The role of small nuclear RNAs in RNA splicing

The role of small nuclear Michael Salk Institute,

,

RNAs in RNA splicing

McKeown San Diego,

USA

Recent genetic and biochemical experiments have revealed an intimate and dynamic role for small nuclear RNAs fsnRNAs) in multiple steps of RNA-splicing reactions. Both snRNA-substrate and snRNA-snRNA interactions are involved. These interactions concern not only splice site and branch point definition, but also the catalytic reactions of the first and second steps of splicing. Studies reveal a striking conservation between snRNA interactions and interactions found in RNAs encoded by genes with group II self-splicing introns. Current

Opinion

in Cell Biology

1993, 5448-454

with the pre-mRNA that are of particular interest in this review.

Introduction

Almost all mRNAs in eukaryotes are produced by splicing of intron-containing precursors. In both the group II self-splicing introns and the introns in pre-mRNAs, the chemical reactions of the RNA are the same (Fig. 1; reviewed in [1,2]). In the first stage of splicing, the 2’ hydroxyl group of a single nucleotide near the 3’ end of the intron carries out a nucleophilic attack on the phosphodiester bond joining the last base of the first exon with the first base of the intron. The result is the formation of a free 5’ exon and a ‘lariat’ structure in which the 5’ end of the intron is attached, in a 2’-5’ phospodiester bond, near the 3’ end of the intron. During the second stage, the 3’ hydroxyl group of the first exon carries out a nucleophilic attack on the phospodiester bond joining the 3’ nucleotide of the intron to the 5’ nucleotide of the second exon. This reaction joins the first and second exons via a 3’-5’ phosphodiester bond and releases the lariat intron. The sequences involved in these reactions are highly conserved in Saccbaromyces cerevtie and sufficiently conserved in metazoans that consensus sequences can be derived (Fig. 1). Although the group II self-splicing introns carry out these reactions on their own, pre-mRNA splicing requires a complex of proteins and RNA~ called the spliceosome (reviewed in [1,2] >. Particularly striking is the involvement of a series of snFNPs, each containing a defined snRNA (Ul, U2, U4, U5 and U6). Figure 2 outlines the movement of snRNps into and out of the spkeosome in coordination with the reactions at the substrate RNA. SpeciIicaIIy, the Ul snRNA enters first, followed by U2. This is followed by the entry of a n-i-snRNP containing the U4, U5 and ~6 snRNPs. Shortly after this, U4 snRNA dissociates from U6 snRNA, to which it has been base paired, and the splicing reaction itself begins. It is the dynamic interactions of the snR~As with each other and

In the discussion that follows, I discuss genetic and biochemical data from yeast and mammalian systems that give new insights into the role of snRNAs in the splicing mechanism. I will not attempt to cover much recent work on the proteins that are clearly important for many of the actions of the RN4.s. In general, genetic data indicating the importance of RNA-RNA interactions take the form of compensatory mutations altering potentially base-paired nucleotides, such that either mutation alone leads to a loss of function, while the pair of mutations together restores function. Such tests of function can be performed in vivo or in r+tro. The biochemical data involve the isolation of RNA-RNA complexes, particularly by crosslinking between two RNAs. Assuming that key features of the splicing reaction are evolutionarily conserved, I will describe data from multiple systems, as if they all give insights into the same basic process. In some details, this may be incorrect, but in terms of general outline, it probably emphasizes key points. For additional recent reviews and/or alternative perspectives on the subject of this review, see Steitz [3] and Weiner [ 41. Earlier data are reviewed by Green [ 11, and Ruby and Abelson [ 21.

RNA-RNA

interactions

The initial step of splicing involves interaction of Ul snRNP with pre-mRNA Complementarity and base pairing between the 5’ end of Ul RNA and the conserved sequences of the 5’ splice site are critical for this step (Fig. 3). In addition, in budding yeast, binding of Ul is effected by mutations in the branch point region, suggesting a role for Ul snRNA in branch point recognition.

Abbreviations snRNP-small

448

nuclear

@ Current

ribonucleoprotein;

Biology

UV-ultraviolet.

