Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.     

Compensatory mutations demonstrate that P8 and P6 are RNA secondary structure elements important for processing of a group I intron.

Compensatory mutations have been constructed which demonstrate that P8 and P6, two of nine proposed base-pairing interactions characteristic of group I introns, exist within the folded structure of the Tetrahymena thermophila rRNA intervening sequence, and that these secondary structure elements are important for splicing in E. coli and self-splicing in vitro. Two-base mutations in the 5' and 3' segments of P8 are predicted to disrupt P8 and a strong splicing-defective phenotype is observed in each case. A compensatory four-base mutation in P8 is predicted to restore pairing, and results in the restoration of splicing activity to nearly wild type levels. Thus, we conclude that P8 exists and is essential for splicing. In contrast to the strong phenotypes generally exhibited by mutations which disrupt RNA secondary structure, a two-base mutation in L8, the loop between P8[5'] and P8[3'], results in only a slight decrease in splicing activity. We also tested P6, a pairing which is proposed to consist of only two base-pairs in this intron. A two-base mutation in P6[3'] reduces splicing activity to a greater extent than does a two-base mutation in P6[5']. Comparison of the activities of these mutants and a compensatory P6 four-base mutant support the existence of P6, and suggest that the P6 pairing may be particularly important in the exon ligation step of splicing.     

Better late than early: delayed translation of intron‐encoded endonuclease I‐TevI is required for efficient splicing of its host group I intron

The td group I intron interrupting the thymidylate synthase (TS) gene of phage T4 is a mobile intron that encodes the homing endonuclease I‐TevI. Efficient RNA splicing of the intron is required to restore function of the TS gene, while expression of I‐TevI from within the intron is required to initiate intron mobility. Three distinct layers of regulation temporally limit I‐TevI expression to late in the T4 infective cycle, yet the biological rationale for stringent regulation has not been tested. Here, we deleted key control elements to deregulate I‐TevI expression at early and middle times post T4 infection. Strikingly, we found that deregulation of I‐TevI, or of a catalytically inactive variant, generated a thymidine‐dependent phenotype that is caused by a reduction in td intron splicing. Prematurely terminating I‐TevI translation restores td splicing, full‐length TS synthesis, and rescues the thymidine‐dependent phenotype. We suggest that stringent translational control of I‐TevI evolved to prevent the ribosome from disrupting key structural elements of the td intron that are required for splicing and TS function at early and middle times post T4 infection. Analogous translational regulatory mechanisms in unrelated intron‐open reading frame arrangements may also function to limit deleterious consequences on splicing and host gene function.     

Functional analyses of positions across the 5' splice site of the trypanosomatid spliced leader RNA. Implications for base-pair interaction with U5 and U6 snRNAs

In this study, we have used a genetic compensatory approach to examine the functional significance of the previously proposed interaction of spliced leader (SL) RNA with U5 small nuclear RNA (snRNA) (Dungan, J. D., Watkins, K. P., and Agabian, N. (1996) EMBO J. 15, 4016-4029; Xu, Y.-X., Ben Shlomo, H., and Michaeli, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8473-8478) and the interaction of the SL RNA intron with U6 snRNA analogous to cis-splicing. Mutations were introduced at positions -4, -1, +1, +4, +5, and +7/+8 relative to the SL RNA 5' splice site that were proposed to interact with U5 and U6 snRNAs. All mutants exhibited altered splicing phenotypes compared with the parental strain, showing the importance of these intron and exon positions for trans-splicing. Surprisingly, mutation at invariant +1 position did not abolish splicing completely, unlike cis-splicing, but position +2 had the most severe effect on trans-splicing. Compensatory mutations were introduced in U5 and U6 snRNAs to examine whether the defects resulted from failure to interact with these snRNAs by base pairing. Suppression was observed only for positions +5 and +7/+8 with U5 compensatory mutations and for position +5 with a U6 compensatory mutation, supporting the existence of a base pair interaction of U5 and U6 with the SL RNA intron region. The failure to suppress the other SL RNA mutants by the U5 compensatory mutations suggests that another factor(s) interacts with these key SL RNA positions.     

In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.     

A Tyrosyl-tRNA Synthetase Suppresses Structural Defects in the Two Major Helical Domains of the Group I Intron Catalytic Core

TheNeurospora crassamitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used anEscherichia coligenetic assay based on the phage T4tdintron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long- range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in thetdintron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.     

Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits.

U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 and I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA.     

Sequence specificity of the P6 pairing for splicing of the group I td intron of phage T4.

Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5' element (P6[5']) of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5'] and P6[3'] compensatory mutations. Second, all P6[5'] and P6[3'] mutants are defective in the first step of splicing, guanosine-dependent 5' splice site cleavage, whereas their activity at the 3' splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5']:[3'] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.     

Suppressor mutations identify box9 as a central nucleotide sequence in the highly ordered structure of intron RNA in yeast mitochondria.

Data presented here lend support to the notion that RNA splicing in yeast mitochondria depends on the formation of hybrid structures involving the well-conserved intron sequences box9 and box2. Starting with the cis-dominant splicing-defective box2 mutant G2590, a G----A transition, we isolated a revertant having a mitochondrial second site suppressor mutation, which restores splicing competence in the presence of the original mutation. Sequence analysis reveals that the suppressor mutation is a C----T transition in box9(5' part). This second mutation compensates for the first one in box2 and restores a box2/box9(5') hybrid. Combined with previous data demonstrating an interaction of the adjacent sequence box9(3' part) with the upstream box9c sequence in intron 4, the central role of box9 in the formation of the intron 4, the central role of box9 in the formation of the intron 4 RNA high order structure becomes evident.     

