ANDY NEWMAN

Activity in the spliceosomeIn the active spliceosome, U2, U5 and U6 small nuclear

RNAs form a network of interactions with the RNA substrate.U6 snRNA is closely implicated in the catalysis of splicing.

Splicing of nuclear mRNA precursors occurs in spliceo-somes, complex ribonucleoprotein particles built upfrom five spliceosomal small nuclear RNAs (snRNAs U1,U2, U4, U5 and U6) and a large number of proteins.Nuclear pre-mRNA splicing is mechanistically similar tothe autocatalytic excision of group II introns, whichhave been found in genes of plant and animalorganelles and bacteria. In group II introns, splice-siterecognition and catalysis are achieved by the formationof a characteristic intramolecular network of secondaryand tertiary RNA structures.

Both group II and spliceosomal pathways involve twosuccessive phosphotransfer reactions. In the first reac-tion, the 2'OH of a specific adenosine residue near the3' end of the intron attacks the 5' splice site, generatinga free upstream exon and a 'lariat' intermediate. In thesecond reaction, the 3'OH of the upstream exon attacksthe 3' splice site, resulting in ligation of the two exonsand release of the excised intron lariat. In these lariatmolecules, the 5' end of the intron is joined to thebranchpoint adenosine via a 2'-5' phosphodiester bond.

These mechanistic similarities have led to speculationthat pre-mRNA splicing might be evolutionarily relatedto the group II self-splicing process, and could alsoinvolve catalysis by RNA (see [1-3] for reviews). If thiswere the case, it might be expected that the character-istic RNA structures of group II introns would be recap-itulated in the spliceosome by interactions amongst thesnRNAs and the substrate. There is now a good deal ofexperimental evidence that this is the case.

U1 and U4 snRNAs have important functions in thespliceosome prior to the catalytic steps of splicing - forexample, U1 plays a crucial role in 5'-splice-site recog-nition - but it is currently believed that U2, U5 and U6are the only snRNAs directly involved in the reactionsthat take place in the active spliceosome. Of these, U6snRNA is the component that is most closely implicatedin spliceosomal catalysis. U6 is the most highly con-served of the spliceosomal snRNAs, and single basemutations in two short, invariant motifs - ACAGAGand AGC - in the central region of U6 can specificallyblock the first or second trans-esterification reaction.When U6 enters the spliceosome it is extensively base-paired to U4 snRNA, but this interaction is disruptedprior to the first catalytic step of splicing.

Mutational analysis in yeast has shown that some of theU6 sequences exposed by the removal of U4 snRNA

interact instead with a conserved element in U2 snRNA,very close to the motif in U2 that interacts with theintron branchpoint. This interaction between U6 andU2 snRNAs places the invariant ACAGAG and AGCelements of U6 snRNA in the vicinity of the intronbranchpoint adenosine, which contributes the nucleo-phile for the first trans-esterification reaction. Photo-chemical cross-linking experiments have shown that theU6 snRNA motif ACAGAG lies close to the pre-mRNA5' splice site. This suggests a structural model in whichthe U2-U6 interaction brings together the pre-mRNA 5'splice site and branchpoint adenosine, tethered by U6and U2 respectively, for the first trans-esterification reac-tion [1-3]. Intriguingly, the helix formed between U2and U6 bears a striking resemblance to domain V ofgroup II self-splicing introns, which is known to becrucial for autocatalytic activity.

Two recent genetic studies [4,5] in yeast have confirmedthat the ACAGAG element in U6 snRNA interactsdirectly with conserved intron sequences near the 5'splice site, as suggested by the photochemical cross-linking experiments. Substituting the conserved G atposition 5 in yeast introns activates aberrant 5' cleavagesites in the vicinity of the authentic 5' splice site.Mutational analysis shows that these aberrant cleavagesare caused by illegitimate interactions between theACAGAG motif in U6 snRNA and sequences near theauthentic 5' splice site. At consensus 5' splice sites, theconserved G at intron position 5 base-pairs with theinvariant C (position 48) in the U6 ACAGAG element(Fig. 1) and this interaction contributes to the precisionof 5'-splice-site cleavage [4,5].

