- Email: [email protected]
Pre-mRNA splicing
Pre-mRNA splicing Andrew I Newman MRC Laboratory of Molecular Biology, Cambridge, UK Information from yeast and mammalian pre-mRNA splicing systems has advanced our understanding of the roles of protein factors in the early steps of spliceosome assembly. New results on the stereochemistry of nuclear pre-mRNA splicing and data on the transposition of Group II self-splicing introns in vivo have fuelled the long-running debate on the evolution of introns and RNA splicing. Current Opinion in Genetics and Development 1994, 4:298-304 Introduction Spliceosomes are complex ribonucleoprotein particles whose task is to recognize the correct splice sites in nuclear mRNA precursors and catalyze the accurate removal of intervening sequences. Spliceosomal processing occurs via two distinct transesterification steps and results in the formation of branched lariat RNAs as intermediates and products. This scheme is similar to the mechanism used by Group II introns, and there has been much speculation that these two modes of splicing may be evolutionarily related. Group II introns can act autocatalytically in vitro ~ in other words, they can function without any trans-acting factors. In contrast, the spliceosome has multiple RNA and protein components, many of which perform essential functions, but few of these are understood in any detail. What roles do each of these factors play? Which ones are involved in the catalysis of the transesterification reactions? Why are multiple molecules of ATP consumed in the process? Which factors govern the choice between alternative splice sites in complex pre-mRNAs? In this review, I discuss some recent findings which address some of these questions and the debate over the ancestry of introns. Several recent reviews on RNA splicing cover the background information [1°,2,3].
Spliceosomal proteins One theme to emerge from recent studies of spliceosomal proteins is that at least some of them show striking conservation, even between organisms as distandy related as yeasts and manmaals. Genetic approaches in yeast have been particularly successful. For example, a screen for synthetic lethals was used to identify trans-acting mutations that enhance the phenotype of
a conditional allele of the S N R 1 9 gene (which encodes U1 snRNA in budding yeast) [4]. This led to the identification of an apparent homologue of the much-stuclied mammalian UIA protein. Surprisingly, the yeast U1A gene (MUD1) is not essential for splicing or viability in the presence of wild type U1 snRNA. Its function is unclear, but it may be to fold or maintain U1 snRNA in an active configuration [4]. The yeast SMD1 gene was isolated fortuitously by virtue of its close linkage to the splicing factor gene PRP38. The SMD1 protein is strikingly similar to the D1 polypeptide from human snRNP particles, and in yeast this protein performs an essential function in splicing. Furthermore, the human D1 polypeptide can complement the splicing deficiency and lethality phenotypes associated with a yeast s m d l null allele [5,6]. The early steps in the spliceosome assembly pathway have been studied intensively in an effort to understand how pre-mRNAs are recognized by spliceosoreal components and conmdtted to splicing and how the 5" and 3' boundaries of introns are identified. Earlier work in yeast showed that an association between the 5' and 3' encls of the intron is formed in the commitment complex, the earliest functional intermediate in spliceosome assembly. ATP-independent U1 snRNPcontaining complexes (E complexes) analogous to the commitment complex in yeast have also been identified in mammalian extracts; they contain U2AF and several spliceosome-associated proteins (SAPs) [7]. Efficient E complex formation requires both 5' and 3' splice sites, implying that, as in yeast, there is a functional interaction between components bound to the two ends of the intron at this early stage [8]. Biochemical analysis of HeLa cell nuclear splicing extracts has led to the identification of five chromatographic fractions required for the assembly of ATPdependent pre-spliceosomes. These fractions contain U1 and U2 snRNPs, U2AF (U2 auxiliary factor) and
Abbreviations hnRNP--heterogeneous nuclear ribonucleoprotein; RRM---RNA recognition motif; SAP--spliceosome-associatedprotein; snRNA--small nuclear RNA; snRNP--small nuclear ribonucleoprotein. 298
© Current Biology Ltd ISSN 0959-437X
Pre-mRNA splicing Newman 299 two activities named SF1 and SF3. The SF3 activity can be fractionated into two components, SF3a and SF3b. SF3a consists of three polypeptides with molecular weights of 60, 66 and 120 kDa, which correspond to three 17S U2 snRNP-specific proteins [9"]. The 60kDa SF3a protein has now been found to be immunologically related to the yeast splicing factor PRP9 [9",10], which is required for pre-spliceosome formation in yeast and interacts with the product of the SPP91 gene [11]. SPP91 turns out to be a previously identified gene, PRP21 [12]. Furthermore, SPP91 (PRP21) interacts directly with PRPll, and mutations in PRP9 and P R P l l can act synergistically, indicating that there is a close functional relationship between these proteins [13"]. These results suggest that the PRP9 and PRPll proteins can interact with SPP91 (PRP21) to form a threemolecule complex [13"]. Analysis of manmmlian SAPs shows that SAP62 is related to the yeast PRPll protein and that SAP61 is related to PRP9 [14"]. There is also evidence for a specific interaction between SAP62 and the pre-spliceosomal component SAP114 [14"]. In summary, these findings establish striking parallels between PRPs 9, 11 and 21 in yeast, the mammalian pre-spliceosomal SAPs 61, 62 and 114 and the SF3a trimeric splicing factor in HeLa cells [9",10-12,13",14"]. Furthermore, they define a group of conserved proteins involved in recruitment of U2 snRNP into the spliceosome. Additional genetic evidence suggests that PRP5 (a RNA helicase-like protein containing the DEAD motif, in the one-letter anaino acid code) is also involved in the recruitment of U2 snRNP to the pre-spliceosome, as mutations in PRP5, PRPg, PRP11 and PRP21 act synergistically with each other and in some cases with mutations in U2 snRNA [15"!. It is still not understood why ATP is required for U2 snRNP recruitment. Perhaps it reflects the activity of a helicase [16] or the requirement for an energy-driven conformational change, but, in any case, it is clear that the yeast PRP5 protein is involved in this step [15"]. There is an additional requirement for ATP later in the splicing pathway after the U4/5/6 tri-snRNP has joined the spliceosome, both before and after the first catalytic step of splicing. Again the precise role of ATP hydrolysis at this stage has not been defined. The yeast PRP2 and PRP16 factors are closely related proteins, required for the first and second catalytic steps, respectively. They contain sequence motifs found in RNA-dependent ATPases and RNA helicases. Purified PRP2 does exhibit RNA-dependent ATPase activity [17], and PRP2-dependent ATP hydrolysis is required for the first catalytic step of splicing [18]. So far, however, no RNA helicase activity has been demonstrated for these proteins. PRP16 mediates an ATP-dependent conformational change in the spliceosome before the second catalytic step and, like PRP2, it has an RNA-dependent ATPase activity, but no proven helicase activity [19]. Perhaps these factors are indeed RNA helicases with stringent co-factor requirements or substrate specificities. Alternatively, they may be important in the fidelity of splicing, as there is evidence for a pathway under the genetic control of PRP16which discards in-
correctly branched intermediates in an ATP-dependent fashion [201. Genetic approaches in yeast have also led to the identification of some of the protein factors involved in the second catalytic step of splicing. Synthetic lethality screens have been used to identify trans-acting mutations that enhance the phenotypes of conditional alleles of the SNR7 gene, which encodes U5 snRNA [211. The S£U4 and SLU7 genes are required only for the second catalytic step, and SLU4 corresponds to PRP17. Both S£U4 and SLU7 interact genetically with PRP16 and PRP18, defining a set of factors with related functions in the second catalytic step. The SLU7 protein must be involved in 3" splice site utilization, as mutations in SLU7 affect 3' splice site choice in c/s-competition experiments [22]. The PRP18 protein is a U5 snRNP-associated factor involved in the second catalytic step of splicing and is rather unusual, in that strains bearing only a null allele of PRP18 show temperature-sensitive growth. Splicing intermediates that accumulate in vitro after depletion of PRP18 are rapidly converted into products after addition of purified PRP18 protein to the reaction. This conversion is not dependent on ATP, so although the function of PRP18 is still obscure it presumably acts after the PRP16-dependent step [23,24]. The mechanism that underlies the accurate and efficient recognition of the AG dinucleotide at 3' splice sites has remained unresolved. Earlier results suggested that the 3' splice site is identified in mammalian introns via a scanning mechanism that searches for the first AG 3' of the branchpoint polypyrimidine tract. More recent data support this idea and also indicate that closely spaced AG dinucleotides can compete for recognition [25]. In these cases, competitive strength is influenced by the identity of the preceding nucleotide in the order CAG=UAG>AAG>GAG. This order corresponds to the preference at this position at natural 3' splice sites [25].
