.

Stress stimuli result in the MKK3/6-p38-mediated cytoplasmic accumulationof hnRNP A1. A concomitant decrease in the nuclear hnRNP A1 to SR protein ratio correlates with a change in the alternative splicing pattern of areporter mRNA, suggesting that signaling mechanisms regulate pre-mRNAsplicing by altering the subcellular distribution of antagonistic splicing factors.

20. Karpova AY, Howley PM, Ronco LV: Dual utilization of anacceptor/donor site governs the alternative splicing of the IRF-3gene. Genes Dev 2000, 14:2813-2818.

21. Kuroyanagi H, Kimura T, Wada K, Hisamoto N, Matsumoto K,Hagiwara M: SPK-1, a C. elegans SR protein kinase homologue, isessential for embryogenesis and required for germlinedevelopment. Mech Dev 2000, 99:51-64.

22. Longman D, Johnstone IL, Cáceres JF: Functional characterization • of SR and SR-related genes in Caenorhabditis elegans. EMBO J

2000, 19:1625-1637.RNAi with C. elegans SR protein genes reveals that loss of SF2/ASF, but notother SR proteins, causes lethality. Simultaneous RNAi with multiple SR pro-teins causes developmental defects or lethality. The results are consistent withpartial SR protein redundancy in splicing. Several other splicing factor proteinhomologs are analyzed by RNAi, some of which show distinct phenotypes.

23. Kawano T, Fujita M, Sakamoto H: Unique and redundantfunctions of SR proteins, a conserved family of splicing factors,in Caenorhabditis elegans development. Mech Dev 2000,95:67-76.

24. Ring HZ, Lis JT: The SR protein B52/SRp55 is essential forDrosophila development. Mol Cell Biol 1994, 14:7499-7506.

25. Peng X, Mount SM: Genetic enhancement of RNA-processingdefects by a dominant mutation in B52, the Drosophila genefor an SR protein splicing factor. Mol Cell Biol 1995,15:6273-6282.

26. Hoffman BE, Lis JT: Pre-mRNA splicing by the essential Drosophila • protein B52: tissue and target specificity. Mol Cell Biol 2000,

20:181-186.In vitro splicing using extracts from tissues of Drosophila null mutants forB52, the human homolog of SRp55, suggests that in brain tissue, whereB52 is the predominant SR protein, splicing is compromised. However, inother tissues, sufficient levels of other SR proteins apparently complementthe B52 depletion and splicing is not affected.

27. Sun H, Chasin LA: Multiple splicing defects in an intronic falseexon. Mol Cell Biol 2000, 20:6414-6425.

28. Chen S, Anderson K, Moore MJ: Evidence for a linear search in • bimolecular 3′′ splice site AG selection. Proc Natl Acad Sci USA

2000, 97:593-598.The authors present evidence that 3′ss selection occurs by a 5′ to 3′directional search for splice sites. They also propose that although there is a

linear search for a 3′ss when the AG is more than 35 nucleotides from theBPS, the selection of a 3′ss AG is random when the AG is less than 13–22nucleotides from the BPS.

29. Förch P, Puig O, Kedersha N, Martínez C, Granneman S, Séraphin B, • Anderson P, Valcárcel J: The apoptosis-promoting factor TIA-1 is a

regulator of alternative pre-mRNA splicing. Mol Cell 2000,6:1089-1098.

TIA-1, a protein previously implicated in apoptosis and homologous to theyeast Nam8 splicing factor, is shown to bind to uridine-rich stretches down-stream of certain 5′ss. The levels of the protein influence Drosophila msl-2pre-mRNA alternative splicing in vitro, as well as alternative splicing of themammalian apoptotic gene Fas in vivo.

30. Spingola M, Ares M Jr: A yeast intronic splicing enhancer and • Nam8p are required for Mer1p-activated splicing. Mol Cell 2000,

6:329-338. Mer1p-dependent splicing enhancers are identified in three yeast intronswhose splicing is activated during meiosis. The U1 snRNP protein Nam8p,which is normally not required for general splicing, is indispensable forMer1p/enhancer-dependent splicing of these meiosis-specific introns.

31. Barnard DC, Patton JG: Identification and characterization of anovel serine-arginine-rich splicing regulatory protein. Mol Cell Biol2000, 20:3049-3057.

32. Del Gatto-Konczak F, Bourgeois CF, Le Guiner C, Kister L,Gesnel M-C, Stévenin J, Breathnach R: The RNA-binding proteinTIA-1 is a novel mammalian splicing regulator acting throughintron sequences adjacent to a 5′′ splice site. Mol Cell Biol 2000,20:6287-6299.

33. McCullough AJ, Berget SM: An intronic splicing enhancer binds U1snRNPs to enhance splicing and select 5′′ splice sites. Mol CellBiol 2000, 20:9225-9235.

34. Rutz B, Séraphin B: A dual role for BBP/ScSF1 in nuclear pre-mRNA retention and splicing. EMBO J 2000, 19:1873-1886.

35. Chou M-Y, Underwood JG, Nikolic J, Luu MHT, Black DL: Multisite • RNA binding and release of polypyrimidine tract binding protein

during the regulation of c-src neural-specific splicing. Mol Cell2000, 5:949-957.

Neural-specific splicing of c-src is derepressed in neuronal cell extracts bythe ATP-dependent dissociation of polypyrimidine tract-binding protein(PTB) from intronic elements.

