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RNA Interactions in mRNA Splicing

  1. Andrew J Newman

Published Online: 14 MAR 2008

DOI: 10.1002/9780470015902.a0000881.pub2

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Newman, A. J. 2008. RNA Interactions in mRNA Splicing. eLS. .

Author Information

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

Publication History

  1. Published Online: 14 MAR 2008

Overview

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Splicing of messenger ribonucleic acid (mRNA) precursors occurs via a two-step transesterification pathway in complex ribonucleoprotein machines called spliceosomes. The transesterification reactions generate branched or ‘lariat’ forms of the intron as intermediates and products and the splicing pathway involves an obligatory bimolecular intermediate consisting of 5′ exon and intron-3′ exon RNA species. Spliceosomes contain several smaller ribonucleoprotein assemblies (small nuclear ribonucleoprotein particles called U1, U2, U4/U6 and U5 snRNPs) and an array of additional protein components of diverse functions. Each snRNP subassembly contains a small nuclear RNA (snRNA) molecule – two in the case of the U4/U6 snRNP – and multiple protein factors. The snRNAs contain short phylogenetically conserved motifs that interact to form a dynamic network of snRNA and substrate sequences. These interactions play crucial roles in splice site recognition, juxtaposition of the reactive species for catalysis and perhaps in the chemistry of the transesterification reactions. See also Messenger RNA: Interaction with Ribosomes, and mRNA Splicing: Regulated and Differential

Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Comparison of spliceosomal snRNAs from distantly related eukaryotes reveals striking conservation of primary sequences and secondary structures. Short, invariant motifs in these snRNAs are involved in critical base pairing contacts with conserved sequences in the pre-mRNA or in other spliceosomal snRNAs. These contacts produce a fluid network of RNA–RNA interactions that is remodelled as the spliceosome is assembled and activated (Figure 1). It is extraordinary that the results from a wide range of experimental approaches in eukaryotes as diverse as yeast and mammals can be accommodated in a single scheme of interactions. Powerful, independent support for this network of RNA contacts has followed from the discovery in metazoan organisms of a novel, minor type of spliceosome responsible for splicing of rare pre-mRNAs with unusual splice sites. Although most of the snRNA components of this minor class of spliceosome are quite distinct in sequence from the major spliceosome's snRNAs, they nevertheless form a strikingly similar network of snRNAsnRNA and snRNA–intron contacts. See also Messenger RNA in Eukaryotes, and Spliceosomal Machinery

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Figure 1. Cartoon showing the dynamic network of RNA interactions at three successive stages of formation and activation of a conventional (U2-type) cis-spliceosome (precatalytic, first-step catalysis and second-step catalysis, respectively). ‘*’ Indicates a non-Watson–Crick interaction between the G residues at the 5′ and 3′ ends of the intron. See the text for detailed explanation. Modified with permission from Nilsen 1998. Copyright © 1998 Cold Spring Harbor Laboratory Press.

Interactions between U1 snRNA and the 5′ Splice Site

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

A short invariant sequence ACUUAC at the 5′ end of U1 snRNA is complementary to the intron 5′ splice site consensus sequence GURAGU (R=A or G). Genetic and biochemical studies in budding yeast (Saccharomyces) and mammalian systems have demonstrated that these two sequences interact by base pairing (Figure 1). For the majority of introns the integrity of the 5′-terminal U1 snRNA sequence is essential for splicing in vitro. Site-specific photochemical crosslinking experiments have shown that the U1:5′ splice site interaction is the earliest snRNA–pre-mRNA contact detected in splicing reactions (Wyatt et al., 1992; Sontheimer and Steitz, 1993). A U1 snRNP:pre-mRNA complex, called a ‘commitment complex’, is the first defined biochemical entity on the spliceosome assembly pathway (Seraphin and Rosbash, 1989). See also Messenger RNA Splicing Signals, and mRNA Splicing: Role of snRNAs