Alternative splicing and SR proteins Earlier experiments using biochemically fractionated mammalian splicing extracts showed that essential components of the splicing machinery can have profound effects on splice site choice in alternatively spliced pre-mRNAs. For example, the essential human splicing factor SF2/ASF and hnRNP A1 regulate alternative splicing in vitro by affecting 5' splice site selection [26]. SF2/ASF is a member of the SR phosphoprotein family [27], which is a group of structurally related and evolutionarily conserved splicing factors. These factors share an amino-terminal RNA recognition motif (RRM) and a carboxy-terminal domain rich in alternating Set (S) and Arg (R) residues. In reconstitution assays, any member of the family can restore splicing activity to SR protein defcient extracts, and two distinct SR proteins, SF2 and SC35, have indistinguishable effects on 5' and 3" splice site selection [28]. Despite this apparent similarity, it is clear that these proteins are not function-
300
Chromosomes and expression mechanisms ally equivalent. When given the appropriate substrates, different SR proteins can elicit different patterns of alternative splice site utilization [29]. Recent evidence indicates that SR proteins play a role in the alternative splicing of doublesex(dsx) pre-mRNA in Drosophila. Female-specific splicing of this transcript is regulated by the products of the transformer (tra) and transformer-2 (tra-2) genes, which both contain SR domains. The tra and tra-2 proteins recruit members of the SR family of general splicing factors to a regulatory element in the female-specific exon and thus activate female-specific splicing [30"]. A commitment assay has been used to exanaine SR protein function in this process and, rather surprisingly, it turns out that individual SR proteins can conmait pre-mRNAs to the splicing pathway; pre-incubation of pre-mRNA with an SR protein allows it to be spliced preferentially when splicing extract and an excess of competitor pre-mRNA are added subsequendy [31"]. Different SR proteins can commit different pre-mRNAs to splicing with pronounced substrate specificity, but the molecular basis for these effects is not currently known. U2AF binds to the pyrimidine tract upstream of the 3' splice site and is required for targeting of U2 snRNP to the branch-site on higher eukaryote pre-mRNAs. Human U2AF consists of two associated polypeptides with molecular weights of 35 and 65 kDa; only the 65 kDa protein is required to restore activity to U2AFdepleted extracts. The sequence of the U2AF65 cDNA reveals that the protein has both an RNA-binding domain and an SR domain, which is essential for activity [32]. Recendy, a cDNA clone carrying the Drosophila counterpart of U2AF65 has been isolated, and germline transformation experiments have shown that the gene is essential for viability [33]. The fission yeast UZAF65 homologue is also an essential gene product [34], and both of these U2AF65 homologues have extensive similarities with mammalian U2AF65. In Drosophila, the product of the Sex-lethal (Sxl) gene operates a splice site switch by activating a female-specific 3' splice site in the first intron of tra pre-mRNA and repressing an alternative default site. U2AF and Sxl both bind polypyrimidine tracts, but the SR domain, which enables U2AF to activate the nearby 3' splice site, is absent from Sxl. The Sxl protein inhibits the use of the default site by preventing the binding of U2AF. This enables U2AF to activate the lower affinity female-specific site instead [35]. Several reports have drawn attention to the phosphorylation and dephosphorylation of splicing factors as a potential mechanism for regulating their activity. Studies on the effects of ATP analogues, phosphatase inhibitors and purified protein phosphatases have shown that at least two different serine/threonine phosphatases are essential for the catalytic steps of splicing [36,37]. The Ul-specific 70 kDa protein (U1 70K) is an SR protein that is phosphorylated in vivo on multiple serine residues [38,39]. Thiophosphorylation of U1 70K (which gives products resistant to dephosphorylation by protein phosphatases) inhibits splicing,
although spliceosome assembly is unaffected [381. Purified spliceosomal snRNPs contain a kinase activity that phosphorylates serine residues on U1 70K and in the SR-rich domain of the ASF/SF2 splicing factor 139].
RNA interactions in the spliceosome Information about RNA-based interactions and the possible molecular basis for catalysis in the spliceosome has accumulated rapidly over the past two years [1",2,3] and this continues to be an active area. The picture that emerges is one of a complex and dynamic network of interactions between substrate and snRNA sequences. A new and unexpected interaction has recently been proposed between the G nucleotides at the 5' and 3' ends of introns [40]. The evidence for this interaction rests on the observation that there can be reciprocal suppression of mutations at these two positions in the yeast actin gene intron. When present singly, these mutations prevent the second catalytic step of splicing and so cause accumulation of lariat intermediates. Reciprocal suppression in the double mutant is allele-specific, implying that a specific interaction involving these two nucleotides is important for the second catalytic step
[401. It is well known that U1 snRNA base-pairs with conserved intron sequences at the 5" splice site in premRNAs, but recent findings reveal a more extensive repertoire of interactions for this snRNA. Genetic data indicate that, in fission yeast, U1 snRNA can also interact with the 3' splice site AG by base-pairing via the universally conserved CU (positions seven and eight) just downstream of the 5' splice site interaction region. Mutations at C7 are lethal, whereas mutations at U8 cause the cells to grow slowly and accumulate premRNAs, consistent with a general role for these two nucleotides in fission yeast splicing [41]. In contrast, mutants at these positions in budding yeast are fully functional, ruling out the possibility that there is an essential base-pairing interaction between the U1 snRNA and 3" splice sites [42]. Instead, these positions in U1 snRNA might play a part in 5' splice site selection by interacting with 5' exon sequences [42]. The interaction between the U1 snRNAand the 5' splice site can also extend downstream into the intron. Efficient splicing of the MER2 intron in budding yeast normally requires the meiosis-specific MER1 protein. An extragenic suppressor that bypassed the requirement for MER1 turned out to be an allele of the gene for U1 snRNA with a single base change that allowed pairing with position eight of the intron [43]. It is thought that only three snRNAs (U2, U5 and U6) contribute functionally once catalysis begins in the spliceosome. Earlier genetic evidence established an interaction between the conserved nine-nucleotide loop I sequence in yeast U5 snRNA and exon nucleotides adjacent to the 5' and 3" splice sites [44]. The interaction between the U5 loop motif and sequences upstream of the 5' splice site has been confirmed bio-
Pre-mRNA splicing Newman
(a) Spliceosome
Exon 1
SI)
(b) Group I intron
R.
Exon 1 5'
3' ~ J
-OU .... P = O(S) pre-mRNA
(S)O ~ P""alO" j~p.
Exon 2 ~ 3 '
G I'()11
Exon 1
% 5'
5'
Rl,
tl
Exon 1 5"
Exon2 n 3 ,
5'Y~GpZ
~~ 1st slep
X HO 3'
Substrale
l st step
Intermediates
X 3'OH
O-
,,,x
s°"
Exon 2 GpZ I Y
(S)O = P'"gqlSy
3.1G
Exon 2
CpZ Intron-exon2 lariat intermediate
~[/
Aclive sile rearranppement and splice site exchange
t l
Exon 1 5'
St, ..,"
- ~ -
5'
X
Exon2 n 3 '
~.
(S)O ~ P ~91Z ~ GpY ~
-rG
5'
X
13'
I
(S)O = P~'"'u O-
(SlO =
Exon 2
Lariat inlron
H
Spliced mRNA
Rp
P .... iI O -
S •Exon , 2
s' Z ~ 3 ' ~ G r O
2nd step
Exon 1
R~
Xr
3'
G
ti
Exon 1 5'
St,
3 0 ~ , , , "O. Exon2
2ncl step
Spliced mRNA
Splice site exchange
Exon 1 X rOH
(S)O = P ' ~ Z ~ A
Sp
' Z ~ 3 '
GpY ~
G
3'OH
Linear intron © 1994 Currenl Opinion in Genelics and Developmenl
Fig. 1. Stereochemistry of the two steps in pre-mRNA splicing. (a) Spliceosomal splicing occurs via two distinct transesterification steps at different active sites. (b) Group I intron self-splicing, in which both steps of splicing are thought to occur at a single active site. The mechanism and configuration of the first step differ substantially between the spliceosome and Group h they show opposite phosphorothioate diastereomer preferences (Sp versus Rp) and use different reactive nucleophiles (2'OH versus 3'OH). In contrast, the second reaction steps are stereochemically similar: both pathways show preference for the Sp phosphorothioate diastereomer, use of a 3'-OH as the nucleophile and the presence of a conserved guanosine (G) attached through a 3' oxygen to the phosphate at the 3' splice site. The non-bridging position (S) at which sulphur is not significantly inhibitory, along with the corresponding phosphorothioate diastereomer (Rp or Sp) is indicated for the subslrate at each step. The diastereomer that results is indicated for the product of each step. X, Y and Z indicate nucleotide positions that are not highly conserved. Adapted from [53"'].
301
302
Chromosomesand expressionmechanisms chemically in HeLa cell nuclear extracts. [451. In these experiments, ligase technology [46] was used to introduce a modified nucleotide (4-thiouridine), carrying a photo-activated cross-linking group, into the exon sequence two nucleotides upstream of the 5' splice site in a pre-mRNA substrate. Selective photoactivation of the 4-thiouridine residue resulted in crosslinks to the U5 snRNA loop sequence and to the U5 snRNP protein p220 (the mammalian counterpart of the yeast PRP8 gene produc0. These interactions between the U5 loop sequence and exon sequences at the splice sites have striking parallels in autocatalytic introns [44,45,47]. A good deal of evidence is consistent with the idea that U6 snRNA plays an important part in the catalytic steps of splicing. Cross-linking experiments have shown that an invariant sequence motif in U6 snRNA (nucleotides 47-53 [ACAGAGA] in yeast U6 snRNA) is in close proximity to the 5' splice site in the active spliceosome [48,49]. Mutations in this U6 snRNA motif can specifically block the first or second catalytic steps of splicing, and there is genetic evidence in yeast to support a model in which a U2-U6 interaction juxtaposes the invariant residues in U6 with the branchpoint [50]. The structure produced by this U2-U6 interaction resembles domain V of group II introns, which is the most conserved domain and is thought to contribute to catalysis [51].