36. Carstens RP, Wagner EJ, Garcia-Blanco MA: An intronic splicingsilencer causes skipping of the IIIb exon of fibroblast growthfactor receptor 2 through involvement of polypyrimidine tractbinding protein. Mol Cell Biol 2000, 20:7388-7400.

37. Markovtsov V, Nikolic JM, Goldman JA, Turck CW, Chou M-Y,Black DL: Cooperative assembly of an hnRNP complex inducedby a tissue-specific homolog of polypyrimidine tract bindingprotein. Mol Cell Biol 2000, 20:7463-7479.

38. Polydorides AD, Okano HJ, Yang YYL, Stefani G, Darnell RB: A brain-enriched polypyrimidine tract-binding protein antagonizes theability of Nova to regulate neuron-specific alternative splicing.Proc Natl Acad Sci USA 2000, 97:6350-6355.

39. Croft L, Schandorff S, Clark F, Burrage K, Arctander P, Mattick JS:ISIS, the intron information system, reveals the high frequency ofalternative splicing in the human genome. Nat Genet 2000,24:340-341.

40. Mironov AA, Fickett JW, Gelfand MS: Frequent alternative splicingof human genes. Genome Res 2000, 9:1288-1293.

41. Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, •• Dixon JE, Zipursky SL: Drosophila Dscam is an axon guidance

receptor exhibiting extraordinary molecular diversity. Cell 2000,101:671-684.

Alternative splicing of Drosophila Dscam pre-mRNA can potentially generate 38,016 Dscam protein isoforms. This report exemplifies the complex regulatory mechanisms involved in alternative splicing.

42. Phillips AV, Cooper TA: RNA processing and human disease. CellMol Life Sci 2000, 57:235-249.

43. Ars E, Serra E, Garcia J, Kruyer H, Gaona A, Lazaro C, Estivill X: • Mutations affecting mRNA splicing are the most common

molecular defects in patients with neurofibromatosis type 1. HumMol Genet 2000, 9:237-247.

A systematic analysis of cDNAs from patients with NF1 reveals that out of44 different mutations, 19 (43%) result in aberrantly spliced transcripts,whereas only 13 (30%) were predicted to affect mRNA splicing when themutations were analyzed at the DNA level. This study highlights the

308 Nucleus and gene expression

prevalence of splicing mutations in human disease and the importance ofstudying mutations at both the genomic and RNA levels.

44. Teraoka SN, Telatar M, Becker-Catania S, Liang T, Önengüt S, Tolun A,Chessa L, Sanal O, Bernatowska E, Gatti RA, Concannon P: Splicingdefects in the ataxia-telangiectasia gene, ATM: Underlying mutationsand consequences. Am J Hum Genet 1999, 64:1617-1631.

45. Liu H-X, Cartegni L, Zhang MQ, Krainer AR: A mechanism for exon •• skipping caused by nonsense or missense mutations in BRCA1

and other genes. Nat Genet 2001, 27:55-58.A nonsense mutation in BRCA1 that causes inappropriate exon skippingdoes so by disrupting an ESE, rather than by altering the translational reading frame. Many nonsense or missense mutations probably disrupt splicing by a similar mechanism.

46. Jiang Z, Côté J, Kwon JM, Goate AM, Wu JY: Aberrant splicing oftau pre-mRNA caused by intronic mutations associated with theinherited dementia frontotemporal dementia with parkinsonismlinked to chromosome 17. Mol Cell Biol 2000, 20:4036-4048.

47. Monani UR, Coovert DD, Burghes AH: Animal models of spinalmuscular atrophy. Hum Mol Genet 2000, 9:2451-2457.

48. Lorson CL, Androphy EJ: An exonic enhancer is required forinclusion of an essential exon in the SMA-determining gene SMN.Hum Mol Genet 2000, 9:259-265.

49. Hofman Y, Lorson CL, Stamm S, Androphy EJ, Wirth B: Htra2-ββ 1stimulates an exonic splicing enhancer and can restore full-lengthSMN expression to survival of motor neuron 2 (SMN2). Proc NatlAcad Sci USA 2000, 97:9618-9623.

50. Lacerra G, Sierakowska H, Carestia C, Fucharoen S, Summerton J, • Weller D, Kole R: Restoration of hemoglobin A synthesis in

erythroid cells from peripheral blood of thalassemic patients. ProcNatl Acad Sci USA 2000, 97:9591-9596.

Cells from thalassemic patients with defective alleles of β-globin were treatedwith antisense modified oligonucleotides complementary to aberrant β-globinsplice sites, resulting in the restoration of correct splicing and protein production in a cell type that could eventually be targeted for clinical treatment.

51. International Human Genome Sequencing Consortium: Initialsequencing and analysis of the human genome. Nature 2001,409:860-921.

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 thehuman genome. Science 2001, 291:1304-1351.

53. Chua K, Reed R: An upstream AG determines whether a• downstream AG is selected during catalytic step II of splicing. Mol

Cell Biol 2001, 21:1509-1514.A model for 3′ss selection, based on studies of splicing of pre-mRNAs containing two AG 3′ss dinucleotides spaced at varying distances from theBPS, proposes a multistep mechanism for AG selection.

54. Wang H-Y, Xu X, Ding J-H, Bermingham JR Jr, Fu X-D: SC35 plays a•• role in T cell development and alternative splicing of CD45. Mol

Cell 2001, 7:331-342.The conditional deletion of the SR protein SC35 in mouse T cells indicatesthat SC35 is important for T cell maturation and also influences the regulation of a T cell-specific alternative splicing pathway.

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