1、RNA Structure and Function 447Towards understanding the catalytic corestructure of the spliceosomeS.E. Butcher1and D.A. BrowDepartments of Biochemistry and Biomolecular Chemistry, University of WisconsinMadison, Madison, WI 53706, U.S.A.AbstractThe spliceosome catalyses the splicing of nuclear pre-m
2、RNA (precursor mRNA) in eukaryotes. Pre-mRNAsplicing is essential to remove internal non-coding regions of pre-mRNA (introns) and to join the remainingsegments (exons) into mRNA before translation. The spliceosome is a complex assembly of five RNAs (U1,U2, U4, U5 and U6) and many dozens of associate
3、d proteins. Although a high-resolution structure of thespliceosome is not yet available, inroads have been made towards understanding its structure and function.There is growing evidence suggesting that U2 and U6 RNAs, of the five, may contribute to the catalysis ofpre-mRNA splicing. In this review,
4、 recent progress towards understanding the structure and function of U2and U6 RNAs is summarized.Components of the catalytic coreMost of the eukaryotic protein-coding genes have introns.Before the translation of the RNA transcript, these in-trons must be accurately removed and the exons splicedtoget
5、her. This process proceeds through two steps: (i) attackof the 2prime-hydroxyl of an intronic branch point adenosine atthe 5prime-splice site, resulting in formation of an intermediate2prime5primebranched lariat intron3prime-exon, (ii) attack of the 3prime-hy-droxyl of the 5prime-exon at the 3prime-
6、splice site, producing theligated exons and liberating the 2prime5primebranched lariat intron.The macromolecular machine that catalyses these steps is thespliceosome,amega-DaltonassemblyofRNAsandproteins.The spliceosome undergoes a cascade of assembly events anddynamic rearrangements before forming
7、an active complexon pre-mRNA (precursor-mRNA) 1, during which timetheU1andU4RNAsareeitherreleasedordestabilizedtotheextent that they are only weakly associated with the complex1. The U5 RNA, which positions the exons for ligation 2,isprobablynotinvolveddirectlyincatalysis,becausemuchofits sequence i
8、s dispensable in vitro 3. Therefore, despite thecomplexity of the spliceosome, which can involve more thana hundred different proteins 4, there are few componentsknown to interact directly with the pre-mRNA substratethat are ideal candidates for catalysing the splicing reaction.These include the Prp
9、8 protein that has no recognizablestructural motifs within its sequence and is remarkably large(2400 amino acids) and highly conserved (62% identity be-tween yeast and humans) 2 and the base-paired complex ofU2 and U6 RNAs that directly base-pair to the intron 3.This review will focus on the structu
10、re and function of theU2 and U6 RNAs of the spliceosomal catalytic core 3.Key words: catalytic core, NMR, structure, spliceosome, U2 RNA, U6 RNA.Abbreviations used: ISL, intramolecular stemloop; pre-mRNA, precursor mRNA.1To whom correspondence should be made (email butchernmrfam.wisc.edu).RNA and me
11、tal-ion requirementsBydirectlycontactingthepre-mRNA,theU2andU6RNAsare certain to reside near the active site of the spliceosome.However, are the RNAs themselves responsible for chem-istry? In other words, “is the spliceosome a ribozyme?” 5.Intriguing parallels exist between the spliceosome and group
12、II self-splicing introns, which are true ribozymes. Valadkhanand Manley 6 investigated the catalytic potential of a pro-tein-free preparation of U2U6 RNA and found that indeedthe human U2U6 RNA complex is capable of stimulating aslow, inefficient reaction that mimics the first step of splicing6. The
13、 protein-free reaction is further stimulated by a con-served pseudouridine that serves to remodel the structure ofthe branch site helix 7,8. The slow protein-free reaction isreminiscent of the peptidyl transfer reaction rate observedwith deproteinized ribosomes 9. It was not until theribosome crysta
14、l structure was solved, however, that it wasrealized that ribosomes utilize an RNA core to catalyse thechemical steps of translation 10.RNA is also likely to dominate the catalytic core ofthe spliceosome, and the similarities between the group IIribozymes and the spliceosome reinforce this idea. Mec
15、h-anistic analyses indicate that both group II introns and thespliceosome employ identical reaction pathways and stereo-chemistry 11. Additionally, both require metal ion as acofactor and utilize the same catalytic strategies, in whichmagnesium ion co-ordinates with the 3prime-oxyanion leavinggroups
16、 to stabilize the build-up of negative charge in thetransition state for both steps of splicing 12,13. Thesemechanistic similarities have led to the hypothesis that thespliceosome and group II introns evolved from a commonmolecular ancestor 12.Linandco-workers14haveelucidatedanessentialmetal-ion-bin
17、ding site within the U6 RNA at the U80 pro-Sphosphate oxygen of a highly conserved ISL (intramolecularC2005 Biochemical Society448 Biochemical Society Transactions (2005) Volume 33, part 3Figure 1 Similarities between domain 5 of group II self-splicingintrons and the U6 ISLMetal-binding sites are in