Catalysis and intron ancestry The multiple similarities between spliceosomal and Group II intron splicing have sustained a lively and continuing debate about the possible evolutionary relationships between spliceosomal snRNAs and autocatalytic introns [47], and support the widely held belief that catalysis in the spliceosome is likely to be'RNAbased. A general model has recently been proposed for a two-metal-ion phosphoryl transfer mechanism for RNA-catalyzed reactions in spliceosomes and autocatalytic introns [52"]. According to this model, catalysis is facilitated by two divalent metal cations as in protein-catalyzed phosphoryl transfer reactions such as the 3',5'-exonuclease activity of Escbericbia coli DNA polymerase I. The model proposes that the role of the RNA is to position the two catalytic metal ions and orient the substrates via three specific binding sites. Ligase technology [46] has recently been used to make pre-mRNAs with single chiral phosphorothioate linkages at either the 5' or 3' splice site in order to follow the stereochemical course of the two catalytic steps [53%54]. These experiments showed that both catalytic steps proceed with inversion of the stereochemical configuration of the phosphate at the splice site, consistent with a transesterification mechanism involving 'in-line' SN2 nucleophilic displacement reactions (see Fig. 1). Also, as both steps are inhibited by the Rp phosphorothioate diastereomer, but not by Sl,, the
spliceosome probably shifts between two active sites for catalysis of the two transesterifications [53"'1. This is in contrast to the situation in Group I introns, where both steps of splicing are thought to occur at a single active site (Fig. 1). The discovery of Group II introns in cyanobacteria and purple bacteria [55"1, which are thought to be the ancestors of chloroplasts and mitochondria, has resulted in further insights into the ancestry of RNA splicing pathways. This fnding fits in with Cavalier-Smith's theory for the origin of spliceosomal introns [561, which suggested that they are degenerate descendants of Group II introns initially resident in the genomes of endosymbiotic purple bacteria. New data on Group II intron mobility are also consistent with CavalierSmith's scheme. It has been known for some time that Group II introns can use reverse splicing in vitro to move to allelic and non-allelic locations by processes known as homing and transposition [57,58]. Transposition of Group II introns has recently been shown to occur in vivo in the mitochondria of Podospora and yeast [59,60]. Transposition of the aI1 intron in the cox1 gene of yeast mitochondria bears all the hallmarks of an RNA-mediated event: the sequences flanking the intron are not co-transposed to the insertion site, and mutations which compromise splicing activity also prevent intron mobility [59]. This intron and its downstream companion intron a12 encode reverse transcriptases [61] that are highly specific for molecules containing the a l l and a12 introns. Group II-like introns in protomitochondrial endosymbionts could have invaded the nuclear genome via reverse splicing, reverse transcription and recombination with the genomic DNA. The development of the spliceosomal machinery by fragmentation of a self-splicing nuclear intron into transacting snRNAs would then have allowed the resident introns to lose their capacity for self-splicing.
Conclusions Significant progress has been made recently in identifying proteins involved in pre-spliceosome assembly and in defining a network of RNA-based interactions in the active spliceosome. The use of new technology for making site-specific modifications of large RNA molecules has made important contributions in this area and should continue to yield new insights into the interactions of the RNA and protein components of the spliceosome. The stereochemistry of the transesterification events in the spliceosome has been investigated using splicing substrates that contain single chiral phosphorothioate linkages. The data indicate that both steps of splicing occur as single SN2 nucleophilic displacement reactions and that the machinery probably shifts between two active sites in catalysis of the two steps. It will be very interesting to see whether Group II introns behave in a similar way.
Pre-mRNA splicing N e w m a n
Acknowledgements I would like to thank my colleagues, particularly Jim Haseloff, Kiyoshi Nagai, Ingrid Kelly and Jean Beggs, for advice and encouragement. Work on pre-ml~IA splicing in my laboratory is suppoaed by the Medical Research Council.
13. •
LEGRAIN P, CHAPON C: Interaction between PRPII and SPP91 Yeast Splicing Factors and Characterisation of a PRP9-PRPII-SPPgl Complex. Science 1993, 262:108-110. This paper presents genetic evidence for a three-protein complex involved in yeast pre-spliceosome formation. Protein-protein interactions are assayed in vivo using a two-hybrid system based on GAL4-mediated activation of a reporter gene. 14. •
References and recommended reading Papers of particular interest, published within the annual peri~.l of review, have been highlighted as: ,, of special interest *• of outstanding interest 1. MOOREM, QUERY C, SHARP P: Splicing of Precursors to • mRNA by the Spliceosome. In The RNA World. Cold Spring Harbor: Cold Spring Harlx)r Laboratory Press; 1993:303-357. Comprehensive and up-to-date review of pre-mRNA splicing, covering mechanism and stereochemistry, spliceosome assembly and the roles of snRNA and protein comlxments of the splicing machinery. 2.
RYMONI)1], llOSBASH M: Yeast Pre-mRNA Splicing. In The Molecular a n d Celhdar BIologv o f the )'east Saccbaromyces. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1992:143-192. 3. LAMONDA: The Spliceosome. Bloessays 1993, 15:595-603. 4. LIAO X, TANGJ, ROSBASH M: An Enhancer Screen Identifies a Gene that Encodes the Yeast UI snRNP A Protein: Implications for snRNP Protein Function in Pre-mRNA Splicing. Genes Dev 1993, 7:419-428. 5. RYMOND13: Convergent Transcripts of the Yeast PRP38-SMDI Locus Encode Two Essential Splicing Factors, Including the DI Core Polypeptide of Small Nuclear Ribonucleoprotein Particles. Proc Natl Acad Sol USA 1993, 90:848--852. 6. RYMONDB, ROKEACH L, HOCH S: Human snRNP Polypeptide DI Promotes Pre-mRNA Splicing in Yeast and Defines Nonessential Yeast Smdlp Sequences. Nucleic Acids Res 1993, 21:3501-3505. 7. BENNI-71"I" M, MICHAHD S, KINGS'IONJ, REED R: Protein Components Specifically Associated with Prespliceosome and Spliceosome Complexes. Genes Dev 1992, 6:1986--2000. 8. MICHAtIDS, REED R: A Functional Association Between the 5' and 3' Splice Sites is Established in the Earliest PreSpliceosome Complex (E) in Mammals. Genes Dev 1993, 7:10(O-1020. 9. BROSl R, GRONING K, BEHRENSS, LL'IHRMANNR, KR.~MERA: ln* teraction of Mammalian Splicing Factor SF3a with U2 snRNP and Relation of Its 60-kD Subunlt to Yeast PRP9. Science 1993, 262:102-105. This paper presents a biochemical analysis of the components involved in mammalian pre-spliceo.~)me assembly, showing that splicing factor SF3a consists of three 17S U2 snRNP-specific polypeptides which play a role in the incorporation of U2 snRNP into the prespliceosome. Immunological cross-reactivity between yeast splicing factor PRP9 and one of the SF3a proteins (see also [10-12,13•-15•]) is demonstrated. 10.
11.
12.
BEHRENSS, GALL%'qON F, LEGRAIN P, LUHRMANN R: Evidence that the 60-kDa Protein of 17S U2 Small Nuclear Ribonucleoprotein is Immunologicaliy and Functionally Related to the Yeast PRP9 Splicing Factor and is Required for the Efficient Formation of Prespliceosomes. Proc Naa Acad Set USA 1993, 90:8229--8233. LEGRAIN P, CHAPON C, GAI.I~SON F: Interactions Between PRP9 and SPP91 Splicing Factors Identify a Protein Complex Required in Prespliceosom¢ Assembly. Genes Dev 1993, 7:1390-I 399. ARENA."; J, ABEl.SON J: The Saccharomyce$ cerevtslae PRP21 Gene Product is an Integral Component of the Prespliceosome. Proc Naa Acad Sol USA 1993, 90:6771-6775.
BENNETTM, REEDR: Correspondence Between a Mammalian Spliceosome Component and an Essential Yeast Splicing Factor. Science 1993, 262:10%108. An analysis of SAPs in mammalian extracts shows that SAP62 is probably the functional homologue of yeast PRPll. The paper a'iso shows immunological cross reactivity between SAP61 and yeast PRP9 and protein-protein interactions between SAP62 and SAPI14. 15. .
RuIsvS, CHANG T, ABEl.SONJ: Four Yeast Spliceosomal Proteins (PRP5, PRP9, PRPI1 and PRP21) Interact to Promote U2 snRNP Binding to Pre-mRNA. Genes Dev 1993, 7:1909-1925. This paper presents biochemical and genetic evidence for interactions between four yeast proteins involved in pre-spliceosome formation, including the helicase-like factor PRP5, and suggests that these proteins interact physically and/or act concertedly to promote U2 snRNP binding to the pre-mRNA. 16.
LIAO X, COLOr H, WANG Y, ROSBASH M: Requirements for U2 snRNP Addition to Yeast Pre-mRNA. Nucleic Acids Res 1992, 20:4237-4245.
17.
KIM S, SMITH J, CLAUDEA, LIN R: The Purified Yeast PremRNA Splicing Factor PRP2 is an RNA-Dependent NTPase. EMBO J 1992, 11:2319--2326.
18.
KIM S, LIN R: Pre-mRNA Splicing within an Assembled Yeast Spliceosome Requires an RNA-Dependent ATPase and ATP Hydrolysis. Proc Nail A c a d Set USA 1993, 90:888--892.
19.
SCHWERB, GOTHRIE C: PRPI6 is an RNA-Dependent ATPase that Interacts Transiently with the Spliceosome. Nature 1991, 349:494--499.
20.
BURGESSS, GI.rI'NRIE C: A Mechanism to Enhance mRNA Splicing Fidelity: The RNA-Dependent ATPase Prpl6 Governs Usage of a Discard Pathway for Aberrant Lariat Intermediates. Cell 1993, 73:1377-1391.
21.
