Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.

The conserved terminal guanosine of a group I intron can help prevent reopening of the ligated exons.

The highly conserved terminal guanosine of the Tetrahymena group I intron was mutated to an adenosine. The intron still excised itself but more slowly than the wild-type. At very low concentrations of GTP only ligated exons were produced, but at high concentrations of GTP unligated exons accumulated and the 3' exon acquired a GTP at its 5' end. A series of experiments suggested that GTP was primarily reopening the ligated exons, rather than directly cleaving the 3' splice site. 5' Truncated forms of the wild-type intron can cleave RNA in trans. Cleavage takes place downstream of sequences similar to the last few bases of the 5' exon. The ligated exons would therefore be a potential substrate. We believe that the intron uses the terminal guanosine to compete with exogenous GTP until the ligated exons have dissociated from their binding site. Other interactions between intron sequences which are known to aid in 3' splice-site recognition may assist in this process.

A comprehensive characterization of a group IB intron and its encoded maturase reveals that protein-assisted splicing requires an almost intact intron RNA.

The group I intron (AnCOB) of the mitochondrial apocytochrome b gene from Aspergillus nidulans encodes a bi-functional maturase protein that is also a DNA endonuclease. Although the AnCOB intron self-splices, the encoded maturase protein greatly facilitates splicing, in part, by stabilizing RNA tertiary structure. To determine their role in self-splicing and in protein-assisted splicing, several peripheral RNA sub-domains in the 313 nucleotide intron were deleted (P2, P9, P9.1) or truncated (P5ab, P6a). The sequence in two helices (P2 and P9) was also inverted. Except for P9, the deleted regions are not highly conserved among group I introns and are often dispensable for catalytic activity. Nevertheless, despite the very tight binding of AnCOB RNA to the maturase and the high activity of the bimolecular complex (the rate of 5' splice-site cleavage was >20 min(-1) with guanosine as the cofactor), the intron was surprisingly sensitive to these modifications. Several mutations inactivated splicing completely and virtually all impaired splicing to varying degrees. Mutants containing comparatively small deletions in various regions of the intron significantly decreased binding affinity (generally >10(4)-fold), indicating that none of the domains that remained constitutes the primary recognition site of the maturase. The data argue that tight binding requires tertiary interactions that can be maintained by only a relatively intact intron RNA, and that the binding mechanism of the maturase differs from those of two other well-characterized group I intron splicing factors, CYT-18 and Cpb2. A model is proposed in which the protein promotes widespread cooperative folding of an RNA lacking extensive initial tertiary structure.

The maturase encoded by a group I intron from Aspergillus nidulans stabilizes RNA tertiary structure and promotes rapid splicing.

The AnCOB group I intron from Aspergillus nidulans self-splices, providing the Mg2+ concentration is >/= 15 mM. The splicing reaction is greatly stimulated by a maturase protein encoded within the intron itself. An initial structural and biochemical analysis of the splicing reaction has now been performed. The maturase bound rapidly to the precursor RNA (kon approximately 3 x 10(9) M(-1) min(-1)) and remained tightly bound (koff /= 150 mM) was tenfold slower, in part because of the existence of an equilibrium between folded and partially folded RNA. In contrast, the maturase very effectively stabilized tertiary structure in 5 mM Mg2+, a noticeable example being an interaction between the P8 helix and a GNRA sequence that constitutes the L2 terminal loop of the P2 helix. Formation of the 5' splice-site recognition helix was assisted by either the maturase or high concentrations of Mg2+. The maturase was required during splicing so it is not a true chaperone. However, RNase protection assays and kinetic studies suggest that the maturase recognizes and facilitates folding of an intron with limited tertiary structure and even incomplete secondary structure.

Close relationship between certain nuclear and mitochondrial introns. Implications for the mechanism of RNA splicing.

We present the first indication of a direct relationship between a nuclear and a mitochondrial splicing system. The intron in the precursor of the large, nuclearly coded ribosomal RNA of two species of Tetrahymena possesses all the features of a class of fungal mitochondrial introns. Sequences conserved in mitochondrial introns of different fungal species are also found in the same order in these Tetrahymena nuclear introns, and the intron RNA can be folded to form a secondary structure similar to that proposed for mitochondrial introns by Davies et al. (1982). This "core" secondary structure brings the ends of the intron together. Furthermore, the first intron in the precursor of the large, nuclearly coded rRNA of Physarum polycephalum also has the characteristic conserved sequences and core RNA secondary structure. The limited sequence data available suggest that the intron in the large rRNA of chloroplasts in Chlamydomonas reinhardtii also resembles the mitochondrial introns. Tetrahymena large nuclear rRNA introns also have an internal sequence that can act as an adaptor by pairing with upstream and downstream exon sequences adjacent to the splice junctions to precisely align the splice junctions. These nuclear introns therefore fit the model of the role of intron RNA in the splicing process that was proposed by Davies et al. (1982), suggesting that the mechanisms of splicing may be very similar in these apparently diverse systems. It is therefore probable that the RNA secondary structures for which there is good evidence in the case of mitochondrial introns will be found to form the basis of active site structure and precise alignment in splicing and cyclization of the Tetrahymena intron "ribozyme".