Ltd ISSN 0955-0674

The role of snRNAs in RNA splicing

McKeown

(a)

Polypyrimidine

k)

a

5’ It-OH

cd)

a Exonl

Exon

2

Recent data from fission yeast also suggest that Ul RNA base pairing to the 3’.AG is important for splicing introns where initiation of splicing requires the presence of the 3’ splice site AG [ 5.1. Direct biochemical evidence for interactions between Ul and the 5’ splice site has been gathered using various methods of crosslinking [6*,7**,8-1. Crosslinks detected by ultraviolet light (UV) or psoralen map in the intron or near the 5’ splice site of the premRNA and near the 5’ end of the Ul RNA, consistent with the molecular genetic data [6*,7**]. Part of Ul must also be near position - 2 of the first exon as a photo-activatable reagent at that position becomes crosslinked to Ul RNA [8*].

U2 snRNP is the next snRNP to enter the splicing complex (Figs 2 and 3). A key determinant for U2 binding is the sequence at the putative branch point. In both yeast and mammalian systems, U2 binding involves specific base pairing between U2 RNA and the branch point (Fig. 3). A key fact of this interaction is that the A residue, which will serve as the branch point, does not base pair

Fig. 1. A schematic representation of pre-mRNA splicing. (a) An outline of the basic structures in a simple premRNA. Metazoan intron consensus sequences are shown. (bHd) describe the pre-mRNA splicing steps, which lead to the formation of mature RNA and the lariat intron. The bold ‘A’ in the branch point sequence represents the adenosine nucleotide that carries out the nucleophilic attack on the 5’ splice site.

and bulges out of the surrounding helix. The production of a stable complex containing Ul, U2 and pre-mRNA requires ATP in z&o. Both UV and psoralen crosslinking experiments show that U2 RNA is located near the branch point sequence during in vitro splicing reactions [ 6*,7**]. Surprisingly, Wassarman and Steitz [7**], using psoralen crosslinking, observed an interaction between U2 and the branch point even in the absence of ATP, suggesting that ATP is normally involved in stabilization of the U2-branchpoint interaction and not in its formation.

After the formation of a stable U2containing complex at the branch point, the U4, U5 and ~6 snRNPs enter the spliceosome as a u-i-snRNP particle. The U5 snRNP is thought to be involved in 3’ splice site interactions (either via its RNA or via associated proteins). Genetic evidence in budding yeast suggests a role for the U5 snRNA in 5’ splice site identification and cleavage [p]. Comparison of structures and sequences lead to the hypothesis that a particular U5 loop sequence corresponds to loop D3 of group II introns, a region believed to be involved in

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The role of snRNAs in RNA splicing

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binding exonic portions of the intronexon junctions [lo**]. This hypothesis suggests an interaction of the US loop with both 5’ and 3’ splice sites (Fig. 4). Genetic experiments using mutations constructed in uifro or selected for suppression of specific point mutations are all in agreement with the predictions of the hypothesis. These results suggest that pairing between nucleotides 5 and 6 of the loop and positions -3 and - 2 of exon 1 plays a role in choosing the 5’ cleavage site. In addition, mutations that increase the strength of potential base pairing between positions 3 and 4 of the loop and the first two nucleotides of the second exon increase the the efficiency of 3’ splice site cleavage for introns that otherwise do not show 3’ cleavage. Pairing always occurs at the same position relative to cleavage, consistent with a role in cleavage site definition. Note that the high variability of exonic sequences, and the fact that the importance of the 3’ and 5’ exon pairing interactions of U5 is demonstrated on debilitated splice sites, suggest that these interactions themselves may not supply all of the information on splice site location all of the time. With regard to the problem of exon variability, it has been hypothesized that the ~5 loop is particularly U-rich, in order to take advantage of the promiscuity of potential base pairing interactions that can be achieved by U residues [3]. One difference between the US-substrate interaction and the group II RNA interactions is that the 5’ and 3’ cleavage sites (relative to the loop sequence) are one base displaced from each other in mRNA splicing, while they are in the same place in group II splicing [lo-]. The involvement of U5 in 5’ splice site identification or cleavage is also supported by crosslinking of U5 snRNP to preRNA in splicing reactions in lttro. Two US-substrate complexes are observed in psoralen crosslinking experiments. One forms (and disappears) with kinetics similar to those for Ul binding to the 5’ splice site, while the other appears later in the splicing reaction