FRANKD, PAI"I'ERSONB, GLrrHRIE C: Synthetic Lethal Mutations Suggest Interactions Between U5 Small Nuclear RNA and Four Proteins Required for the Second Step of Splicing. Mol Cell Bfol 1992, 12:5197-5205.
22.
FRANKD, GIYrHRIE C: An Essential Splicing Factor, SLU'7, Mediates 3" Splice Site Choice in Yeast. Genes Dev 1992, 6:2112-2124.
23.
HOROWrtx D, ABELSON J: Stages in the Second Reaction of Pre-mRaNA Splicing: the Final Step is ATP Independent. Genes Dev 1993, 7:320--329.
24.
HOROWlTZD, ABELSONJ: A U5 Small Nuclear Ribonucleoprotein Particle Protein Involved Only in the Second Step of Pre-mRNA Splicing in SaccharomFce$ cem'vlMae. Mol Cett BIol 1993, 13:2959-2970.
25.
SMrrHC, CHU T, NAOAL-GINARD13: Scanning and Competition between AGs Are Involved in 3' Splice Site Selection in Mammalian Introns. Mol Cell BIol 1993, 13:4939-4952.
26.
MAYEDAA, KRAINERA: Regulation of Alternative Pre-mRNA Splicing by hnRNP A1 and Splicing Factor SF2. Cell 1992, 68:365-375.
27.
Z.MILERA, LANE W, STOLK J, ROTH M: SR Proteins: a Conserved Family of Pre-mRNA Splicing Factors. Genes Dev 1992, 6:837-847.
28.
Fu X, MAYEDAA, MANIATISM, KRAINERA: General Splicing Factors SF2 and SC35 Have Equivalent Activities In Vitro, and Both Affect Alternative 5' and 3' Splice Site Selection. Proc Nail Acad Sci USA 1992, 89:11224--11228.
29.
ZAHLERA, NEUGEBAUERK, LANE W, ROTH M: Distinct Functions of SR Proteins in Alternative Pre-mRNA Splicing. Science 1993, 260:219-222.
303
304
Chromosomesand expression mechanisms 30. •
TIAN'M, M^NnAllS T: A Splicing Enhancer Complex Cow trois Alternative Splicing of doublesex Prc-tI1RNA. Cell 1993, 74:105--114. Biochemical methods are used here to study the regulation of female-specific splicing of ~ pre-mRNA by the tra and tra-2 proteins. The data indicate a role for wa, tra-2 and other SR proteins in the formation of a complex which forms on a regulatory site in dsx pre-mRNA, and commits the splicing machinery to use the female-specific splice site. 31. Fu X: Specific Commitment of Different Pre-mRNAs to Splic• ing by Single SR Proteins. Nature 1993, 365:82--85. Purified SR proteins produced in a baculovirus expression system are sufficient to commit pre-mRiNAs to splicing, implying that they can efficiendy recruit other spliceosomal components. Different SR proteins are shown to have different pre-mRNA substrate specificities, but it is not known how they achieve their effects. 32.
33.
34. 35.
36.
ZAMOREP, PKI'TONJ, GREEN M: Cloning and Domain Structure of the Mammalian Splicing Factor U2AE. Nature 1992, 355:609--614. KANAARR, ROCHE S, I{,EALLE, GREEN M, RIO D: The Conserved Pre-mRNA Splicing Factor U2AF from Drosophila: Requirement for Viability. Science 1993, 262:569-573. POTASHKINJ, NAIK K, WEN'IX-HtlNqXRK: U2AF Homolog Required for Splicing in Vlvo. Science 1993, 262:573-575. VAI.Ci~.RCELJ, SINGH R, ZAMORE P, GREEN M: The Protein Sex-lethal Antagonizes the Splicing Factor U2AF to Regulate Alternative Splicing of Transformer Pre-mRNA. Nature 1993, 362:171-175. MERMOLtDJ, COHEN P, LAMONt:) A: Ser/Thr Protein Phosphatases are Required for Both Catalytic Steps of Pre-mRNA Splicing. Nucleic Acids Res 1992, 20:5263-5269.
37.
TAZI J, DAUGERON M, CKfHALA G, BRUNEL C, JEANI"EUR P: Adenosine Phosphorothioates (ATP(xS and ATP'rS) Differentially Affect the Two Steps of Mammalian Pre-mRNA Splicing. J Btol Chem 1992, 267:4322-4326.
38.
TAZI J, KORNS'I'ADT U, ROSSI F, JEAN'I'EIJR P, CATHAI.A G, BRONELC, LOHRMANNR: Thiophosphorylation of U I-70K Protein Inhibits Pre-mRNA Splicing. Nature 1993, 363:283-286.
39.
WOPPMANN A, WILL C, KORNS'r,~DT U, Zuo P, MANLEY J, LOHRMANN R: Identification of an snRNP-Associated Kinase Activity that Phosphorylates Arginine/Serine Rich Domains Typical of Splicing Factors. Nucleic Acids Res 1993, 21:2815--2822.
40.
PARKER R, SILICIANO P: Evidence for an Essential NonWatson-Crick Interaction between the First and Last Nucleotides of a Nuclear Pre-mRNA Intron. Nature 1993, 361:660--662.
47.
WEINERA: mRNA Splicing and Autocatalytic Introns: Distant Cousins or the Products of Chemical Determinism? Cell 1993, 72:161-164.
48.
WASSARMAND, SlXII'ZJ: Interactions of Small Nuclear RNAs with Precursor Messenger RNA During tn Vitro Splicing. Science 1992, 257:1918-1925.
49.
SAWAH, ABELC,ON J: Evidence for a Base-Pairing Interaction between U6 Small Nuclear RNA and the 5' Splice Site During the Splicing Reaction in Yeast. Proc Naa Acad Set USA 1992, 89:11269-11273.
50.
MADHANIH, GU'II-IRIE C: A Novel Base-Pairing Interaction between 02 and U6 snRNAs Suggests a Mechanism for the Catalytic Activation of the Spliceosome. Cell 1992, 71:803-817.
51.
KOCHJ, lk.)ULANGER S, DIB-HA.U S, HEBP,AR S, PERLMAN P: Group II Introns Deleted for Multiple Substructures Retain Self-Splicing Activity. Mol Cell Bfol 1992, 12:1950-1958.
52. S'rEI17_T, STI-.TI'ZJ: A General Two-Metal-Ion Mechanism for • Catalytic RNA. Proc Natl A c a d Sci USA 1993, 90:6498--6502. The model presented in this paper is based on protein-catalyzed phosphoryl transfer reactions, such as the Y,5'-exonuclease of DNA polymerase 1. Catalytic RNAs, such as self-splicing introns and the spliceo~me, are proposed to position two divalent met',d kms in an active site, with three specific binding sites to properly orient the substrates. 53. *•
MOORE M, SHARP P: Evidence for Two Active Sites in the Spliceosome Provided by Stereochemistry of Pre-mRNA Splicing. Nature 1993, 365:364-368. Ligase technology 1461 is used to make synthetic pre-mRNAs cont:dning single chiral phosphorothioates at the splice sites, and these are used to investigate the stereochemical course of the catalytic steps in splicing. The paper provides strong evidence that both steps of premRNA splicing occur as single 'in-line' SN2 nucleophilic displacement reactions. 54.
MASCHHOFFK, PAtX3,E-rU"R: The Stereochemical Course of the First Step of Pre-mRNA Splicing. Nucle,'c Acids Res 1993, 21:5456-5462.
55.
FERATJ, MICHELF: Group 11 Self-Splicing introns in Bacteria. Nature 1993, 364:358-361. PCR is used to isolate Group 11 introns from purple bacteria, the probable ancestors of mitochondria in eukaryotes. This observation is consistent with a recent hypothesis [561 for the origin of nuclear spliceosomal introns, which suggests that they originate from Group II self-splicing introns in proto-mitochondrial endosymbionts.
•
56.
CAVALIER-SMI'I'I-IT: Intron Phylogeny: a New Hypothesis. Trends Genet 1991, 7:145-148.
57.
AtlGtJS'I'INS, MtJEI.LER M, .cK':.HWEYENR: Reverse Self-Splicing of Group I1 Intron RNAs In Vitro. Nature 1990, 343:383--386.
58.
MORLM, SCHMEI.ZER C: Integration of Group It Intron bll into a Foreign RNA by Reversal of the Self-Splicing Reaction In Vitro. Cell 1990, 60:629-636.
cerevlslae Does Not Require Base Pairing with UI snR.NA. Cell 1993, 73:803---812.
59.
NANDABALANK, PRICE L, ROEDERG: Mutations in U1 snRNA Bypass the Requirement for a Cell Type-Specific RNA Splicing Factor. Cell 1993, 73:407-415.
MUELLERM, ALLMAIERM, ESKE.~;R, SCHWEYEN R: Transposition of Group II lntron a l l in Yeast and Invasion of Mitochondrial Genes at New Locations. Nature 1993, 366:174-176.
60.
SEI.LEMC, LECELLIERG, BELCOORL: Transposition of a Group II Intron. Nature 1993, 366:176-178.
44.
NEWMANA, NORMAN C: U5 snRNA Interacts with Exon Sequences at 5' and 3' Splice Sites. Cell 1992, 68:743-754.
61.
45.
WYA'rrJ, SONTHEUMERE, S'~.rFz J: Site-Specific Cross-Linking of Mammalian U5 snRN1P to the 5' Splice Site Before the First Step of Pre-mRNA Splicing. Genes Dev 1992, 6:2542-2553.
