Exon Ligation Research Articles

The DEAH Box ATPases Prp16 and Prp43 Cooperate to Proofread 5′ Splice Site Cleavage during Pre-mRNA Splicing

To investigate the mechanisms underlying accurate pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5' splice site cleavage. We inactivated spliceosomes by disrupting a metal-ligand interaction at the catalytic center and discovered that, when the DEAH box ATPase Prp16 was disabled, these spliceosomes catalyzed 5' splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5' splice site cleavage, we found that Prp16 can associate with spliceosomes before 5' splice site cleavage, consistent with a role for Prp16 in proofreading 5' splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH box ATPase Prp43, a spliceosome disassembly factor. These data indicate that spliceosomes distinguish slow substrates and that the mechanisms for establishing the fidelity of 5' splice site cleavage and exon ligation share a common ATP-dependent framework.

Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns

The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites.

Spliceosome discards intermediates via the DEAH box ATPase Prp43p

To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5' exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.

Constraining errors in splice site choice

While even single nucleotide errors in pre‐mRNA splicing can lead to catastrophic consequences, our understanding of fidelity mechanisms in splicing is poor. Genetics studies have established the importance of fidelity mechanisms in splicing and implicated 3 of the 8 spliceosomal DExD/H box ATPases in competing with splicing to enable kinetic proofreading. However, due to a paucity of in vitro assays, it has remained unclear how specificity is established by kinetic proofreading and how rejected substrates are discarded. We have established in vitro that each chemical step in splicing is proofread by a DEAH box ATPase – 5′ splice site cleavage is proofread by Prp16 and exon ligation is proofread by Prp22. In a kinetic proofreading mechanism, specificity can be achieved if the rate of the ATPase and/or the rate of the proofread step varies with the authenticity of the substrate. We have found evidence that both Prp16 and Prp22 enhance specificity by discriminating against slowly splicing substrates. Both Prp16 and Prp22 reject substrates reversibly, necessitating an independent discard activity that we attribute to the DEAH box ATPase Prp43, which normally functions to discard a genuine intron after excision from the precursor. These findings establish that both chemical steps in splicing are proofread by a common ATP‐dependent framework and support a general role for DExD/H box ATPases in fidelity. Support: ACS, NIH.

Mechanisms involved in efficient removal of multiple introns

The vast majority of mammalian pre‐mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre‐mRNA splicing that can interfere with downstream splicing events. These considerations raise the question whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron‐containing pre‐mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions, or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, kinetic trap experiments show that spliceosomal components that are not involved in the initial splicing event remain associated with the pre‐mRNA to ensure efficient subsequent intron removal. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splices sites. NIH funding.

Exon sequences at the splice junctions affect splicing fidelity and alternative splicing

Identification of splice sites is essential for the expression of most eukaryotic genes, allowing accurate splicing of pre-mRNAs. The splice sites are recognized by the splicing machinery based on sequences within the pre-mRNA. Here, we show that the exon sequences at the splice junctions play a significant, previously unrecognized role in the selection of 3' splice sites during the second step of splicing. The influence of the exon sequences was enhanced by the Prp18 mutant Prp18DeltaCR, and the strength of an exon sequence in Prp18DeltaCR splicing predicted its effect in wild-type splicing. Analysis of the kinetics of splicing in vitro demonstrated that 3' splice sites were chosen competitively during the second step, likely at the same time as exon ligation. In wild-type yeast, splice site selection for two genes studied was altered by point mutations in their exon bases, affecting splicing fidelity and alternative splicing. Finally, we note that the degeneracy of the genetic code allows competing 3' splice sites to be eliminated from coding regions, and we suggest that the evolution of the splicing signals and the genetic code are connected.