Functional analysis of U1 snRNA:5′ splice site interactions in yeast, however, has shown that 5′ splice site cleavage can be uncoupled from its interaction with the U1 snRNA (Seraphin and Rosbash, 1990). For example, mutation of the highly conserved G at position 5 in the 5′ splice site sequence can activate cleavage at aberrant 5′ splice sites in the vicinity of the authentic site. However, cleavage at these sites is not suppressed by mutations in U1 snRNA that restore base pairing with the mutant 5′ splice site. This clearly suggests that other recognition factors may be important for precise definition of the 5′ splice site. Interestingly there is a class of pre-mRNAs, exemplified by the Drosophila fushi tarazu gene (ftz) pre-mRNA, that are efficiently spliced even in extracts depleted of U1 snRNA. The U1-independence of ftz pre-mRNA splicing is promoted by specific sequence elements flanking the 5′ splice site that may act to enhance U6 snRNA interactions with the 5′ end of the intron (see later).

Interactions between U2 snRNA and the Branch Site

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

U2 snRNA contains an invariant sequence GUAGUA that is complementary to the highly conserved UACUAAC branchpoint sequence of budding yeast introns. The effects of compensatory mutations in these two elements show that they do indeed interact by base pairing and in the current model for this interaction the branchpoint adenosine nucleophile is unpaired and ‘bulged’ out of the U2 snRNA:intron helix. In fact, the behaviour of pre-mRNAs with alterations of the backbone ribose residues flanking the branchpoint adenosine suggests that bulging of this nucleotide is important for the use of its 2′ hydroxyl as the nucleophile in the first catalytic step (Query et al., 1994). In mammals the branchpoint sequence is quite degenerate, with a consensus sequence YNCURAY. Nevertheless, mutational analysis of U2 snRNA:branch site interactions in the mammalian system has demonstrated a role for base pairing just as in yeast. See also Catalytic RNA, Covalent Nucleophilic Catalysis, and RNA Structure

In both yeast and mammals, multiple protein factors are involved in the adenosine triphosphate (ATP)-dependent binding of U2 snRNP to the intron branch site in the U1 snRNP-containing ‘commitment complex’. Indeed, initial recognition of the branch site sequence is mediated by a sequence-specific RNA-binding protein called BBP for branchpoint bridging protein (Abovich and Rosbash, 1997; Berglund et al., 1997). Addition of U2 snRNP to the U1 snRNP:pre-mRNA complex thus requires the eviction of BBP from the branch site sequence. Functional analysis of U2 snRNA has shown that the interaction between the branch site recognition sequence and the branch site requires a specific conformational change in U2 snRNA, which may be a crucial switch in early spliceosome assembly. Two of the protein factors required for this step are putative ATP-dependent RNA helicases, one of which is hypothesized to act on U2 snRNA to increase the accessibility of the branch site recognition sequence. See also snRNPs: Methods for Purification

Interactions between U5 snRNA and the Exon Borders

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

U5 snRNA has a conserved secondary structure and an essential, invariant 9-nucleotide sequence GCCUUUUAC displayed in an 11-nucleotide loop. Functional analysis and crosslinking experiments in yeast and mammalian systems have shown that the U5 snRNA loop directly contacts exon sequences at the 5′ and 3′ splice sites (Newman and Norman, 1992; Sontheimer and Steitz, 1993). The 5′ exon:U5 interaction is established before 5′ splice site cleavage and persists through both catalytic steps of splicing. In contrast, the interaction between the U5 loop and the 3′ exon occurs only after the first catalytic step. These interactions are thought to align the 5′ and 3′ exons for the second catalytic step in which the 3′ hydroxyl of the 5′ exon attacks the phosphodiester bond at the 3′ splice site (Figure 1). Thus spliceosomes that lack the exon-interaction RNA loop of U5 are unable to carry out the second catalytic step, although 5′ splice site cleavage occurs normally in the absence of U5 snRNA:5′ exon interactions (O'Keefe et al., 1996).