KENNELLJ, MORANJ, PERLMAN P, BUTOW R, LAMBOWI'IXA: Reverse Tran$criptase Activity Associated with Maturasc-Encoding Group It Introns in Yeast Mitochondria. Cell 1993, 73:133-146.
46.
MOOREM, SHARPP: Site-Specific Modification of Pre-mRNA: The 2'-Hydroxyl Groups at the Splice Sites. Science 1992, 256:992-997.
41.
42.
43.
REICH C, VANHOV R, PORlXR G, WISE J: Mutations at the 3' Splice Site Can Be Suppressed by Compensatory Base Changes in UI snRNA in Fission Yeast. Cell 1992, 69:] 159--1169. SI::RAPHINB, KANDELS-Li':VaSS: 3' Splice Site Recognition in S.
AJ Newman, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Spliceosomal proteins One theme to emerge from recent studies of spliceosomal proteins is that at least some of them show striking conservation, even between organisms as distandy related as yeasts and manmaals. Genetic approaches in yeast have been particularly successful. For example, a screen for synthetic lethals was used to identify trans-acting mutations that enhance the phenotype of
a conditional allele of the S N R 1 9 gene (which encodes U1 snRNA in budding yeast) [4]. This led to the identification of an apparent homologue of the much-stuclied mammalian UIA protein. Surprisingly, the yeast U1A gene (MUD1) is not essential for splicing or viability in the presence of wild type U1 snRNA. Its function is unclear, but it may be to fold or maintain U1 snRNA in an active configuration [4]. The yeast SMD1 gene was isolated fortuitously by virtue of its close linkage to the splicing factor gene PRP38. The SMD1 protein is strikingly similar to the D1 polypeptide from human snRNP particles, and in yeast this protein performs an essential function in splicing. Furthermore, the human D1 polypeptide can complement the splicing deficiency and lethality phenotypes associated with a yeast s m d l null allele [5,6]. The early steps in the spliceosome assembly pathway have been studied intensively in an effort to understand how pre-mRNAs are recognized by spliceosoreal components and conmdtted to splicing and how the 5" and 3' boundaries of introns are identified. Earlier work in yeast showed that an association between the 5' and 3' encls of the intron is formed in the commitment complex, the earliest functional intermediate in spliceosome assembly. ATP-independent U1 snRNPcontaining complexes (E complexes) analogous to the commitment complex in yeast have also been identified in mammalian extracts; they contain U2AF and several spliceosome-associated proteins (SAPs) [7]. Efficient E complex formation requires both 5' and 3' splice sites, implying that, as in yeast, there is a functional interaction between components bound to the two ends of the intron at this early stage [8]. Biochemical analysis of HeLa cell nuclear splicing extracts has led to the identification of five chromatographic fractions required for the assembly of ATPdependent pre-spliceosomes. These fractions contain U1 and U2 snRNPs, U2AF (U2 auxiliary factor) and
Abbreviations hnRNP--heterogeneous nuclear ribonucleoprotein; RRM---RNA recognition motif; SAP--spliceosome-associatedprotein; snRNA--small nuclear RNA; snRNP--small nuclear ribonucleoprotein. 298
© Current Biology Ltd ISSN 0959-437X
Pre-mRNA splicing Newman 299 two activities named SF1 and SF3. The SF3 activity can be fractionated into two components, SF3a and SF3b. SF3a consists of three polypeptides with molecular weights of 60, 66 and 120 kDa, which correspond to three 17S U2 snRNP-specific proteins [9"]. The 60kDa SF3a protein has now been found to be immunologically related to the yeast splicing factor PRP9 [9",10], which is required for pre-spliceosome formation in yeast and interacts with the product of the SPP91 gene [11]. SPP91 turns out to be a previously identified gene, PRP21 [12]. Furthermore, SPP91 (PRP21) interacts directly with PRPll, and mutations in PRP9 and P R P l l can act synergistically, indicating that there is a close functional relationship between these proteins [13"]. These results suggest that the PRP9 and PRPll proteins can interact with SPP91 (PRP21) to form a threemolecule complex [13"]. Analysis of manmmlian SAPs shows that SAP62 is related to the yeast PRPll protein and that SAP61 is related to PRP9 [14"]. There is also evidence for a specific interaction between SAP62 and the pre-spliceosomal component SAP114 [14"]. In summary, these findings establish striking parallels between PRPs 9, 11 and 21 in yeast, the mammalian pre-spliceosomal SAPs 61, 62 and 114 and the SF3a trimeric splicing factor in HeLa cells [9",10-12,13",14"]. Furthermore, they define a group of conserved proteins involved in recruitment of U2 snRNP into the spliceosome. Additional genetic evidence suggests that PRP5 (a RNA helicase-like protein containing the DEAD motif, in the one-letter anaino acid code) is also involved in the recruitment of U2 snRNP to the pre-spliceosome, as mutations in PRP5, PRPg, PRP11 and PRP21 act synergistically with each other and in some cases with mutations in U2 snRNA [15"!. It is still not understood why ATP is required for U2 snRNP recruitment. Perhaps it reflects the activity of a helicase [16] or the requirement for an energy-driven conformational change, but, in any case, it is clear that the yeast PRP5 protein is involved in this step [15"]. There is an additional requirement for ATP later in the splicing pathway after the U4/5/6 tri-snRNP has joined the spliceosome, both before and after the first catalytic step of splicing. Again the precise role of ATP hydrolysis at this stage has not been defined. The yeast PRP2 and PRP16 factors are closely related proteins, required for the first and second catalytic steps, respectively. They contain sequence motifs found in RNA-dependent ATPases and RNA helicases. Purified PRP2 does exhibit RNA-dependent ATPase activity [17], and PRP2-dependent ATP hydrolysis is required for the first catalytic step of splicing [18]. So far, however, no RNA helicase activity has been demonstrated for these proteins. PRP16 mediates an ATP-dependent conformational change in the spliceosome before the second catalytic step and, like PRP2, it has an RNA-dependent ATPase activity, but no proven helicase activity [19]. Perhaps these factors are indeed RNA helicases with stringent co-factor requirements or substrate specificities. Alternatively, they may be important in the fidelity of splicing, as there is evidence for a pathway under the genetic control of PRP16which discards in-
correctly branched intermediates in an ATP-dependent fashion [201. Genetic approaches in yeast have also led to the identification of some of the protein factors involved in the second catalytic step of splicing. Synthetic lethality screens have been used to identify trans-acting mutations that enhance the phenotypes of conditional alleles of the SNR7 gene, which encodes U5 snRNA [211. The S£U4 and SLU7 genes are required only for the second catalytic step, and SLU4 corresponds to PRP17. Both S£U4 and SLU7 interact genetically with PRP16 and PRP18, defining a set of factors with related functions in the second catalytic step. The SLU7 protein must be involved in 3" splice site utilization, as mutations in SLU7 affect 3' splice site choice in c/s-competition experiments [22]. The PRP18 protein is a U5 snRNP-associated factor involved in the second catalytic step of splicing and is rather unusual, in that strains bearing only a null allele of PRP18 show temperature-sensitive growth. Splicing intermediates that accumulate in vitro after depletion of PRP18 are rapidly converted into products after addition of purified PRP18 protein to the reaction. This conversion is not dependent on ATP, so although the function of PRP18 is still obscure it presumably acts after the PRP16-dependent step [23,24]. The mechanism that underlies the accurate and efficient recognition of the AG dinucleotide at 3' splice sites has remained unresolved. Earlier results suggested that the 3' splice site is identified in mammalian introns via a scanning mechanism that searches for the first AG 3' of the branchpoint polypyrimidine tract. More recent data support this idea and also indicate that closely spaced AG dinucleotides can compete for recognition [25]. In these cases, competitive strength is influenced by the identity of the preceding nucleotide in the order CAG=UAG>AAG>GAG. This order corresponds to the preference at this position at natural 3' splice sites [25].