18、dicated with an asterisk. The AGC triad isshown in red for both sequences and also in the U6 ISL NMR structure(Protein Data Bank code 1XHP). The U80 nucleotide is represented as aspace-filling model.stemloop)structure(Figure1).Itisnotknownifthismetal-ion-binding site also helps to co-ordinate the sa
19、me metalion that is involved in stabilizing the 3prime-oxyanion. However,sulphur substitution of the U80 pro-S phosphate oxygenatomissufficienttoaltersplicingchemistrytosuchanextentthat it becomes entirely dependent on the addition of metalions that can co-ordinate with sulphur, such as cadmium14. N
20、MR experiments indicate that metal ions readily co-ordinatewiththeU80pro-SphosphateoxygenintheisolatedU6 ISL domain 15. Furthermore, the structure of the U6ISL is not altered when the U80 pro-S phosphate oxygen issubstituted with sulphur 16.Structural analysis of U2U6 RNAWe have determined the NMR s
21、tructure of the U6 ISL(Figure 1) 15,16. Interesting features of this RNA includea pentaloop that makes a GNRA-type fold and thesefolds often mediate RNA tertiary interactions 17 ormay function as protein-recognition sites 18. Adjacent tothe metal-binding site at U80 is a conserved, protonatedC67+A79
22、 wobble pair with a pKanear neutrality 15.The unprotonated state of A79 favours metal ion binding,and metal ion binding, in turn, lowers the pKa15. Thisobservation raises the interesting possibility that protonuptake could regulate splicing by influencing the binding ofa required metal ion. Addition
23、ally, proton uptake results in asignificant conformational change in the U6 ISL. At higherFigure 2 Secondary-structure representations of possible foldsfor the U2U6 four-helix junction(A) Secondary structure of the U2U6 complex as determined by NMR.U2U6 helices Ia, II and III are shown, as is the U6
24、 sequence that pairswith the 5prime-splice site (5prime-ss) and the U2 sequence that pairs with thebranch point (b.p.) of the intron. (B, C) Possible patterns of coaxialstacking for the four observed helices. Sites of UV-induced cross-linkingare shown with jagged arrows. The metal-binding site is ma
25、rked withan asterisk in all Figure parts.pH values, U80 stacks above the unprotonated A79 base.At lower pH values, U80 is flipped out of the helix andthe protonated A79 base stacks upon G81 19. These twoconformational states exist in equilibrium and inter-converton the micro- to millisecond timescal
26、e 19.An atomic-level model of the U2U6 RNA structureis required to understand how it could participate in thesplicing reaction. We therefore used NMR to analyse the hy-drogen-bonding patterns for a number of protein-free U2U6 RNA complexes of up to 110 nt total length 20. All thecomplexes studied fo
27、rmed a four-helix junction (Figure 2A).The observed four-helix junction forms an extended U6ISL structure 20 (Figure 1). This was unexpected, sincethe extended U6 ISL sequesters a highly conserved AGCsequence that has been shown to participate in the formationof an intermolecular U2U6 helix, helix 1
28、b 21,22. Helix 1bis essential for splicing; however, mutagenesis experimentssuggest an additional role for the AGC triad beyond helix1b formation 21,22. Mechanistic studies suggest that aconformational change in the spliceosome may be rate-limiting for the second catalytic step of splicing 12. Wehyp
29、othesize that the four-helix junction may play a role inthe first but not second step of splicing and that proteins willbe required for helix 1b formation and remodelling of theintrinsic U2U6 structure 20.There are several interesting implications of the observedfour-way junction fold. First, the ex
30、tended U6 ISL in thisstructure closely resembles domain 5, the catalytic core ofC2005 Biochemical SocietyRNA Structure and Function 449group II self-splicing introns, particularly with respect tothe conserved AGC sequence (Figure 1). This observationlends further support to the hypothesis that the s
31、pliceosomeand group II introns share a common molecular ancestor.Secondly, four-way junctions form coaxial helical stacks thatcould juxtapose catalytically essential elements, as observedinthehairpinribozyme23.Weutilizedthecrystalstructureof a hairpin ribozyme four-way junction 24 to model suchan in
32、teraction 20, and the resulting model was satisfyingin that it predicted a close proximity between the U6 ISLmetal-binding site and the intron-binding regions of U2 andU6 (Figure 2B). However, UV cross-linking studies 25,26report tertiary interactions between U2 and U6 that areconsistentwithapreviou
33、slyidentifiedgeneticinteraction27butinconsistentwithamodelthatjuxtaposestheU6ISLandtheintron-bindingregionoftheU2U6complex.Adifferentpattern of coaxial stacking probably explains these results(Figure 2C). It is possible that these two coaxial stackingpatterns are formed at different points during th
34、e splicing re-action, and the switch between them is one of the conform-ational changes required for spliceosome activation.References1 Brow, D.A. (2002) Annu. Rev. Genet. 36, 3333602 Turner, I.A., Norman, C.M., Churcher, M.J. and Newman, A.J. (2004)Biochem. Soc. Trans. 32, 9289313 Villa, T., Pleiss
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