Since exon sequences encode a variety of protein sequences, most of the RNA sequence information that specifies the splice sites is found within the intron. Given that the U5 exon-interaction loop sequence is invariant, how is base pairing between U5 and diverse exon sequences achieved and maintained? Site-specific RNA–protein crosslinking data suggest that a highly conserved U5 snRNP protein encoded by the PRP8 gene in yeast plays crucial roles in maintaining contacts between exon sequences and U5 snRNP, possibly by stabilizing intrinsically weak (often non-Watson–Crick) base pairing between exons and the U5 snRNA loop (Teigelkamp et al., 1995). The Prp8 protein (and its mammalian orthologue p220 or hPrp8) makes contact with 5′ and 3′ exon sequences even in spliceosomes lacking the exon interaction loop of U5 snRNA. Contact with the Prp8 protein may be the primary interaction that anchors or tethers the 5′ exon splicing intermediate, while base pairing between U5 snRNA loop and the exons may assist in aligning the 5′ and 3′ exons for the second catalytic step of splicing (Dix et al., 1998). See also Base Pairing in RNA: Unusual Patterns

Interactions between U6 snRNA and the 5′ Splice Site

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

An interaction between U6 snRNA and the 5′ splice site was first identified by ultraviolet (UV) crosslinking in yeast and mammalian splicing extracts (Sawa and Abelson, 1992; Sawa and Shimura, 1992). Mapping of the crosslinks led to an explicit model for this interaction, which was then confirmed by making compensatory mutations in both RNA partners. Specifically, the first three nucleotides of the invariant ACAGAG sequence in U6 snRNA (nucleotides 47–52 of yeast U6) base pair with positions 4–6 of the 5′ splice site, including the highly conserved G at intron position 5 (Kandels-Lewis and Seraphin, 1993; Lesser and Guthrie, 1993). Clearly this interaction is mutually exclusive with the base pairing between U1 snRNA and the 5′ splice site, which is known to occur early in spliceosome assembly (Figure 1).

After identification of the 5′ end of the intron by U1 snRNA the 5′ splice site is transferred to U6 snRNA (Konforti and Konarska, 1994; Konforti et al., 1993), which then determines the precise phosphodiester bond to be attacked. This explains why aberrant 5′ splice site cleavage caused by mutation of the highly conserved G at intron position 5 is not suppressed by the compensating mutation in U1 snRNA: the interaction between the U6 snRNA ACAGAG sequence and the mutated 5′ splice site is still destabilized. Genetic data suggest that the U6 snRNA:5′ splice site contact may extend to an interaction between the invariant G (intron position 1) and U6 G52, the last nucleotide of the ACAGAG sequence (Luukkonen and Seraphin, 1998). In any case it is clear that the U6 snRNA:5′ splice site interaction is crucial for the specificity of the first catalytic step. Site-specific crosslinking experiments in mammalian splicing extracts have shown that contact between the 5′ splice site and U6 snRNA continues after the first catalytic step, since a crosslink then becomes detectable between the second position of the intron (U in the invariant GU sequence) and the last A of the ACAGAG sequence (Sontheimer and Steitz, 1993).

The replacement of U1 snRNA by U6 snRNA at the 5′ splice site is ATP-dependent and genetic data have implicated the Prp28 factor (a putative ATP-dependent RNA helicase) in this step. The mammalian homologue of the Prp28 protein is a U4/U6.U5 triple-snRNP protein. However, it is currently unclear whether Prp28 acts by unwinding the U1 snRNA:5′ splice site helix, by promoting formation of the U6 snRNA:5′ splice site interaction or by some other means (Staley and Guthrie, 1998).