McKeown

as the first disappears [7**]. Use of photo-activatable crosslinking agents in the substrate RN& has allowed a direct mapping of a probable interaction in the first complex, specifically an activatable crosslinking agent at position - 2 of the exon is most often linked to the U residue at position 5 of the loop [@I. This interaction is ATP-dependent. A base pair between these two positions has been suggested based on the yeast data [ 1p.l. The second complex shows psoralen crosslinking of position 4 of the loop to bases downstrea of position 5 of the ’ intron. This is consistent with a base pairing interaction between bases 5 to 9 of the loop and positions 1 to 5 of the intron (Fig. 4). In addition, U5 also crosslinks to excised lariat RNA, again indicative of an interaction with intron sequences. As two complexes with different crosslink sites are formed, and because the kinetics of formation of the two are consistent with a precursor/product relationship, it has been suggested that an early step in splicing involves U5 interactions with exon 1 followed by a significant conformational change such that the U5 loop now interacts with sequences within the intron [ 3,7**,8*]. Also entering the spliceosome with ~5 are the U4 and U6 snRNPs, which start out base paired to each other (Fig. 5). After spliceosome assembly, but before the chemical steps of splicing, this extensive base pairing is broken. Recent data suggest that the regions of U6 that were paired with U4 now enter into new interactions with U2 snRNA [11**,12*]. A likely interpretation of these data suggests that the stem I region now forms two short helixes with the region of ~2 just 5’ to the branch point binding sequence and the stem II region forms a relatively lengthy stem loop. In addition, the 3’ end of ~6 appears to be capable of forming a helix with the 5’ end of U2 [ ll**]. This structure can be drawn in a way that is strikingly similar to a structure forrmd by the group IL4 self-splicing introns. The idea of a ‘dynamic interaction between ~2, ~4, U6 and pre-mRNA is supported by psoralen crosslinking experiments showing that U2/U4/U6 complexes form early in the splicing reaction and then decrease in abundance, while later in the reaction U2/Ub/premRNA complexes are observed [7**]. The U2/Ub/pre-mRNA interaction positions a hexa nucleotide region of ~6 critical for the splicing reaction (the ~6 hexanucleotide sequence) [ 13,141 in the region of the potential branch point (Fig. 5). Crosslinking experiments show that this region is also close to the 5’ splice site in pre-mRNA-containing spliceosomes. Specifically, psoralen and UV crosslinking map sites of CrosslInking between a region of the pre-mRNA just downstream of the 5’ splice site and a region of ~6 just upstream of the hexanucleotide sequence [ 6*,7**]. A similar UV crosslink appears to occur between ~6 and the excised lariat [ 6*,7*=,15*]. Based on the mapped position of crosslinks it is possible to draw at least two possible base pairing interactions between ~6 and the 5’ portion of the intron. Both of these involve some potential competition with putative U5 base pairs within the intron. One set of specific bases involved in interactions was determined for UGpre-mRNA complexes [7**], while the other set was

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Fig. 4. Interactions between Ul, U5 and the splice sites. (a) The U5 loop sequence [IO**]. (b) Base pairing of both Ul and U5 to the 5’ and 3’ splice sites in a hypothetical

from yeast numbered as in Newman and Norman early splicing complex. This particular arrangement of base pairs is suggested by Steitz 131 using data for U5 from Newman and Norman IlO** and Wyatt et al. [Sol. (c) A potential base pairing of U5 to the pre-mRNA that can occur after Ul has become dissociated from the pre-mRNA. Note that this arrangement, based on data from Wassarman and Steitz PI has U5 pairing with the 5’ end of the intron, not with the exon.

determined from U&lariat complexes [ 1591. One of the two pairing configurations is consistent with both sets of data, but it is also possible that there is a conformational shift between the first and second steps of splicing. All of these data place the splicing-critical nucleotides of the ~6 hexanucleotide sequence near both the branch point and the 5’ splice site of pre-rnRNA The multiple structural and functional similarities between features of the group II introns and the snRNAs are consistent with the hypothesis that the snPNAs together perform many or all of the functions of the group II introns. As group II introns are self splicing, corollary of this hypothesis is that the snRNAs themselves are the catalytic agents that facilitate the splicing reaction. Certainly the snR~~s are responsible for helping to align key positions of the pre-mRNA for splicing by several means: identifying and juxtaposing 3’ and 5’ splice site sequences; identifying branch point sequences; positioning the branch point A such that it bulges out of a helical region; and positioning the ~6 hexanucleotide near the branch point and 5’ splice site regions. A simplified and tentative outline of the movement of the snRNAs during the splicing reaction would go as follows.