Alternative splicing and SR proteins Earlier experiments using biochemically fractionated mammalian splicing extracts showed that essential components of the splicing machinery can have profound effects on splice site choice in alternatively spliced pre-mRNAs. For example, the essential human splicing factor SF2/ASF and hnRNP A1 regulate alternative splicing in vitro by affecting 5' splice site selection [26]. SF2/ASF is a member of the SR phosphoprotein family [27], which is a group of structurally related and evolutionarily conserved splicing factors. These factors share an amino-terminal RNA recognition motif (RRM) and a carboxy-terminal domain rich in alternating Set (S) and Arg (R) residues. In reconstitution assays, any member of the family can restore splicing activity to SR protein defcient extracts, and two distinct SR proteins, SF2 and SC35, have indistinguishable effects on 5' and 3" splice site selection [28]. Despite this apparent similarity, it is clear that these proteins are not function-
300
Chromosomes and expression mechanisms ally equivalent. When given the appropriate substrates, different SR proteins can elicit different patterns of alternative splice site utilization [29]. Recent evidence indicates that SR proteins play a role in the alternative splicing of doublesex(dsx) pre-mRNA in Drosophila. Female-specific splicing of this transcript is regulated by the products of the transformer (tra) and transformer-2 (tra-2) genes, which both contain SR domains. The tra and tra-2 proteins recruit members of the SR family of general splicing factors to a regulatory element in the female-specific exon and thus activate female-specific splicing [30"]. A commitment assay has been used to exanaine SR protein function in this process and, rather surprisingly, it turns out that individual SR proteins can conmait pre-mRNAs to the splicing pathway; pre-incubation of pre-mRNA with an SR protein allows it to be spliced preferentially when splicing extract and an excess of competitor pre-mRNA are added subsequendy [31"]. Different SR proteins can commit different pre-mRNAs to splicing with pronounced substrate specificity, but the molecular basis for these effects is not currently known. U2AF binds to the pyrimidine tract upstream of the 3' splice site and is required for targeting of U2 snRNP to the branch-site on higher eukaryote pre-mRNAs. Human U2AF consists of two associated polypeptides with molecular weights of 35 and 65 kDa; only the 65 kDa protein is required to restore activity to U2AFdepleted extracts. The sequence of the U2AF65 cDNA reveals that the protein has both an RNA-binding domain and an SR domain, which is essential for activity [32]. Recendy, a cDNA clone carrying the Drosophila counterpart of U2AF65 has been isolated, and germline transformation experiments have shown that the gene is essential for viability [33]. The fission yeast UZAF65 homologue is also an essential gene product [34], and both of these U2AF65 homologues have extensive similarities with mammalian U2AF65. In Drosophila, the product of the Sex-lethal (Sxl) gene operates a splice site switch by activating a female-specific 3' splice site in the first intron of tra pre-mRNA and repressing an alternative default site. U2AF and Sxl both bind polypyrimidine tracts, but the SR domain, which enables U2AF to activate the nearby 3' splice site, is absent from Sxl. The Sxl protein inhibits the use of the default site by preventing the binding of U2AF. This enables U2AF to activate the lower affinity female-specific site instead [35]. Several reports have drawn attention to the phosphorylation and dephosphorylation of splicing factors as a potential mechanism for regulating their activity. Studies on the effects of ATP analogues, phosphatase inhibitors and purified protein phosphatases have shown that at least two different serine/threonine phosphatases are essential for the catalytic steps of splicing [36,37]. The Ul-specific 70 kDa protein (U1 70K) is an SR protein that is phosphorylated in vivo on multiple serine residues [38,39]. Thiophosphorylation of U1 70K (which gives products resistant to dephosphorylation by protein phosphatases) inhibits splicing,
although spliceosome assembly is unaffected [381. Purified spliceosomal snRNPs contain a kinase activity that phosphorylates serine residues on U1 70K and in the SR-rich domain of the ASF/SF2 splicing factor 139].
RNA interactions in the spliceosome Information about RNA-based interactions and the possible molecular basis for catalysis in the spliceosome has accumulated rapidly over the past two years [1",2,3] and this continues to be an active area. The picture that emerges is one of a complex and dynamic network of interactions between substrate and snRNA sequences. A new and unexpected interaction has recently been proposed between the G nucleotides at the 5' and 3' ends of introns [40]. The evidence for this interaction rests on the observation that there can be reciprocal suppression of mutations at these two positions in the yeast actin gene intron. When present singly, these mutations prevent the second catalytic step of splicing and so cause accumulation of lariat intermediates. Reciprocal suppression in the double mutant is allele-specific, implying that a specific interaction involving these two nucleotides is important for the second catalytic step
[401. It is well known that U1 snRNA base-pairs with conserved intron sequences at the 5" splice site in premRNAs, but recent findings reveal a more extensive repertoire of interactions for this snRNA. Genetic data indicate that, in fission yeast, U1 snRNA can also interact with the 3' splice site AG by base-pairing via the universally conserved CU (positions seven and eight) just downstream of the 5' splice site interaction region. Mutations at C7 are lethal, whereas mutations at U8 cause the cells to grow slowly and accumulate premRNAs, consistent with a general role for these two nucleotides in fission yeast splicing [41]. In contrast, mutants at these positions in budding yeast are fully functional, ruling out the possibility that there is an essential base-pairing interaction between the U1 snRNA and 3" splice sites [42]. Instead, these positions in U1 snRNA might play a part in 5' splice site selection by interacting with 5' exon sequences [42]. The interaction between the U1 snRNAand the 5' splice site can also extend downstream into the intron. Efficient splicing of the MER2 intron in budding yeast normally requires the meiosis-specific MER1 protein. An extragenic suppressor that bypassed the requirement for MER1 turned out to be an allele of the gene for U1 snRNA with a single base change that allowed pairing with position eight of the intron [43]. It is thought that only three snRNAs (U2, U5 and U6) contribute functionally once catalysis begins in the spliceosome. Earlier genetic evidence established an interaction between the conserved nine-nucleotide loop I sequence in yeast U5 snRNA and exon nucleotides adjacent to the 5' and 3" splice sites [44]. The interaction between the U5 loop motif and sequences upstream of the 5' splice site has been confirmed bio-
Pre-mRNA splicing Newman
(a) Spliceosome
Exon 1
SI)
(b) Group I intron
R.
Exon 1 5'
3' ~ J
-OU .... P = O(S) pre-mRNA
(S)O ~ P""alO" j~p.
Exon 2 ~ 3 '
G I'()11
Exon 1
% 5'
5'
Rl,
tl
Exon 1 5"
Exon2 n 3 ,
5'Y~GpZ
~~ 1st slep
X HO 3'
Substrale
l st step
Intermediates
X 3'OH
O-
,,,x
s°"
Exon 2 GpZ I Y
(S)O = P'"gqlSy
3.1G
Exon 2
CpZ Intron-exon2 lariat intermediate
~[/
Aclive sile rearranppement and splice site exchange
t l
Exon 1 5'
St, ..,"
- ~ -
5'
X
Exon2 n 3 '
~.
(S)O ~ P ~91Z ~ GpY ~
-rG
5'
X
13'
I
(S)O = P~'"'u O-
(SlO =
Exon 2
Lariat inlron
H
Spliced mRNA
Rp
P .... iI O -
S •Exon , 2
s' Z ~ 3 ' ~ G r O
2nd step
Exon 1
R~
Xr
3'
G
ti
Exon 1 5'
St,
3 0 ~ , , , "O. Exon2
2ncl step
Spliced mRNA
Splice site exchange
Exon 1 X rOH
(S)O = P ' ~ Z ~ A
Sp
' Z ~ 3 '
GpY ~
G
3'OH
Linear intron © 1994 Currenl Opinion in Genelics and Developmenl
Fig. 1. Stereochemistry of the two steps in pre-mRNA splicing. (a) Spliceosomal splicing occurs via two distinct transesterification steps at different active sites. (b) Group I intron self-splicing, in which both steps of splicing are thought to occur at a single active site. The mechanism and configuration of the first step differ substantially between the spliceosome and Group h they show opposite phosphorothioate diastereomer preferences (Sp versus Rp) and use different reactive nucleophiles (2'OH versus 3'OH). In contrast, the second reaction steps are stereochemically similar: both pathways show preference for the Sp phosphorothioate diastereomer, use of a 3'-OH as the nucleophile and the presence of a conserved guanosine (G) attached through a 3' oxygen to the phosphate at the 3' splice site. The non-bridging position (S) at which sulphur is not significantly inhibitory, along with the corresponding phosphorothioate diastereomer (Rp or Sp) is indicated for the subslrate at each step. The diastereomer that results is indicated for the product of each step. X, Y and Z indicate nucleotide positions that are not highly conserved. Adapted from [53"'].
301
302
Chromosomesand expressionmechanisms chemically in HeLa cell nuclear extracts. [451. In these experiments, ligase technology [46] was used to introduce a modified nucleotide (4-thiouridine), carrying a photo-activated cross-linking group, into the exon sequence two nucleotides upstream of the 5' splice site in a pre-mRNA substrate. Selective photoactivation of the 4-thiouridine residue resulted in crosslinks to the U5 snRNA loop sequence and to the U5 snRNP protein p220 (the mammalian counterpart of the yeast PRP8 gene produc0. These interactions between the U5 loop sequence and exon sequences at the splice sites have striking parallels in autocatalytic introns [44,45,47]. A good deal of evidence is consistent with the idea that U6 snRNA plays an important part in the catalytic steps of splicing. Cross-linking experiments have shown that an invariant sequence motif in U6 snRNA (nucleotides 47-53 [ACAGAGA] in yeast U6 snRNA) is in close proximity to the 5' splice site in the active spliceosome [48,49]. Mutations in this U6 snRNA motif can specifically block the first or second catalytic steps of splicing, and there is genetic evidence in yeast to support a model in which a U2-U6 interaction juxtaposes the invariant residues in U6 with the branchpoint [50]. The structure produced by this U2-U6 interaction resembles domain V of group II introns, which is the most conserved domain and is thought to contribute to catalysis [51].