Both photochemical cross-linking and genetic datasuggest that the close association between U6 and the5' splice site is maintained after the first catalytic stephas been completed. A photo-activatable modified baseincorporated at position 2 in the intron forms, onphoto-activation during splicing, a cross-link with thelast A of the ACAGAG element in the lariat intermediate[6] (Fig. 1). The fact that this cross-link is not pickedup prior to the first catalytic step implies that theU6-5'-splice-site interaction may alter its precise con-formation after 5'-splice-site cleavage. This would fit inwith recent stereochemical data which show that theactive sites for the two catalytic steps in the spliceosomeare non-identical [7].

After the first trans-esterification reaction, the 5'-splice-site-sequence, in association with U6 snRNA, is attached.

© Current Biology 1994, Vol 4 No 5462

RNA SPLICING

DISPATCH 463

Fig. 1. Interacting RNA sequences in an active spliceosome (left) and a self-splicing group II intron (right), just before the second trans-esterification reaction. The sequence CCUU displayed in the U5 snRNA loop is part of the invariant U5 sequence GCCUUUUAC. Thegroup II intron structure shown is based on the consensus sequences for subgroup IIA introns. EBS, exon-binding site; IBS, intron-binding site; e-e', a conserved pairing that may be analogous to the U6-5'-splice-site interaction in the spliceosome. (Modified from [3].)

by a 2'-5' phosphodiester bond to the intron branch-point in the lariat intermediate (Fig. 1). Genetic dataargue that a non-canonical interaction between thefirst and last bases of the intron is required for effi-cient onward processing of the lariat intermediate [8],so it follows that the invariant U6 snRNA motifACAGAG will be juxtaposed with the 3' splice site priorto the second catalytic step (Fig. 1). That an inter-action occurs between U6 snRNA and the 3' splice siteduring splicing is supported by the observation thatsubstituting U6 residue G52 - the final position inthe ACAGAG motif - allows partial suppression of3'-splice-site mutations [4].

Photochemical cross-linking data have been instru-mental in clarifying the role of U5 snRNA in the spliceo-some. Consistent with previous genetic results,cross-linking experiments show that an invariant, single-stranded loop sequence (GCCUUUUAC) in U5 snRNAinteracts with the upstream exon prior to 5'-splice-sitecleavage, and this interaction persists through both cat-alytic steps. This indicates that U5 snRNA provides thesite at which the upstream exon is 'tethered' after it isreleased from the pre-mRNA by 5'-splice-site cleavage[6]. The same invariant loop motif contacts the firstnucleotide of the downstream exon in the lariat inter-mediate, suggesting that U5 snRNA also provides thesurface on which the two exons are aligned for ligationin the second catalytic step [61 (Fig. 1). These findingsadd up to a convincing validation of the notion that thisU5 snRNA loop is the spliceosomal counterpart of thed3 loop in domain I of group II self-splicing introns,which also bears exon-binding sites and is believedto carry out analogous functions. In any case, thesenew results show that U2, U5 and U6 participate in

a network of interactions that plays a crucial partin juxtaposing the reacting sequences in the activespliceosome (Fig. 1).

Some extraordinary observations on the behaviour ofU6 snRNA in a nematode cell-free splicing system havestrengthened the impression that this snRNA is centralto, and may participate in, catalysis of the splicingreactions. In this system, U6 snRNA itself can act asthe branch acceptor, becoming covalently linked tosubstrate sequences. More remarkably still, in somecases the liberated U6 'exon' can proceed through thesecond catalytic step, using an authentic 3' splice siteto produce a 'spliced' U6-exon product [9]. The fre-quency of these aberrant splicing events is enhancedby mutations of highly conserved sequences in the5' region of U6. The simplest interpretation of thisbizarre behaviour is that it results from aberrantpositioning of the authentic 5' splice site in the spliceo-somal active site, which leads to the use of U6 itself asa surrogate splicing substrate. These results furtheremphasize the close relationship between U6 snRNAand catalysis: attack by the 2'OH of the branchpointadenosine on U6 shows that it is, by definition, in thespliceosome's active site for the first phosphotransferreaction [9].