Interactions between U2 and U6 snRNAs

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

After addition of the U4/U6.U5 triple-snRNP particle to the U1.U2 pre-spliceosome, there is a major remodelling of RNA interactions in preparation for the catalytic steps. Initially U4 and U6 snRNAs are extensively base paired together via two intermolecular helices, but these interactions are disrupted and the U6 snRNA sequences can then form new contacts. Mutational and crosslinking studies indicate that an extensively base-paired RNA complex forms between U2 and U6 snRNAs to produce a critical catalytic component of the spliceosome (Madhani and Guthrie, 1992; Sun and Manley, 1995). Together with the U2 snRNA:branch site and U6 snRNA:5′ splice site interactions (see earlier), U2–U6 helix formation may provide a structural basis for directing the adenosine branch site nucleophile to attack the phosphodiester bond at the 5′ splice site. Consistent with this model, analysis of RNA structures in activated spliceosomes using hydroxyl-radical probing shows that conserved U2 and U6 snRNA sequences are closely juxtaposed to the 5′ splice site (Rhode et al., 2006). Recent genetic analysis of U2 snRNA functions in yeast spliceosomes has shown that U2 sequences downstream of the branch-binding site are extensively remodelled during the splicing cycle, switching between two mutually exclusive intramolecular stem structures that promote catalysis and substrate binding/release, respectively (Hilliker et al., 2007; Perriman and Ares, 2007).

Structural analysis of protein-free complexes of U2 and U6 snRNAs has revealed clear similarities with an essential RNA structure from Group II self-splicing introns and the data suggest that the U2–U6 structure could undergo a conformational change between the first and second catalytic steps of splicing (Sashital et al., 2004). A protein-free U2–U6 complex has also been shown to bind a short synthetic RNA containing an intron branch site and to activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction related to the first step of splicing (Valadkhan and Manley, 2001). These findings underscore the importance of specific U2–U6 RNA structures in critical events in the core of the spliceosome.

Interactions between U4 and U6 snRNAs

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

U6 enters the spliceosome as part of a U4/U6.U5 triple-snRNP and is extensively base paired to U4 snRNA: in yeast this interaction involves a total of 24 base pairs in two helical segments. Yet in preparation for catalysis these helices are unwound to allow U6 to interact with U2 snRNA (see earlier). Thereafter, U4 apparently plays no further essential role, since spliceosomes blocked before the first catalytic step and artificially stripped of U4 can subsequently perform the splicing reactions when appropriate protein factors are added back.

Since the unravelling of the U4:U6 helices is a crucial prelude to catalysis, there has been a great deal of interest in the mechanism of this step and the identities of the spliceosome components involved. Three different spliceosomal RNA helicases have been shown to be capable of ATP-dependent unwinding of U4/U6 helices in vitro: the yeast Prp16 and Prp22 and Brr2/Snu246 factors (Laggerbauer et al., 1998; Schwer and Gross, 1998; Wagner et al., 1998; Wang et al., 1998). The human U5 200-kDa factor (the orthologue of the yeast Brr2/Snu246 protein) is thought to be the factor that actually unwinds U4/U6 in the spliceosome. This protein is a stable component of the U4/U6.U5 tri-snRNP and so is in the right molecular environment to act on U4/U6. In contrast, the Prp16 and Prp22 factors are known to associate transiently with spliceosomes and to act later in the splicing pathway, well after the U4/U6 dissociation step. Biochemical studies have shown that the Prp24 RNA binding protein acts to antagonize the U4/U6 snRNP unwinding function of the Brr2 factor in a spliceosome-independent snRNP recycling pathway (Raghunathan and Guthrie, 1998).