Catalysis and intron ancestry The multiple similarities between spliceosomal and Group II intron splicing have sustained a lively and continuing debate about the possible evolutionary relationships between spliceosomal snRNAs and autocatalytic introns [47], and support the widely held belief that catalysis in the spliceosome is likely to be'RNAbased. A general model has recently been proposed for a two-metal-ion phosphoryl transfer mechanism for RNA-catalyzed reactions in spliceosomes and autocatalytic introns [52"]. According to this model, catalysis is facilitated by two divalent metal cations as in protein-catalyzed phosphoryl transfer reactions such as the 3',5'-exonuclease activity of Escbericbia coli DNA polymerase I. The model proposes that the role of the RNA is to position the two catalytic metal ions and orient the substrates via three specific binding sites. Ligase technology [46] has recently been used to make pre-mRNAs with single chiral phosphorothioate linkages at either the 5' or 3' splice site in order to follow the stereochemical course of the two catalytic steps [53%54]. These experiments showed that both catalytic steps proceed with inversion of the stereochemical configuration of the phosphate at the splice site, consistent with a transesterification mechanism involving 'in-line' SN2 nucleophilic displacement reactions (see Fig. 1). Also, as both steps are inhibited by the Rp phosphorothioate diastereomer, but not by Sl,, the
spliceosome probably shifts between two active sites for catalysis of the two transesterifications [53"'1. This is in contrast to the situation in Group I introns, where both steps of splicing are thought to occur at a single active site (Fig. 1). The discovery of Group II introns in cyanobacteria and purple bacteria [55"1, which are thought to be the ancestors of chloroplasts and mitochondria, has resulted in further insights into the ancestry of RNA splicing pathways. This fnding fits in with Cavalier-Smith's theory for the origin of spliceosomal introns [561, which suggested that they are degenerate descendants of Group II introns initially resident in the genomes of endosymbiotic purple bacteria. New data on Group II intron mobility are also consistent with CavalierSmith's scheme. It has been known for some time that Group II introns can use reverse splicing in vitro to move to allelic and non-allelic locations by processes known as homing and transposition [57,58]. Transposition of Group II introns has recently been shown to occur in vivo in the mitochondria of Podospora and yeast [59,60]. Transposition of the aI1 intron in the cox1 gene of yeast mitochondria bears all the hallmarks of an RNA-mediated event: the sequences flanking the intron are not co-transposed to the insertion site, and mutations which compromise splicing activity also prevent intron mobility [59]. This intron and its downstream companion intron a12 encode reverse transcriptases [61] that are highly specific for molecules containing the a l l and a12 introns. Group II-like introns in protomitochondrial endosymbionts could have invaded the nuclear genome via reverse splicing, reverse transcription and recombination with the genomic DNA. The development of the spliceosomal machinery by fragmentation of a self-splicing nuclear intron into transacting snRNAs would then have allowed the resident introns to lose their capacity for self-splicing.
Conclusions Significant progress has been made recently in identifying proteins involved in pre-spliceosome assembly and in defining a network of RNA-based interactions in the active spliceosome. The use of new technology for making site-specific modifications of large RNA molecules has made important contributions in this area and should continue to yield new insights into the interactions of the RNA and protein components of the spliceosome. The stereochemistry of the transesterification events in the spliceosome has been investigated using splicing substrates that contain single chiral phosphorothioate linkages. The data indicate that both steps of splicing occur as single SN2 nucleophilic displacement reactions and that the machinery probably shifts between two active sites in catalysis of the two steps. It will be very interesting to see whether Group II introns behave in a similar way.
Pre-mRNA splicing N e w m a n
Acknowledgements I would like to thank my colleagues, particularly Jim Haseloff, Kiyoshi Nagai, Ingrid Kelly and Jean Beggs, for advice and encouragement. Work on pre-ml~IA splicing in my laboratory is suppoaed by the Medical Research Council.
13. •
LEGRAIN P, CHAPON C: Interaction between PRPII and SPP91 Yeast Splicing Factors and Characterisation of a PRP9-PRPII-SPPgl Complex. Science 1993, 262:108-110. This paper presents genetic evidence for a three-protein complex involved in yeast pre-spliceosome formation. Protein-protein interactions are assayed in vivo using a two-hybrid system based on GAL4-mediated activation of a reporter gene. 14. •
References and recommended reading Papers of particular interest, published within the annual peri~.l of review, have been highlighted as: ,, of special interest *• of outstanding interest 1. MOOREM, QUERY C, SHARP P: Splicing of Precursors to • mRNA by the Spliceosome. In The RNA World. Cold Spring Harbor: Cold Spring Harlx)r Laboratory Press; 1993:303-357. Comprehensive and up-to-date review of pre-mRNA splicing, covering mechanism and stereochemistry, spliceosome assembly and the roles of snRNA and protein comlxments of the splicing machinery. 2.
RYMONI)1], llOSBASH M: Yeast Pre-mRNA Splicing. In The Molecular a n d Celhdar BIologv o f the )'east Saccbaromyces. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1992:143-192. 3. LAMONDA: The Spliceosome. Bloessays 1993, 15:595-603. 4. LIAO X, TANGJ, ROSBASH M: An Enhancer Screen Identifies a Gene that Encodes the Yeast UI snRNP A Protein: Implications for snRNP Protein Function in Pre-mRNA Splicing. Genes Dev 1993, 7:419-428. 5. RYMOND13: Convergent Transcripts of the Yeast PRP38-SMDI Locus Encode Two Essential Splicing Factors, Including the DI Core Polypeptide of Small Nuclear Ribonucleoprotein Particles. Proc Natl Acad Sol USA 1993, 90:848--852. 6. RYMONDB, ROKEACH L, HOCH S: Human snRNP Polypeptide DI Promotes Pre-mRNA Splicing in Yeast and Defines Nonessential Yeast Smdlp Sequences. Nucleic Acids Res 1993, 21:3501-3505. 7. BENNI-71"I" M, MICHAHD S, KINGS'IONJ, REED R: Protein Components Specifically Associated with Prespliceosome and Spliceosome Complexes. Genes Dev 1992, 6:1986--2000. 8. MICHAtIDS, REED R: A Functional Association Between the 5' and 3' Splice Sites is Established in the Earliest PreSpliceosome Complex (E) in Mammals. Genes Dev 1993, 7:10(O-1020. 9. BROSl R, GRONING K, BEHRENSS, LL'IHRMANNR, KR.~MERA: ln* teraction of Mammalian Splicing Factor SF3a with U2 snRNP and Relation of Its 60-kD Subunlt to Yeast PRP9. Science 1993, 262:102-105. This paper presents a biochemical analysis of the components involved in mammalian pre-spliceo.~)me assembly, showing that splicing factor SF3a consists of three 17S U2 snRNP-specific polypeptides which play a role in the incorporation of U2 snRNP into the prespliceosome. Immunological cross-reactivity between yeast splicing factor PRP9 and one of the SF3a proteins (see also [10-12,13•-15•]) is demonstrated. 10.
11.
12.
BEHRENSS, GALL%'qON F, LEGRAIN P, LUHRMANN R: Evidence that the 60-kDa Protein of 17S U2 Small Nuclear Ribonucleoprotein is Immunologicaliy and Functionally Related to the Yeast PRP9 Splicing Factor and is Required for the Efficient Formation of Prespliceosomes. Proc Naa Acad Set USA 1993, 90:8229--8233. LEGRAIN P, CHAPON C, GAI.I~SON F: Interactions Between PRP9 and SPP91 Splicing Factors Identify a Protein Complex Required in Prespliceosom¢ Assembly. Genes Dev 1993, 7:1390-I 399. ARENA."; J, ABEl.SON J: The Saccharomyce$ cerevtslae PRP21 Gene Product is an Integral Component of the Prespliceosome. Proc Naa Acad Sol USA 1993, 90:6771-6775.
BENNETTM, REEDR: Correspondence Between a Mammalian Spliceosome Component and an Essential Yeast Splicing Factor. Science 1993, 262:10%108. An analysis of SAPs in mammalian extracts shows that SAP62 is probably the functional homologue of yeast PRPll. The paper a'iso shows immunological cross reactivity between SAP61 and yeast PRP9 and protein-protein interactions between SAP62 and SAPI14. 15. .
RuIsvS, CHANG T, ABEl.SONJ: Four Yeast Spliceosomal Proteins (PRP5, PRP9, PRPI1 and PRP21) Interact to Promote U2 snRNP Binding to Pre-mRNA. Genes Dev 1993, 7:1909-1925. This paper presents biochemical and genetic evidence for interactions between four yeast proteins involved in pre-spliceosome formation, including the helicase-like factor PRP5, and suggests that these proteins interact physically and/or act concertedly to promote U2 snRNP binding to the pre-mRNA. 16.
LIAO X, COLOr H, WANG Y, ROSBASH M: Requirements for U2 snRNP Addition to Yeast Pre-mRNA. Nucleic Acids Res 1992, 20:4237-4245.
17.
KIM S, SMITH J, CLAUDEA, LIN R: The Purified Yeast PremRNA Splicing Factor PRP2 is an RNA-Dependent NTPase. EMBO J 1992, 11:2319--2326.
18.
KIM S, LIN R: Pre-mRNA Splicing within an Assembled Yeast Spliceosome Requires an RNA-Dependent ATPase and ATP Hydrolysis. Proc Nail A c a d Set USA 1993, 90:888--892.
19.
SCHWERB, GOTHRIE C: PRPI6 is an RNA-Dependent ATPase that Interacts Transiently with the Spliceosome. Nature 1991, 349:494--499.
20.
BURGESSS, GI.rI'NRIE C: A Mechanism to Enhance mRNA Splicing Fidelity: The RNA-Dependent ATPase Prpl6 Governs Usage of a Discard Pathway for Aberrant Lariat Intermediates. Cell 1993, 73:1377-1391.
21.
FRANKD, PAI"I'ERSONB, GLrrHRIE C: Synthetic Lethal Mutations Suggest Interactions Between U5 Small Nuclear RNA and Four Proteins Required for the Second Step of Splicing. Mol Cell Bfol 1992, 12:5197-5205.
22.
FRANKD, GIYrHRIE C: An Essential Splicing Factor, SLU'7, Mediates 3" Splice Site Choice in Yeast. Genes Dev 1992, 6:2112-2124.
23.
HOROWrtx D, ABELSON J: Stages in the Second Reaction of Pre-mRaNA Splicing: the Final Step is ATP Independent. Genes Dev 1993, 7:320--329.
24.