In conclusion, the evidence for a catalytic role for U6snRNA is getting stronger all the time, but a precisedescription of the catalytic mechanism must await amore detailed three-dimensional picture of the core ofthe spliceosome. It should be particularly instructive toidentify the spliceosomal binding sites for the two metalions that are hypothesized to perform crucial rolesduring catalysis [10].

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References1. WEINER AM: mRNA splicing and autocatalytic introns: distant

cousins or the products of chemical determinism? Cell 1993,72:161-164.

2. LAMOND Al: A glimpse into the spliceosome. Curr Biol 1993,3:62-64

3. WISE JA: Guides to the heart of the spliceosome. Science 1993,262:1978-1979.

4. LESSER CF, GUTHRIE C: Mutations in U6 snRNA that alter splicesite specificity: implications for the active site. Science 1993,262:1982-1988.

5. KANDELS-LEWIS S, SRAPHIN B: Role of U6 snRNA in 5' splice siteselection. Science 1993, 262:2035-2039.

6. SONTHEIMER EJ, STEITZ JA: The U5 and U6 small nuclear RNAs asactive site components of the spliceosome. Science 1993,262:1989-1996.

7. MOORE MJ, SHARP PA: Evidence for two active sites in

the spliceosome provided by stereochemistry of pre-mRNAsplicing. Nature 1993, 365:364-368.

8. PARKER R, SILICIANO PG: Evidence for an essentialnon-Watson-Crick interaction between the first and lastnucleotides of a nuclear pre-mRNA intron. Nature 1993,361:660-662.

9. Yu YT, MARONEY PA, NIILSEN TW: Functional reconstitution ofU6 snRNA in nematode cis- and trans-splicing: U6 can serveas both a branch acceptor and a 5' exon. Cell 1993, 75:1049-1059.

10. STEITZ TA, STEITZ JA: A general two-metal-ion mechanismfor catalytic RNA. Proc Natl Acad Sci USA 1993,90:6498-6502.

Andy Newman, MRC Laboratory of Molecular Biology,Cambridge CB2 2QH, UK.

Andy Newman is among the authors of reviews on Chromosomes and Expression Mechanisms,edited by Bruce Stillman and Michael Green, in

THE APRIL 1994 ISSUE (VOL. 4, NO. 2) OFCURRENT OPINION IN GENETICS & DEVELOPMENT

Pre-mRNA splicing by Andrew J. NewmanDNA replication: enzymology and mechanisms by Zvi Kelman and Mike O'Donnell

Intergenic DNA and the sequence requirements for replication initiation in eukaryotes by Bonita J. BrewerMammalian telomere dynamics: healing, fragmentation shortening and stabilization by Carol W. Greider

Repair of UV-damaged DNA by mammalian cells and Saccharomyces cerevisiaeby Abdelilah Aboussekhra and Richard D. Wood

Recombination genes and proteins by Hazel J. Dunderdale and Stephen C. WestAssembly of nucleosomes: do multiple assembly factors mean multiple mechanisms?

by Paul D. Kaufman and Michael R. BotchanThe role of activators in assembly of RNA polymerase II transcription complexes

by Roderick Hori and Michael CareyThe transcription of chromatin templates by Alan P. Wolffe

Role of DNA methylation in the regulation of transcription by Sharon Eden and Howard CedarChromatin domains as potential units of eukaryotic gene function by Niall Dillon and Frank Grosveld

Parental imprinting of autosomal mammalian genes by Argiris EfstratiadisPosition-effect variegation and the new biology of heterochromatin by Gary H. Karpen

X-chromosome inactivaton and the Xist gene by Sohaila RastanmRNA nuclear export by David J. Elliott, Frangoise Stutz, Alain Lescure and Michael Rosbash

mRNA translation: influence of the 5' and 3' untranslated regions by Nahum SonenbergMechanisms and origins of RNA editing by Brian K. Adler and Stephen L. Hajduk