Interactions between the Intron Ends

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Paradoxically, mutation of the highly conserved G at intron position 1 does not block 5′ splice site cleavage but blocks the second catalytic step, leading to accumulation of ‘dead-end’ splicing intermediates. Mutation of the highly conserved G at the 3′ end of the intron also prevents 3′ splice site cleavage and likewise results in accumulation of intermediates. Surprisingly, specific combinations of point mutations at the first and last positions of the intron display reciprocal suppression. So, for example, a G-to-A mutation at the 5′ end of the intron, when combined with a G-to-C mutation at the 3′ end, results in a substantial level of accurate splicing to produce functional mRNA (Parker and Siliciano, 1993). The fact that only particular combinations of intron terminal nucleotides allow reciprocal suppression strongly suggests that there may be a direct interaction between these two residues that is crucial for the second catalytic step. Substitution of inosine (which lacks the exocyclic N2 amino group) for the intron-terminal guanosines does not significantly affect the second catalytic step. This information suggests that the G residues at the intron termini may interact via a symmetric N1–carbonyl noncanonical base pair, and that this contact is important for the second transesterification. Significantly, I·I and A·C base pairs would be essentially isosteric with the proposed non-Watson–Crick G·G base pair (Scadden and Smith, 1995). The occurrence of a rare class of natural introns with A–C termini (see later) supports the notion that structurally ‘compatible’ intron ends are functionally significant. Possibly this interaction contributes some specificity to the second catalytic step by excluding sterically incompatible 3′ intron termini from the spliceosome's active site.

Role of Pre-mRNA Secondary Structure in Splicing

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Although the short, conserved sequences at the splice sites and branch site are the primary determinants of splice site utilization, other intron sequences can enhance or inhibit splicing by formation of specific secondary structure. In higher eukaryotes there are relatively few cases where intron secondary structure has been shown to affect splice site selection, perhaps reflecting a widespread dependence on protein-based splicing ‘enhancers’ in these organisms. In the yeast Saccharomyces, in contrast, many introns contain short regions of complementarity downstream of the 5′ splice site and upstream of the branch site. Base pairing between these sequences has been demonstrated directly in living cells. Formation of secondary structure via these complementary elements enhances the formation of U1 snRNP-containing commitment complexes. This results in increased splicing efficiency and can influence splice site choice in competition experiments (Libri et al., 1995; Charpentier and Rosbash, 1996).

Usually in pre-mRNA splicing an upstream 5′ splice site is paired with a downstream 3′ splice site. In rare cases, however, a 5′ splice site can be paired with an upstream 3′ splice site and this is believed to account for a low level of ‘scrambled’ mRNAs – in which the exons are misordered – and for the production of circular mRNAs. Circular mRNAs derived from the SRY gene are found in adult mouse testis, probably owing to the unusual secondary structure of the pre-mRNA, which may enhance splicing to the upstream 3′ splice site: the SRY pre-mRNA in testis contains long complementary sequences at its termini. In contrast a downstream promoter is used in genital ridge cells so that the 5′ complementary sequence is absent, and these cells produce only linear SRY mRNAs. Secondary structure-dependent exon circularization has been directly demonstrated in mammalian splicing reactions in vitro (Pasman et al., 1996). See also SRY, Sex Determination and Gonadal Differentiation, and HMG Domain Superfamily of DNA-bending Proteins: HMG, UBF, TCF, LEF, SOX, SRY and Related Proteins

Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

A variant spliceosome has been identified in metazoan cells: these particles consist of novel snRNA components functionally analogous to U1, U2, U4 and U6 snRNAs but contain U5 snRNA in common with conventional U2-containing spliceosomes. Splicing in these variant spliceosomes proceeds by a two-step transesterification pathway identical to that in conventional U2-type spliceosomes. These unusual spliceosomes are responsible for the splicing of pre-mRNAs containing rare introns with distinctive tightly conserved splice sites (/ATATCCTTT and YAC/ or /GTATCCTTT and YAG/) and a highly conserved branch site sequence TCCTTAAC (branch site in bold in Figure 1). These sequences are recognized by base-pairing interactions with the low-abundance snRNAs U11 (analogous to U1 snRNA) and U12 (analogous to U2 snRNA). The U12 snRNA:branch site interaction is predicted to bulge the branch site adenosine as in the case of U2 snRNA (Hall and Padgett, 1994, 1996; Tarn and Steitz, 1996a; Dietrich et al., 1997; Kolossova and Padgett, 1997; Wu and Krainer, 1997).