Processing of mitochondrial RNA in Aspergillus nidulans.

Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.

Mitochondrial Four-Point Crosses in ASPERGILLUS NIDULANS : Mapping of a Suppressor of a Mitochondrially Inherited Cold-Sensitive Mutation.

Four-point mitochondrial crosses were conducted in heterokaryons of Aspergillus nidulans. The mutations used were (oliA1), conferring resistance to oligomycin, (camA112), conferring resistance to chloramphenicol; (cs-67), conferring cold-sensitivity, and ( sumD16), a suppressor of (cs-67). Initially, the crosses were conducted by observing the segregation of extranuclear markers in heterokaryotic sectors emerging from the original point of heterokaryosis. This showed that (camA112), (cs-67) and (sumD16) were linked but were probably all unlinked to (oliA1). Second, four-point crosses were conducted using a double marker selection technique, in which (camA112 ) and (oliA1) were always set in repulsion and the frequency of the phenotypes produced by the segregation of the mutant and wild-type alleles of (cs-67) and (sumD) were observed in (camA112 oliA1) recombinants. From these results we concluded that (camA112 ), (cs-67) and (sumD16) were linked and probably mapped in the order given. It was observed that the two nuclear types of conidia from a heterokaryon often had a dissimilar frequency distribution of the segregants of a mitochondrial cross.

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review.

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.

Deletion of P9 and stem-loop structures downstream from the catalytic core affects both 5' and 3' splicing activities in a group-I intron.

The P9 stem-loop is one of the conserved structural elements found in all group-I introns. Using two deletion mutants in this region of the Tetrahymena thermophilia large ribosomal subunit intron, we show that removal of the P9 element, either alone, or together with the non-conserved downstream P9.1 and P9.2 elements, results in an intron incapable of the first step of the splicing reaction at a low concentration of Mg2+. The mutant introns also require high concentrations of Mg2+ for the second step in splicing, as well as hydrolysis reactions, suggesting that P9, as well as P9.1 and P9.2, are important structural elements in the final folded form of the intron. In addition, RNase-T1-mediated-structure-probing experiments demonstrated that the loss of P9, P9.1 and P9.2 changes the structural context of the region binding the 5' splice site. The deletions lead to less efficient recognition of the 3' splice site and an accumulation of unligated exons. These observations support the view that the P9, P9.1 and P9.2 stem-loops play an important role in the binding of the 3' splice site.

Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes.

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

The mitochondrial ribosomal RNA molecules of Aspergillus nidulans.

The 16S and 23S mitochondrial rRNAs of Aspergillus nidulans have been identified by Northern hybridisation and the ends of the molecules mapped onto the mitochondrial genome by S1 nuclease analysis. The results show that both the rRNA molecules are longer than originally reported, forcing a reassessment of the potential secondary structures that can form in the terminal regions. In particular, structures resembling the 5.8S- and 4.5S-like domains of the bacterial large rRNA can now be recognised within the A. nidulans 23S molecule. The new 5' termini of the 16S and 23S genes lie within conserved 18-bp sequences that may be promoters but are more likely to be processing signals that cleave the mature rRNAs from larger precursor molecules. The new end of the 23S gene abuts the 5' end of the threonine-tRNA gene.

A modified two primer approach to oligonucleotide-directed in vitro mutagenesis.

Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E. coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L. casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila. We have used a modification of the method described by Norris et al. [1]. A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors. We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system. A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies.

Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution.

The group I intron from the rRNA precursor of Tetrahymena undergoes self-splicing. The intron RNA catalyst contains about 400 phosphate groups. Their role in catalysis has been investigated using phosphorothioate substituted RNA. In such RNA one of the peripheral oxygens of the phosphodiesters is replaced with sulfur. Incorporation of adenosine 5' phosphorothioate in either the 5' or 3' half of the ribozyme blocked splicing whereas incorporation of uridine 5' phosphorothioate only blocked splicing if the substitution was in the 3' half of the molecule. Modification-interference assays located two major and three minor inhibitory phosphorothioate substitutions suggesting that the corresponding phosphates play a significant role in self-splicing. These are all located in the most highly conserved region of the intron.

Two group I introns with a C.G basepair at the 5' splice-site instead of the very highly conserved U.G basepair: is selection post-translational?

In virtually all of the 200 group I introns sequenced thus far, the specificity of 5' splice-site cleavage is determined by a basepair between a uracil base at the end of the 5' exon and a guanine in an intron guide sequence which pairs with the nucleotides flanking the splice-site. It has been reported that two introns in the cytochrome oxidase subunit I gene of Aspergillus nidulans and Podospora anserina are exceptions to this rule and have a C.G basepair in this position. We have confirmed the initial reports and shown for one of them that RNA editing does not convert the C to a U. Both introns autocatalytically cleave the 5' splice-site. Mutation of the C to U in one intron reduces the requirement for Mg2+ and leads to an increase in the rate of cleavage. As the C base encodes a highly conserved amino acid, we propose that it is selected post-translationally at the level of protein function, despite its inferior splicing activity.