HOROWlTZD, ABELSONJ: A U5 Small Nuclear Ribonucleoprotein Particle Protein Involved Only in the Second Step of Pre-mRNA Splicing in SaccharomFce$ cem'vlMae. Mol Cett BIol 1993, 13:2959-2970.
25.
SMrrHC, CHU T, NAOAL-GINARD13: Scanning and Competition between AGs Are Involved in 3' Splice Site Selection in Mammalian Introns. Mol Cell BIol 1993, 13:4939-4952.
26.
MAYEDAA, KRAINERA: Regulation of Alternative Pre-mRNA Splicing by hnRNP A1 and Splicing Factor SF2. Cell 1992, 68:365-375.
27.
Z.MILERA, LANE W, STOLK J, ROTH M: SR Proteins: a Conserved Family of Pre-mRNA Splicing Factors. Genes Dev 1992, 6:837-847.
28.
Fu X, MAYEDAA, MANIATISM, KRAINERA: General Splicing Factors SF2 and SC35 Have Equivalent Activities In Vitro, and Both Affect Alternative 5' and 3' Splice Site Selection. Proc Nail Acad Sci USA 1992, 89:11224--11228.
29.
ZAHLERA, NEUGEBAUERK, LANE W, ROTH M: Distinct Functions of SR Proteins in Alternative Pre-mRNA Splicing. Science 1993, 260:219-222.
303
304
Chromosomesand expression mechanisms 30. •
TIAN'M, M^NnAllS T: A Splicing Enhancer Complex Cow trois Alternative Splicing of doublesex Prc-tI1RNA. Cell 1993, 74:105--114. Biochemical methods are used here to study the regulation of female-specific splicing of ~ pre-mRNA by the tra and tra-2 proteins. The data indicate a role for wa, tra-2 and other SR proteins in the formation of a complex which forms on a regulatory site in dsx pre-mRNA, and commits the splicing machinery to use the female-specific splice site. 31. Fu X: Specific Commitment of Different Pre-mRNAs to Splic• ing by Single SR Proteins. Nature 1993, 365:82--85. Purified SR proteins produced in a baculovirus expression system are sufficient to commit pre-mRiNAs to splicing, implying that they can efficiendy recruit other spliceosomal components. Different SR proteins are shown to have different pre-mRNA substrate specificities, but it is not known how they achieve their effects. 32.
33.
34. 35.
36.
ZAMOREP, PKI'TONJ, GREEN M: Cloning and Domain Structure of the Mammalian Splicing Factor U2AE. Nature 1992, 355:609--614. KANAARR, ROCHE S, I{,EALLE, GREEN M, RIO D: The Conserved Pre-mRNA Splicing Factor U2AF from Drosophila: Requirement for Viability. Science 1993, 262:569-573. POTASHKINJ, NAIK K, WEN'IX-HtlNqXRK: U2AF Homolog Required for Splicing in Vlvo. Science 1993, 262:573-575. VAI.Ci~.RCELJ, SINGH R, ZAMORE P, GREEN M: The Protein Sex-lethal Antagonizes the Splicing Factor U2AF to Regulate Alternative Splicing of Transformer Pre-mRNA. Nature 1993, 362:171-175. MERMOLtDJ, COHEN P, LAMONt:) A: Ser/Thr Protein Phosphatases are Required for Both Catalytic Steps of Pre-mRNA Splicing. Nucleic Acids Res 1992, 20:5263-5269.
37.
TAZI J, DAUGERON M, CKfHALA G, BRUNEL C, JEANI"EUR P: Adenosine Phosphorothioates (ATP(xS and ATP'rS) Differentially Affect the Two Steps of Mammalian Pre-mRNA Splicing. J Btol Chem 1992, 267:4322-4326.
38.
TAZI J, KORNS'I'ADT U, ROSSI F, JEAN'I'EIJR P, CATHAI.A G, BRONELC, LOHRMANNR: Thiophosphorylation of U I-70K Protein Inhibits Pre-mRNA Splicing. Nature 1993, 363:283-286.
39.
WOPPMANN A, WILL C, KORNS'r,~DT U, Zuo P, MANLEY J, LOHRMANN R: Identification of an snRNP-Associated Kinase Activity that Phosphorylates Arginine/Serine Rich Domains Typical of Splicing Factors. Nucleic Acids Res 1993, 21:2815--2822.
40.
PARKER R, SILICIANO P: Evidence for an Essential NonWatson-Crick Interaction between the First and Last Nucleotides of a Nuclear Pre-mRNA Intron. Nature 1993, 361:660--662.
47.
WEINERA: mRNA Splicing and Autocatalytic Introns: Distant Cousins or the Products of Chemical Determinism? Cell 1993, 72:161-164.
48.
WASSARMAND, SlXII'ZJ: Interactions of Small Nuclear RNAs with Precursor Messenger RNA During tn Vitro Splicing. Science 1992, 257:1918-1925.
49.
SAWAH, ABELC,ON J: Evidence for a Base-Pairing Interaction between U6 Small Nuclear RNA and the 5' Splice Site During the Splicing Reaction in Yeast. Proc Naa Acad Set USA 1992, 89:11269-11273.
50.
MADHANIH, GU'II-IRIE C: A Novel Base-Pairing Interaction between 02 and U6 snRNAs Suggests a Mechanism for the Catalytic Activation of the Spliceosome. Cell 1992, 71:803-817.
51.
KOCHJ, lk.)ULANGER S, DIB-HA.U S, HEBP,AR S, PERLMAN P: Group II Introns Deleted for Multiple Substructures Retain Self-Splicing Activity. Mol Cell Bfol 1992, 12:1950-1958.
52. S'rEI17_T, STI-.TI'ZJ: A General Two-Metal-Ion Mechanism for • Catalytic RNA. Proc Natl A c a d Sci USA 1993, 90:6498--6502. The model presented in this paper is based on protein-catalyzed phosphoryl transfer reactions, such as the Y,5'-exonuclease of DNA polymerase 1. Catalytic RNAs, such as self-splicing introns and the spliceo~me, are proposed to position two divalent met',d kms in an active site, with three specific binding sites to properly orient the substrates. 53. *•
MOORE M, SHARP P: Evidence for Two Active Sites in the Spliceosome Provided by Stereochemistry of Pre-mRNA Splicing. Nature 1993, 365:364-368. Ligase technology 1461 is used to make synthetic pre-mRNAs cont:dning single chiral phosphorothioates at the splice sites, and these are used to investigate the stereochemical course of the catalytic steps in splicing. The paper provides strong evidence that both steps of premRNA splicing occur as single 'in-line' SN2 nucleophilic displacement reactions. 54.
MASCHHOFFK, PAtX3,E-rU"R: The Stereochemical Course of the First Step of Pre-mRNA Splicing. Nucle,'c Acids Res 1993, 21:5456-5462.
55.
FERATJ, MICHELF: Group 11 Self-Splicing introns in Bacteria. Nature 1993, 364:358-361. PCR is used to isolate Group 11 introns from purple bacteria, the probable ancestors of mitochondria in eukaryotes. This observation is consistent with a recent hypothesis [561 for the origin of nuclear spliceosomal introns, which suggests that they originate from Group II self-splicing introns in proto-mitochondrial endosymbionts.
•
56.
CAVALIER-SMI'I'I-IT: Intron Phylogeny: a New Hypothesis. Trends Genet 1991, 7:145-148.
57.
AtlGtJS'I'INS, MtJEI.LER M, .cK':.HWEYENR: Reverse Self-Splicing of Group I1 Intron RNAs In Vitro. Nature 1990, 343:383--386.
58.
MORLM, SCHMEI.ZER C: Integration of Group It Intron bll into a Foreign RNA by Reversal of the Self-Splicing Reaction In Vitro. Cell 1990, 60:629-636.
cerevlslae Does Not Require Base Pairing with UI snR.NA. Cell 1993, 73:803---812.
59.
NANDABALANK, PRICE L, ROEDERG: Mutations in U1 snRNA Bypass the Requirement for a Cell Type-Specific RNA Splicing Factor. Cell 1993, 73:407-415.
MUELLERM, ALLMAIERM, ESKE.~;R, SCHWEYEN R: Transposition of Group II lntron a l l in Yeast and Invasion of Mitochondrial Genes at New Locations. Nature 1993, 366:174-176.
60.
SEI.LEMC, LECELLIERG, BELCOORL: Transposition of a Group II Intron. Nature 1993, 366:176-178.
44.
NEWMANA, NORMAN C: U5 snRNA Interacts with Exon Sequences at 5' and 3' Splice Sites. Cell 1992, 68:743-754.
61.
45.
WYA'rrJ, SONTHEUMERE, S'~.rFz J: Site-Specific Cross-Linking of Mammalian U5 snRN1P to the 5' Splice Site Before the First Step of Pre-mRNA Splicing. Genes Dev 1992, 6:2542-2553.
KENNELLJ, MORANJ, PERLMAN P, BUTOW R, LAMBOWI'IXA: Reverse Tran$criptase Activity Associated with Maturasc-Encoding Group It Introns in Yeast Mitochondria. Cell 1993, 73:133-146.
46.
MOOREM, SHARPP: Site-Specific Modification of Pre-mRNA: The 2'-Hydroxyl Groups at the Splice Sites. Science 1992, 256:992-997.
41.
42.
43.
REICH C, VANHOV R, PORlXR G, WISE J: Mutations at the 3' Splice Site Can Be Suppressed by Compensatory Base Changes in UI snRNA in Fission Yeast. Cell 1992, 69:] 159--1169. SI::RAPHINB, KANDELS-Li':VaSS: 3' Splice Site Recognition in S.
AJ Newman, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.