The two remaining snRNA components of U12-type spliceosomes are also of low abundance in mammalian cells and appear to be functionally analogous to U4 and U6 snRNAs in U2-type spliceosomes (Tarn and Steitz, 1996b; Incorvaia and Padgett, 1998). Interestingly, although these novel snRNAs are not extensively similar in sequence to their U2-type spliceosome counterparts, they form a base-paired complex just like U4/U6 and almost certainly dissociate in the assembled U12-type spliceosome. Comparison of the U2-type and U12-type U6 species shows that there are clear patches of sequence identity and these correspond precisely to those nucleotides that have been demonstrated to be critical for U6 function in U2-type spliceosomes. Furthermore, the U12-type U6 snRNA can interact with U12 snRNA to form a base-paired structure similar to U2–U6 helix I. These compelling similarities between U2-type and U12-type spliceosomes add up to a convincing independent validation of the current picture of snRNA interactions in spliceosomes.

RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Whereas most eukaryotes use spliceosomes to splice together exons derived from the same pre-mRNA via cis-splicing, some more primitive organisms use trans-splicing to splice a leader sequence on to the 5′ ends of mRNAs. Nematodes such as Caenorhabditis and Ascaris and kinetoplastid organisms such as Trypanosoma employ both cis- and trans-splicing in mRNA biogenesis. In trans-splicing the spliced leader sequence (SL) is derived from a trans-splicing specific snRNP, the SL RNP. Analysis of the snRNA components of nematode trans-spliceosomes shows that this process involves U2, U4, U5 and U6 snRNAs in addition to the SL RNP (Maroney et al., 1996), but U1 does not seem to be required. U5 snRNA interacts with the SL exon before and after the first catalytic step, suggesting that as in cis-spliceosomes U5 is involved in aligning the exons for the second catalytic step (Maroney et al., 1996). A sequence near the 3′ end of U6 snRNA (which is not thought to interact with any of the other snRNAs) interacts with a complementary sequence in the SL RNA (Hannon et al., 1992). This interaction results in the formation of a tetra-snRNP containing U4/U6.SL.U5 that is the likely equivalent of the cis-spliceosomal tri-snRNP. Thus trans-spliceosomes are probably made by addition of the tetra-snRNP containing the SL RNA to a U2 snRNP complex associated with the trans-splice acceptor RNA. See also Messenger RNA in Eukaryotes, and Microinjection of mRNA into Somatic Cells

Trans-splicing in trypanosomes involves a U5-like snRNA that interacts with the SL RNA and the free SL exon splicing intermediate via a conserved loop sequence, in a manner that precisely parallels the U5 snRNA:exon interactions in cis-spliceosomes (Dungan et al., 1996). Indeed, the trypanosome U5-like snRNA is found in a snRNP particle together with a protein highly homologous to the Prp8/hPrp8 U5 snRNP protein, emphasizing that the basic functions of U5 snRNP are conserved between cis- and trans-splicing (Lucke et al., 1997).

RNA Conformational Rearrangements in the Spliceosome

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Analysis of the RNA interactions in spliceosomes has shown that there is a complex and dynamic pattern of contacts among the snRNAs and between snRNA and substrate sequences. The spliceosome is clearly a highly fluid structure, yet the multiple conformational rearrangements of its RNA components appear to occur in a controlled fashion. Which factors and mechanisms orchestrate these rearrangements?