Important 2'-hydroxyl groups within the core of a group I intron.

The catalytic activity of a group I intron is dependent on a core structure, much of which is not exposed to solvent. In order to study the structure of the core, an efficient bimolecular reaction has been developed: the 5'-component is a molecule of about 300 bases which contains the 5'-splice-site and terminates in the loop established by P8, and the 3'-component is a 24 base long oligoribonucleotide which includes the 3'-regions of the P8 and P7 helices with their joining region, J8/7. J8/7 is thought to play several roles including binding the helix containing the 5'-splice-site. P7 forms a major portion of the guanosine binding site required for splicing. We have modified the bimolecular system to make it amenable to kinetic analysis and have used it to study the role of the ribose sugars in the oligomer. Multiple deoxyribonucleotide substitution in the J8/7 region completely blocked 5'-splice-site cleavage even though the Kd was only reduced about 5-fold. This supports the idea that the ribose phosphate backbone in J8/7 plays a key role in catalysis. Individual substitutions at G303 and A306 reduced the rate of catalysis 5-10-fold. The G303 substitution blocked GTP-independent hydrolysis of the 5'-splice-site. The region spanning the junction of P8 and J8/7 was also highly sensitive to multiple deoxyribonucleotide substitution; however, only in the case of C298 did an individual substitution have any effect on cleavage. Deoxyribonucleotide substitution in the 3'-section of P7 was less severe, although kcat/Km in low GTP was down 70-fold.

A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron.

RNA polymerases can synthesize RNA containing phosphorothioate linkages in which a sulfur replaces one of the nonbridging oxygens. Only the Rp isomer is generated during transcription. A Rp phosphorothioate at the 5' splice-site of the Tetrahymena group I intron does not inhibit splicing (McSwiggen, J.A. and Cech, T.R. (1989) Science 244, 679). Transcription of mutants in which the first base of the 3' exon, U+1, was mutated to C or G, in the presence, respectively, of either cytosine or guanosine thiotriphosphate, introduced a phosphorothioate at the 3' splice-site. In both cases exon ligation was blocked. In the phosphorothioate substituted U+1G mutant, a new 3' splice-site was selected one base downstream of the correct site; despite the fact that the correct site was selected with very high fidelity in unsubstituted RNA. In contrast, the exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site. Chirality was reversed during transesterification as in 5' splice-site cleavage (vide supra). This suggests that one non-bridging oxygen is particularly crucial for both splicing reactions.

Nuclear and mitochondrial suppression of a mitochondrially inherited cold-sensitive mutation in Aspergillus nidulans.

Partial suppressors of a mitochondrially inherited mutation, [cs-67], conferring cold-sensitivity at 20 degrees C were identified. These mapped at one mitochondrial and four unlinked nuclear loci. Most suppressors partially restored the cytochrome aa3 deficiency of the cold-sensitive strain at 20 degrees C. Strains carrying two or more suppressors and [cs-67] showed considerably impaired growth. This effect was temperature-dependent, being more severe at 37 degrees C, and was not expressed in the presence of the [cs-67+] allele. The cytochrome oxidase activity of one of these strains was no more heat-sensitive than that of the wild-type implying that these mutations did not directly modify cytochrome oxidase. The wild-type strain grown in the presence of chloramphenicol and the cold-sensitive strain grown at 20 degrees C had similar cytochrome spectra and mitochondrial membrane protein profiles on sodium dodecyl sulphate gradient acrylamide gels. [cs-67] conferred pleiotropically a low level of resistance to paramomycin at 37 degrees C. It is suggested that [cs-67] and the suppressors act at the level of the mitochondrial ribosome.

Self-splicing activity of the mitochondrial group-I introns from Aspergillus nidulans and related introns from other species.

The mitochondrial genome of Aspergillus nidulans contains several group-I introns. Each one has been assayed for its ability to self-splice in vitro in the absence of proteins. The intron from the apocytochrome b gene is unusual among subgroup IB4 introns in being able to self-splice, unlike a similar intron from Saccharomyces cerevisiae. The first intron in the cytochrome oxidase subunit-1 gene self-splices but only correctly completes the first step of splicing; cryptic 3' splice-sites are recognized instead and these are also used at a low frequency in vivo. The highly homologous intron from Podospora anserina completes both steps in vitro. The remaining introns do not self-splice. The correlation between subgroup category, the likely presence of specific tertiary interactions, and self-splicing activity is discussed.

A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease.

Some group I introns self-splice in vitro, but almost all are thought to be assisted by proteins in vivo. Mutational analysis has shown that the splicing of certain group I introns depends upon a maturase protein encoded by the intron itself. However the effect of a protein on splicing can be indirect. We now provide evidence that a mitochondrial intron-encoded protein from Aspergillus nidulans directly facilitates splicing in vitro. This demonstrates that a maturase is an RNA splicing protein. The protein-assisted reaction is as fast as that of any other known group I intron. Interestingly the protein is also a DNA endonuclease, an activity required for intron mobilization. Mobile elements frequently encode proteins that promote their propagation. Intron-encoded proteins that also assist RNA splicing would facilitate both the transposition and horizontal transmission of introns.