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.     

Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis

Of 97 nondirected T4 thymidylate synthase-defective (td) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, tdN57 and tdN47, fell within phylogenetically conserved pairings, with tdN57 disrupting the exon I-internal guide pairing (P1) in the 5' domain and tdN47 destabilizing the P9 helix in the 3' domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4tdN57 and T4tdN47, both of which are impaired in cleavage at the 5' and 3' splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.     

Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion

Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence. This sequence is in the 5' part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5' splice site. Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation (sup-) compensating for the effect of the primary box9- mutation. The sup- mutation alters a sequence in intron 4,293 bp upstream from the box9- primary mutation. The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9- and sup- are compensatory base pair exchanges (G----A and C----U, respectively). Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing.     

Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements.

U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end. The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing. Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24. Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix. These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle.     

A conserved base pair within helix P4 of the Tetrahymena ribozyme helps to form the tertiary structure required for self-splicing.

Site-specific mutagenesis of the self-splicing Tetrahymena intron has been used to investigate the function of C109-G212, a conserved base pair in the P4 stem of group I introns. Mutation of C109 to G affects splicing only slightly, whereas mutation of G212 to A or C reduces the rate of splicing substantially (500-fold reduction in kcat/Km under standard in vitro splicing conditions for the G212C mutant). Splicing activity of the compensatory double mutant (C109G:G212C) is intermediate between those of the two single mutants. Thus, the stability of the P4 stem as well as the identity of the base at position 212 are important for self-splicing. Single and double mutants containing the G212C substitution have a decreased temperature optimum for self-splicing and are partially Mg2+ suppressible, both indicative of structural destabilization. Chemical structure mapping indicates that the mutations do not redirect the global folding of the RNA, but affect the structure locally and at one other site (A183) that is distant in the secondary structure. We propose that, in addition to its pairing in P4, G212 is involved in a base triplet or an alternate base pair that contributes to the catalytically active tertiary structure of the ribozyme.     

Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.     

Analyzing the evolution of RNA secondary structures in vertebrate introns using Kimura's model of compensatory fitness interactions

Previous studies have shown that splicing efficiency, and thus maturation of pre-mRNA, depends on the correct folding of the RNA molecule into a secondary or higher order structure. When disrupted by a mutation, aberrant folding may result in a lower splicing efficiency. However, the structure can be restored by a second, compensatory mutation. Here, we present a logistic regression approach to analyze the evolutionary dynamics of RNA secondary structures. We apply our approach to a set of computationally predicted RNA secondary structures in vertebrate introns. Our results are consistent with the hypothesis of a negative influence of the physical distance between pairing nucleotides on the occurrence of covariations, as predicted by Kimura's model of compensatory evolution. We also confirm the hypothesis that longer local secondary structure elements (helices) can accommodate a larger number of covariations, wobbles, and mismatches. Furthermore, we find that wobbles and mismatches are more frequent in the middle of a helix, whereas covariations occur preferentially at the helix ends. The GC content is a major determinant of this pattern.     

Selection of the 3′-splice site in group I introns

A model for selection of 3′-splice sites in splicing of RNA precursors containing group I introns is presented. The key feature of this model is a newly identified tertiary interaction between the catalytic core of the intron and the 3′-splice site. This tertiary pairing would bring the 3′-splice site into the core of the intron, which is known to contain RNA sequences and structures essential for catalyzing the splicing reactions. The proposed tertiary interaction can coexist with P10, a pairing between 3′-exon sequences and the ‘internal guide sequence’ near the 5′-end of the intron. The model predicts that three RNA-RNA interactions are important in selection of 3′-splice sites: (i) binding of intron sequences with the core; (ii) pairing of exon sequences with the internal guide sequence; and (iii) binding of the terminal guanosine to an unknown site within the core.     

Splicing of the adenovirus-2 E1A 13S mRNA requires a minimal intron length and specific intron signals.

The adenovirus E1A region encodes three overlapping mRNAs, designated 9S, 12S and 13S. They differ from each other with regard to the length of the intron which is removed by RNA splicing. We have constructed E1A genes with deletions and insertions in the intervening sequence that is common to all three E1A mRNAs, in a search for signals which influence splicing of the 13S mRNA. Mutant plasmids were transfected into HeLa cells and the transiently expressed E1A mRNAs characterized by the S1 protection assay. The results show that five upstream and 20 downstream nucleotides are sufficient to allow for a correct utilization of the 5'-splice junction for the E1A 13S mRNA. Moreover, we show that a minimal intron length of 78 nucleotides is required for efficient 13S mRNA splicing. The ability of mutants with large intron deletions to maturate a 13S mRNA could partially be restored by expanding the intron length with phage lambda sequences. However, in no case was the normal splicing efficiency obtained with these mutants. In contrast, one mutant in which sequences from the authentic 13S mRNA intron were used to expand the intron expressed almost normal levels of 13S mRNA, thus suggesting that signals which specifically promote 13S mRNA splicing exist.