The Role of Exon Sequences in C Complex Spliceosome Structure

Pre-mRNA splicing is catalyzed by a large ribonucleoprotein complex called the spliceosome. Previous electron microscopy reconstruction of C complex spliceosomes arrested between the two chemical steps of splicing revealed an averaged core structure consisting of three primary domains surrounding a central cavity. Here we characterize the involvement of pre-mRNA in this structured core of C complex by protection mapping. We find that the 3' end of the cleaved 5′ exon and intron sequences flanking the branched lariat are buried in the complex. Upstream regions of the 5′ exon and the entire 3′ exon, including the mutant 3′ splice site, are accessible and can be removed by nucleolytic cleavage. Furthermore, we show that the second-step active site of the complex, which is arrested by a 3′ splice site mutation, can accept a normal 3′ splice site in trans to catalyze exon ligation. Removal of the accessible exon regions alters the protein composition of the complex, but the core spliceosome proteins associated with the uridine-rich small nuclear ribonucleoproteins U2, U5, and U6 and the Prp19 complex as well as several other proteins remain intact. Two-dimensional averaged images of an exon-trimmed complex closely resemble C complex assembled on full-length pre-mRNA, supporting the hypothesis that the electron microscopy model of C complex reflects the core structure of a catalytically competent particle. Trimming the 3′ exon does, however, alter the distribution of particles that appear to be missing some density, suggesting that the exon plays a role in stabilizing C complex.

Crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of the human spliceosomal DExD/H-box protein hPrp22

The Homo sapiens DExD/H-box protein hPrp22 is a crucial component of the eukaryotic pre-mRNA splicing machinery. Within the splicing cycle, it is involved in the ligation of exons and generation of the lariat and it additionally catalyzes the release of mature mRNA from the spliceosomal U5 snRNP. The yeast homologue of this protein, yPrp22, shows ATP-dependent RNA-helicase activity and is capable of unwinding RNA/RNA duplex molecules. A truncated construct coding for residues 950-1183 of human Prp22, comprising the structurally and functionally uncharacterized C-terminal domain, was cloned into an Escherichia coli expression vector. The protein was subsequently overproduced, purified and crystallized. The crystals obtained diffracted to 2.1 A resolution, belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 78.2, c = 88.4 A, and contained one molecule in the asymmetric unit.

The spliceosome as ribozyme hypothesis takes a second step

The spliceosome is a massive assembly of 5 RNAs and many proteins that, together, catalyze precursor-mRNA (pre-mRNA) splicing. The splicing mechanism involves 2 steps: (i) cleavage of the 5′ exon–intron phosphodiester bond and formation of a new 2′–5′ bond resulting in an intron “lariat,” and (ii) exon ligation (Fig. 1A). This 2-step phosphoryl transfer mechanism is suspiciously identical to the reaction catalyzed by the group II self-splicing introns, which are ribozymes. The precedent for a ribozyme-catalyzed splicing mechanism, combined with the fact that the spliceosome has an RNA core that has been highly conserved for >1 billion years, led researchers to hypothesize that the spliceosome may use an RNA-based catalytic mechanism (1). However, the spliceosome as ribozyme hypothesis has been exceedingly difficult to prove, for 2 major reasons. First, the spliceosome contains many proteins that are essential for splicing (2). Second, the uncatalyzed rate of RNA ligation in vitro is significant when measured over many hours (3). This latter point makes it difficult to establish whether an inefficient reaction is actually caused by a catalytic rate enhancement or occurs spontaneously because of template-driven proximity. This is a particular concern for 1-step phosphoryl transfer reactions. Furthermore, it is possible to serendipitously obtain unrelated activities from RNAs, perhaps because a significant region of RNA conformational space retains some degree of catalytic activity (4). Therefore, it is important to be very cautious when interpreting an inefficient RNA-based reaction. The relevance of using engineered, protein-free RNA systems to study splicing has been recently argued (5, 6). In a recent issue of PNAS, Valadkhan et al. (7) demonstrated that an RNA complex derived from the spliceosome can perform a reaction that resembles splicing (Fig. 1B). This finding is significant because the reaction appears to be identical to the second step of splicing and is ≈10-fold more efficient than previous studies that used similar RNAs (8–10).

