Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.     

A role for branchpoints in splicing in vivo

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.     

Crystal structure of a self-splicing group I intron with both exons

The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here we report the X-ray crystal structure (3.1-A resolution) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asymmetrically positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.     

A tertiary interaction that links active-site domains to the 5' splice site of a group II intron

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.     

Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction

The intervening sequence (IVS) of the Tetrahymena ribosomal RNA precursor is excised as a linear RNA molecule which subsequently cyclizes itself in a protein-independent reaction. Cyclization involves cleavage of the linear IVS RNA 15 nucleotides from its 5' end and formation of a phosphodiester bond between the new 5' phosphate and the original 3'-hydroxyl terminus of the IVS. This recombination mechanism is analogous to that by which splicing of the precursor RNA is achieved. The circular molecules appear to have no direct function in RNA splicing, and we propose the cyclization serves to prevent unwanted RNA from driving the splicing reactions backwards.     

A 3' splice site-binding sequence in the catalytic core of a group I intron

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.     

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.     

Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA

The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.     

The human RNA kinase hClp1 is active on 3' transfer RNA exons and short interfering RNAs

RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer, synthetic siRNAs display a 5' OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5' end. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing and messenger RNA 3'-end formation machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5' end of the 3' exon during human tRNA splicing, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3'-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.     

Human U2 snRNA can function in pre-mRNA splicing in yeast

The removal of introns from messenger RNA precursors requires five small nuclear RNAs (snRNAs), contained within ribonucleoprotein particles (snRNPs), which complex with the pre-mRNA and other associated factors to form the spliceosome. In both yeast and mammals, the U2 snRNA base pairs with sequences surrounding the site of lariat formation. Binding of U2 snRNP to the highly degenerate branchpoint sequence in mammalian introns is absolutely dependent on an auxiliary protein, U2AF, which recognizes a polypyrimidine stretch adjacent to the 3' splice site. The absence of this sequence motif in yeast introns has strengthened arguments that the two systems are fundamentally different. Deletion analyses of the yeast U2 gene have confirmed that the highly conserved 5' domain is essential, although the adjacent approximately 950 nucleotides can be deleted without any phenotypic consequence. A 3'-terminal domain of approximately 100 nucleotides is also required for wild-type growth rates; the highly conserved terminal loop within this domain (loop IV) may provide specific binding contacts for two U2-specific snRNP proteins. We have replaced the single copy yeast U2 (yU2) gene with human U2 (hU2), expecting that weak or no complementation would provide an assay for cloning additional splicing factors, such as U2AF. We report here that hU2 can complement the yeast deletion with surprising efficiency. The interactions governing spliceosome assembly and intron recognition are thus more conserved than previously suspected. Paradoxically, the conserved loop IV sequence is dispensable in yeast.     

Exon shuffling by recombination between self-splicing introns of bacteriophage T4

The organization of genes into exons separated by introns may permit rapid evolution of protein-coding sequences by exon shuffling. Introns could provide non-coding targets for recombination, which would then give rise to novel combinations of exons. Evidence to support this theory is indirect and consists of examples of homologous domains of protein structure encoded in different genes, with introns in conserved positions at the boundaries of these domains. Here, we report the first direct evidence for exon shuffling. Two spontaneous deletion mutations of phage T4 have been characterized by sequencing, and they are clearly the result of recombination between homologous regions of two self-splicing group I introns. As a result of the recombination, exons of different genes are transcribed together, with a hybrid intron between them. One of these introns is proficient in self-splicing.     

Self-splicing introns in tRNA genes of widely divergent bacteria.

The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions. A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts. But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria. Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria. We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp. (beta). The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide. Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.     

Are vertebrate exons scanned during splice-site selection?

Pairwise recognition of splice sites as a result of a scanning mechanism is an attractive model to explain the coordination of vertebrate splicing. Such a mechanism would predict a polarity-of-site recognition in the scanned unit, but no evidence for a polarity gradient across introns has been found. We have suggested that the exon rather than the intron is the unit of recognition in vertebrates and that polyadenylation and splicing factors interact during recognition of 3'-terminal exons. Interaction is reflected in maximal rates of in vitro polyadenylation. If scanning across the exon is operating during this interaction, then insertion of a 5' splice site should depress polyadenylation. Here we report recognition in vitro and in vivo of a 5' splice site situated within a 3'-terminal exon, and a concomitant depression of polyadenylation and ultraviolet crosslinking of a polyadenylation factor. Decreased crosslinking was only found when the 3' and 5' splice sites were within 300 nucleotides of each other. These results are consistent with an exon scanning mechanism for splice-site selection.