Two classes of proteins have attracted particular interest in this regard. The first is a class of ATPases characterized by DExD or DExH sequence motifs that are hypothesized to function as nonprocessive motor proteins that unwind short RNA duplexes. Spliceosomes contain several such putative RNA unwindases, and three of them have been shown to have ATP-dependent RNA unwinding activity using model substrates in vitro (reviewed in Staley and Guthrie, 1998). The activity of these ATPases explains at least in part the requirement for ATP hydrolysis during assembly, activation and remodelling of the spliceosome. Another class of spliceosomal proteins that can directly affect RNA conformation includes RNA annealing factors such as the U2AF65 mammalian factor. U2AF65 has multiple RNA recognition motifs (RRMs) with which it binds to the polypyrimidine tract 3′ of the intron branch site. This positions the U2AF65 RS domain (rich in arginine and serine residues) so that it can promote pairing of U2 snRNA with the branch site (Valcarcel et al., 1996). See also Adenosine Triphosphate, and Motor Proteins

Finally, the U5 snRNP protein Snu114p is a guanosine triphosphatase (GTPase) homologous to the ribosomal GTPase EF-2 (Fabrizio et al., 1997). Biochemical analysis of Snu114p functions suggests that factor has multiple roles in the regulation of spliceosome activation and disassembly of post-splicing complexes, perhaps acting as a classic regulatory G protein (Bartels et al., 2003; Small et al., 2006). See also G Proteins, Peptidyl Transfer on the Ribosome, and Ribosomal Proteins in Eukaryotes

Glossary
snRNA

Small nuclear RNA component of a snRNP (small nuclear ribonucleoprotein). snRNAs are typically 100–300 nucleotides long and can be folded into conserved secondary structures. They contain short conserved sequences that are critical for function.

snRNP

A complex of proteins and one or more snRNAs. Nucleoplasmic snRNPs generally play roles in mRNA processing.

Spliced leader (SL)

A short RNA that donates the 5′ exon (SL exon) in trans-splicing. The SL RNA is incorporated into trans-spliceosomes in the form of an SL RNP.

Spliceosome

A large ribonucleoprotein complex assembled from U1, U2, U4/U6 and U5 snRNPs plus protein cofactors and mRNA precursor. Responsible for intron removal by mRNA splicing.

trans-Splicing

mRNA processing step in which certain pre-mRNAs acquire a 5′-terminal spliced leader sequence from SL RNA.

References

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading

Further Reading

  1. Top of page
  2. Overview
  3. Phylogenetic Conservation of RNA–RNA Interactions in the Spliceosome
  4. Interactions between U1 snRNA and the 5′ Splice Site
  5. Interactions between U2 snRNA and the Branch Site
  6. Interactions between U5 snRNA and the Exon Borders
  7. Interactions between U6 snRNA and the 5′ Splice Site
  8. Interactions between U2 and U6 snRNAs
  9. Interactions between U4 and U6 snRNAs
  10. Interactions between the Intron Ends
  11. Role of Pre-mRNA Secondary Structure in Splicing
  12. Comparison of Interactions in the U12-type and Conventional (U2-type) Spliceosomes
  13. RNA–RNA Interactions Involving Spliced Leader RNAs (SL RNAs)
  14. RNA Conformational Rearrangements in the Spliceosome
  15. References
  16. Further Reading
  • Brow DA (2002) Allosteric cascade of spliceosome activation. Annual Review of Genetics 36: 333360.
  • Butcher SE and Brow DA (2005) Towards understanding the catalytic core structure of the spliceosome. Biochemical Society Transactions 33: 447449.
  • Collins CA and Guthrie C (2000) The question remains: is the spliceosome a ribozyme? Nature Structural Biology 7: 850854.
  • Nilsen TW (1998) RNA–RNA interactions in nuclear pre-mRNA splicing. In: Simons R and Grunberg-Manago M (eds) RNA Structure and Function. New York: Cold Spring Harbor Laboratory Press.
  • Patel AA and Steitz JA (2003) Splicing double: insights from the second spliceosome. Nature Reviews Molecular Cell Biology 4: 960970.
  • Will CL and Luhrmann R (2006) Spliceosome structure and function. In: Cech T and Atkins J (eds) The RNA World. New York: Cold Spring Harbor Laboratory Press.