The Hierarchy of Exon-Junction Complex Assembly by the Spliceosome Explains Key Features of Mammalian Nonsense-Mediated mRNA Decay

Author SummaryThe first step in the expression of eukaryotic protein-coding genes is transcription into a messenger RNA (mRNA) precursor in the nucleus. These precursor mRNAs then undergo maturation through the removal of introns in a process termed splicing. During splicing, the splicing machinery or “spliceosome” deposits a complex of proteins onto the mRNA that accompanies it during post-transcriptional steps in gene expression, including the regulation of mRNA stability, transport out of the nucleus, cellular localisation, and translation. This complex, the exon junction complex (EJC), represents a molecular memory of the splicing process. Understanding the biogenesis of EJCs and their downstream effects helps reveal the basic principles by which the primary steps of mRNA synthesis are coupled to the regulation of gene expression. Here we show that EJCs are assembled in a strictly splicing-dependent manner through an unexpected, coordinated, and hierarchical assembly pathway. Importantly, we show that the EJC recruits the cytoplasmic protein BTZ, which then bridges the complex to an mRNA quality-control machinery called the nonsense-mediated decay pathway that degrades mRNAs containing premature stop codons. This finding suggests that the EJC and bridging by BTZ help determine the stability of mRNA and thus are essential for proper cellular surveillance of mRNA quality.

Evidence that U2/U6 helix I promotes both catalytic steps of pre-mRNA splicing and rearranges in between these steps

During pre-mRNA splicing, the spliceosome must configure the substrate, catalyze 5' splice site cleavage, reposition the substrate, and catalyze exon ligation. The highly conserved U2/U6 helix I, which adjoins sequences that define the reactive sites, has been proposed to configure the substrate for 5' splice site cleavage and promote catalysis. However, a role for this helix at either catalytic step has not been tested rigorously and previous observations question its role at the catalytic steps. Through a comprehensive molecular genetic study of U2/U6 helix I, we found that weakening U2/U6 helix I, but not mutually exclusive structures, compromised splicing of a substrate limited at the catalytic step of 5' splice site cleavage, providing the first compelling evidence that this helix indeed configures the substrate during 5' splice site cleavage. Further, mutations that we proved weaken only U2/U6 helix I suppressed a mutation in PRP16, a DEAH-box ATPase required after 5' splice site cleavage, providing persuasive evidence that helix I is destabilized by Prp16p and suggesting that this structure is unwound between the catalytic steps. Lastly, weakening U2/U6 helix I also compromised splicing of a substrate limited at the catalytic step of exon ligation, providing evidence that U2/U6 helix I reforms and functions during exon ligation. Thus, our data provide evidence for a fundamental and apparently dynamic role for U2/U6 helix I during the catalytic stages of splicing.

A conserved 3′ extension in unusual group II introns is important for efficient second-step splicing

The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3′ extension. Here, we report the discovery of four additional group II introns with a similar 3′ extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47–61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem–loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3′ splice site.

Spliceosomal cleavage generates the 3′ end of telomerase RNA

Telomeres cap the ends of chromosomes and provide a means to complete replication. The DNA portion of telomeres is synthesized by the enzyme telomerase using part of an RNA subunit as a template for reverse transcription. How the mature 3' end of telomerase RNA is generated has so far remained elusive. Here we show that in Schizosaccharomyces pombe telomerase RNA transcripts must be processed to generate functional telomerase. Characterization of the maturation pathway uncovered an unexpected role for the spliceosome, which normally catalyses splicing of pre-messenger RNA. The first spliceosomal cleavage reaction generates the mature 3' end of telomerase RNA (TER1, the functional RNA encoded by the ter1(+) gene), releasing the active form of the RNA without exon ligation. Blocking the first step or permitting completion of splicing generates inactive forms of TER1 and causes progressive telomere shortening. We establish that 3' end processing of TER1 is critical for telomerase function and describe a previously unknown mechanism for RNA maturation that uses the ability of the spliceosome to mediate site-specific cleavage.

The Biflavonoid Isoginkgetin Is a General Inhibitor of Pre-mRNA Splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.

Structure and Function of the Pre-mRNA Splicing Machine

Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.