(Note that the relative order of particular steps, especially during the inferred conformational changes, is not absolutely clear). Ul S-RNA recognizes sequences at the 5’ and 3’ ends of the intron; U2 recognizes the branchpoint region and a stable, ATP-dependent complex is formed; the ~4/U5/U6 tn-snRNP enters with an initial involvement of U5 with 5’ and 3’ exonic sequences followed by a series of conformational changes; Ul binding to the pre-mFWA is destabhzed; U5 binding switches from the 5’ exon to the 5’ end of the intron; the u4/LJ6 base paired region becomes partially unpaired, allowing the production of a U2/Ub/U4 complex; the U~/LJ~ base pairing is eliminated and a U2/~6 complex forms; the region just upstream of the ~6 hexanucleotide sequence base pairs near the 5’ end of the intron; splicing begins and both steps occur rapidly, potentially with additional conformational changes between the two steps of splicing.

Conclusion Our growing knowledge of the complexity of the RNA-RNA interactions involved in the splicing reaction

The role of snRNAs in RNA splicing

McKeown

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Fig. 5. U2, U4, U6 and the splicing reaction. (a) The U4/U6 base pairing interaction using yeast U4 and U6 sequences. Highly conserved residues are shown in capital letters. Bold letters indicate the U6 hexanucleotide sequence. (b) A potential structure between pre-mRNA, U2 and the section of U6 between the arrows in (a), using yeast sequences. Helix la and Helix lb are formed largely from the stem I region of U6 and require displacement of U4. The U6 intramolecular helix forms largely from the part of U6 that was involved in stem II. Thus, from the stand point of U6, essentially all of the base pairing between U4 and U6 can be replaced by intramolecular interactions and U2/U6 interactions. Helix la forms on that part of U2 just upstream of the branch point thereby positioning the branch ‘A’ (bold) and the U6 hexanucleotide (bold) potentially quite close to each other, as suggested by Madhani and Guthrie Ill**l. Chemical crosslinking experiments also indicate that the region just upstream of the U6 hexanucleotide base pairs with the consensus 5’ splice site sequence [6*,7**,15*1. Such an interaction, as drawn, places the U6 hexanucleotide in a region potentially quite near not only the branch ‘A’ but also near the 5’ splice site.

and the realization that S~RNAScan form structures that appear to be both physically and functionally similar to key regions of the group II self-splicing introns strongly support the idea that the catalytic residues involved in splicing will be part of the snFWAs themselves. Determining the exact roles of particular RNA sequences will be

diEcult in the context of mRNA splicing as so many ancillary factors are involved. On the other hand, it is likely that great progress can be made in the study of RNA functions in the group II introns and that this knowledge can be coupled to our growing knowledge of the similarity between the snRNAs and the group II introns such that

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clear and testable hypotheses can be formulated about the functions of specific portions of the snRNAs in the chemistry of the splicing reaction.

Acknowledgements Work on sex differentiation and alternative spiking in the author’s laboratory is supported by a grant from the NIH. The author is aiso pan of Cancer Center Core Grant CA-14195. Thanks to Sanford Mad&an for comments on the manuscript.

References

and recommended

reading

Papers of particuiar interest, published within the annual period of review, have been highiighted as: . of special interest .. of outstanding interest 1.

GREEN MR: Biochemical Mechanisms of Constitutive and Regulated Pre-mRNA SpIicing. Annu Rev Cell Biol 1991, 7:559-599.

2.

RUBY SW,

&net

ABEIX)N

J:

Pre-mRNA Splicing in Yeast. Trends

1991, 7~79-85.

3.

Srsrtz JA Splicing 257:88%889.

4.

WEINER

Takes

a HoRiday.

Science

1992,

AM: mRNA Splicing and Autocatalytic Introns: Distant Cousins or the Products of Chemical Determinism? Cell 1993, 72:161-B%.

5. .

REICH Cl, VANHOY RW. PORTER GL, WISE JA: Mutations at the 3’ Splice Site Can be Suppressed by Compensatory Base Changes in Ul snRNA in Fission Yeast. cell 1992. 69:115%1169. An Important molecuiar genetic demonstration that base pairing between Ul and the 3’ splice site can be important for 3’ splice site dehnition in introns that are AG-dependent for 3’ splice site choice.

6. .

SAWAH, SH~MURA Y: Association of U6 snRNA with the 5’ Splice Site Region in the SpIiceosome. Genes Deu 1992, 6:244-254. The first biochemical data to show that critical parts of ~6 are near the 5’ splice site in the spiiceosome 7. ..

Wm DA, STEW JA Interactions of SmaiI Nudear RNA’s with Precursor Messenger RNA During in Vitro Splicing. Science 1992, 257:191B-1925. An analysis of the interactions between multiple snRNAs and the premRNAs in the spiiceosome, including a definition of the interacting sites for many of the snRIU4components with pre-mRNA

WYATT JR, SONTHE~MEREJ, STE~TZJA Site-Specific CrossLinking of Mammalian US snRNP to the 5’ Splice Site before the First Step of Re-mRNA Splicing. Genes Dev 1992, 6:2542-2553. Use of a photo-activatable crossiinker at position - 2 of the first exon provides biochemical evidence for the hypothesis that U5 interacts with the 5’ exon, as suggested in [ lOa*]. 9. NM AJ, Nom C: Mutations in Yeast U5 snRNA Ai. ter the SpeciEcity of 5’ Splice Site Cleavage. Ceil 1991, 65:115-123. At the time this paper appeared the effect of U5 mutations on 5’ splice site choice was surprising and led to the experiments in [lo**]. 10. NEW?+IAN AJ. NORMANC: U5 snRNA Interacts with Exon Sequences at 5’ and 3’ Splice Sites. Cell 1992, 68:743754. %s paper describes more extensive experiments than [9*] with a substantial amount of data suggesting an interaction between IJ5 and both the 5’ and 3’ exons. In addition, it contains one of the first discussions of the similarity between an snRNA structure and a functionally related structure of a self-splicing intron. 11. MADHANI HD, GUMIUE C: A Novel Base-Pairing Interac.. tion between U2 and U6 snRNAs Suggests a Mechanism for the Catalytic Activation of the SpIiceosome. Cell 1992, 71:803-817. The molecular genetic evidence to support an elegant and elaborate model of the dynamic interactions between U6, U4, U2 and pre-mRNA The paper also contains a strong argument that U2, ~6 and pre-mRNA form a structure similar to one formed by self-splicing introns. 12. MCPHEETERS DS, ABELSON J: Mutational Analysis of the Yeast . U2 snRNA Suggests a Structural SimiIarity to the Catalytic Core of Group I Introns. CeN 1992, 71:81$X331. A biochemical genetic analysis of mutants similar to those examined in uiuo in [ 1l**]. The authors suggest an interaction of U2 with U6 as well as making a proposal that a structure similar to the guanosine.binding pocket of the group 1 self-splicing introns is formed. 13. MA~HANI HD, BORD~NNE R, GUIHKIE C: Multiple Roles for U6 snRNA in the Splicing Pathway. Genes Dev 1990, 8.

.

4:2264-2277.

14.

FAB~UZIOP, ABEL%N J: Two Domains of Yeast U6 SmalI Nuclear RNA Required for Both Steps of Nuclear Precursor Messenger RNA Splicing. Science 1990, 250:404-409. SAWA H, ABE~N J: Evidence for a Base-Pairing Interaction 15. . between U6 SmalI Nuclear RNA and the 5’ Splice Site during the Splicing Reaction in Yeast, Pm Nat1 Acud Sci LISA 1992, 89:1126+11273. A base by base mapping of sites of UV crossiinking between ~6 and the 5’ splice site in yeast, providing evidence for a specific set of base pairing interactions diierent from those suggested by psoralen crosslinking ]7**].

M McKeown, Molecular Biology and Virology Laboratory, The Salk Institute, PO Box 85800, San Diego, California 92186-5800, USA