Flexibility in the site of exon junction complex deposition revealed by functional group and RNA secondary structure alterations in the splicing substrate

The exon junction complex (EJC) is critical for mammalian nonsense-mediated mRNA decay and translational regulation, but the mechanism of its stable deposition on mRNA is unknown. To examine requirements for EJC deposition, we created splicing substrates containing either DNA nucleotides or RNA secondary structure in the 5' exon. Using RNase H protection, toeprinting, and coimmunoprecipitation assays, we found that EJC location shifts upstream when a stretch of DNA or RNA secondary structure appears at the canonical deposition site. These upstream shifts occur prior to exon ligation and are often accompanied by decreases in deposition efficiency. Although the EJC core protein eIF4AIII contacts four ribose 2'OH groups in crystal structures, we demonstrate that three 2'OH groups are sufficient for deposition. Thus, the site of EJC deposition is more flexible than previously appreciated and efficient deposition appears spatially limited.

The assembly of a spliceosomal small nuclear ribonucleoprotein particle

The U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) are essential elements of the spliceosome, the enzyme that catalyzes the excision of introns and the ligation of exons to form a mature mRNA. Since their discovery over a quarter century ago, the structure, assembly and function of spliceosomal snRNPs have been extensively studied. Accordingly, the functions of splicing snRNPs and the role of various nuclear organelles, such as Cajal bodies (CBs), in their nuclear maturation phase have already been excellently reviewed elsewhere. The aim of this review is, then, to briefly outline the structure of snRNPs and to synthesize new and exciting developments in the snRNP biogenesis pathways.

A DExH/D-box Protein Coordinates the Two Steps of Splicing in a Group I Intron

Proteins of the DExH/D family are ATPases that can unwind duplex RNA in vitro. Individual members of this family coordinate many steps in ribonucleoprotein enzyme assembly and catalysis in vivo, but it is largely unknown how the action of these co-factors is specified and precisely timed. As a first step to address this question biochemically, we describe the development of a new protein-dependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast has shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5β group I intron, we show that efficient in vitro splicing of aI5β occurs with only two of these co-factors and, furthermore, they fulfill distinct functions in vitro. The Mrs1p protein stabilizes RNA structure and promotes the first step in splicing. In contrast, a DExH/D protein, Mss116p, acts after the first step and, utilizing ATP hydrolysis, specifically enhances the efficiency of exon ligation. An analysis of Mss116p variants with mutations that impair its RNA-stimulated ATP hydrolysis activity or reduce its ability to unwind duplexes show that the efficiency of ATP hydrolysis is a major determinant in promoting exon ligation. These observations suggest that Mss116p acts in aI5β splicing by catalyzing changes in the structure of the RNA/protein splicing intermediate that promote the second step. More broadly, these observations are consistent with a model in which the “functional-timing” of DExH/D-box protein action can be specified by a specific conformation of its substrate due to the “upstream” activity of other co-factors.

A relaxed active site after exon ligation by the group I intron

During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria

The Pet54p protein is an archetypical example of a dual functioning (‘moonlighting’) protein: it is required for translational activation of the COX3 mRNA and splicing of the aI5β group I intron in the COX1 pre-mRNA in Saccharomyces cerevisiae mitochondria (mt). Genetic and biochemical analyses in yeast are consistent with Pet54p forming a complex with other translational activators that, in an unknown way, associates with the 5′ untranslated leader (UTL) of COX3 mRNA. Likewise, genetic analysis suggests that Pet54p along with another distinct set of proteins facilitate splicing of the aI5β intron, but the function of Pet54 is, also, obscure. In particular, it remains unknown whether Pet54p is a primary RNA-binding protein that specifically recognizes the 5′ UTL and intron RNAs or whether its functional specificity is governed in other ways. Using recombinant protein, we show that Pet54p binds with high specificity and affinity to the aI5β intron and facilitates exon ligation in vitro. In addition, Pet54p binds with similar affinity to the COX3 5′ UTL RNA. Competition experiments show that the COX3 5′UTL and aI5β intron RNAs bind to the same or overlapping surface on Pet54p. Delineation of the Pet54p-binding sites by RNA deletions and RNase footprinting show that Pet54p binds across a similar length sequence in both RNAs. Alignment of the sequences shows significant (56%) similarity and overlap between the binding sites. Given that its role in splicing is likely an acquired function, these data support a model in which Pet54p's splicing function may have resulted from a fortuitous association with the aI5β intron. This association may have lead to the selection of Pet54p variants that increased the efficiency of aI5β splicing and provided a possible means to coregulate COX1 and COX3 expression.