The ability to form full-length intron RNA circles is a general property of nuclear group I introns

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3′ splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5′ splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility.     

Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium Desulfurococcus mobilis

The intron of the 23S rRNA gene of D. mobilis is excised from the pre-23S RNA at specific sites in vivo and subsequently ligated to form a stable circular RNA, with a normal 5'-3' phosphodiester bond, containing the entire intron sequence; 95% of this RNA codes for a protein of 194 amino acids that can be expressed in E. coli. Crude cell extracts from D. mobilis also induce a two-step slicing reaction in vitro, producing the same circular intron RNA but a low yield of ligated exons. Cleavage depends on the RNA structure adjacent to the cleavage site and yields a 3'-terminal phosphate. Splicing is enhanced by GTP, but does not require divalent metal ions. The cleavage and exon-splicing reactions resemble those found for tRNA introns in eukaryotes and a possible structural rationale for this similarity is considered together with its possible implications for the origin of eukaryotic rRNA and tRNA introns.     

Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors

We have previously shown linear and circular splicing intermediates resembling intermediates that result from self-splicing of ribosomal precursor RNA of Tetrahymena to be present in mitochondrial RNA. Here we show that splicing of yeast mitochondrial precursor RNA also occurs in vitro in the absence of mitochondrial proteins. The large ribosomal RNA gene, consisting of the intron and part of the flanking exon regions, was inserted behind the SP6 promoter in a recombinant plasmid and was transcribed in vitro. The resulting RNA shows self-catalyzed splicing via incorporation of GTP at the 5'-end of the excised intron, 5'- to 3'-exon ligation, and intron circularization. When purified mitochondrial RNA is incubated under similar conditions with alpha-32P-GTP, the excised ribosomal intron RNA is also labeled, as well as several other RNA species. Some of these RNAs are derived from excised introns from the multiply split gene coding for cytochrome oxidase subunit I.     

Ribosomal RNA genes of Saccharomyces cerevisiae. II. Physical map and nucleotide sequence of the 5 S ribosomal RNA gene and adjacent intergenic regions.

A DNA fragment containing the structural gene for the 5 S ribosomal RNA and intergenic regions before and after the 35 S ribosomal RNA precursor gene of Saccharomyces cerevisiae has been amplified in a bacterial plasmid and physically mapped by restriction endonuclease cleavage and hybridization to purified yeast 5 S ribosomal RNA. The nucleotide sequence of the DNA fragments carrying the 5 S ribosomal RNA gene and adjacent regions has been determined. The sequence unambiguously identifies the 5 S ribosomal RNA gene, determines its polarity within the ribosomal DNA repeating unit, and reveals the structure of its promoter and termination regions. Partial DNA sequence of the regions near the beginning and end of the 35 S ribosomal RNA gene has also been determined as a preliminary step in establishing the structure of promoter and termination regions for the 35 S ribosomal RNA gene.     

Hydrolytic cleavage by a group I intron ribozyme is dependent on RNA structures not important for splicing.

DiGIR2 is the group I splicing-ribozyme of the mobile twin-ribozyme intron Dir.S956-1, present in Didymium nuclear ribosomal DNA. DiGIR2 is responsible for intron excision, exon ligation, 3'-splice site hydrolysis, and full-length intron RNA circle formation. We recently reported that DiGIR2 splicing (intron excision and exon ligation) competes with hydrolysis and subsequent full-length intron circularization. Here we present experimental evidence that hydrolysis at the 3'-splice site in DiGIR2 is dependent on structural elements within the P9 subdomain not involved in splicing. Whereas the GCGA tetra-loop in P9b was found to be important in hydrolytic cleavage, probably due to tertiary RNA-RNA interactions, the P9.2 hairpin structure was found to be essential for hydrolysis. The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and a consensus kink-turn motif in the proximal stem. We suggest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reaction pathways.     

RNA recognition and cleavage by sequence-specific endoribonucleases

Inactivation of RNA molecules by sequence-specific endoribonucleolytic cleavage is a subtle mechanism by which cells regulate gene expression. Sequence-specific endoribonucleases can recognize and cleave particular phosphodiester bonds confined within hundreds/thousands of chemically similar bonds. Here, we present a comparative analysis of the mechanisms used by endoribonucleases to select and cleave their target RNA molecules. This analysis is based on the very recent molecular details obtained from the structural and/or biochemical studies of nine sequence-specific ribonucleases that target messenger, ribosomal, and transfer RNA molecules. This analysis shows that despite the absence of sequence homologies and the wide diversity of biological sources (prokaryotes, archaea and eukaryotes), the sequence-specific ribonucleases studied here adopt limited structural folds, catalyze their cleavage reactions using a common chemistry and involve a very limited set of amino acids for both RNA binding and processing.     

Modification of ribosomal RNA by ribosome-inactivating proteins from plants.

We have surveyed 14 different toxic and nontoxic ribosome-inactivating proteins from plants for the ability to act on the RNA of the eucaryotic 60 S ribosomal subunit. All of these proteins act to introduce a specific modification into 26-28 S RNA which renders the RNA sensitive to cleavage by aniline. Sequence analysis of the 5'-termini of the fragments produced by ricin and saporin following aniline cleavage indicate that both proteins possess identical specificity. Our observations support the conclusion of Endo and Tsurugi (J. Biol. Chem. 262, 8128-8130, 1987) that ricin is a specific N-glycosidase and we have located the site of this cleavage by direct sequence analysis. Our results further suggest that all plant ribosome-inactivating proteins function as specific N-glycosidases with the same specificity.     

Determination of an RNA structure involved in splicing inhibition of a muscle-specific exon

We have investigated the RNA structure of the region surrounding the muscle-specific exon 6B of the chicken beta-tropomyosin gene. We have used a variety of chemical and enzymatic probes: dimethylsulfate, N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p-tolu enesulfonate) , RNase T1 and RNase V1. Lead acetate was also used to obtain some information on the tertiary structure of this region. Probing the wild-type sequence suggests a model involving one-stem and three-stem-loop structures in and around this exon. Two of these, hairpin I and stem III, have previously been implicated in repression of splicing of the intron following exon 6B in a HeLa nuclear extract. Stem I includes sequences at the beginning of exon 6B and stem III results from interaction of the intron upstream from exon 6B with sequences in the middle of the intron downstream from this exon (the intron whose splicing is repressed). Neither stem I nor stem III directly involves the consensus sequences (5' splice site, branch-point, 3' splice site) of the repressed intron. Probing RNAs that are derepressed for splicing of this intron show that there are structural changes around the 5' splice site and branch-point sequence that correlate with the derepression. This is true, despite the fact that the derepressed RNAs are altered in a region far from these consensus sequences. The most striking structural correlation with splicing capacity of the intron downstream from exon 6B is seen by probing with lead acetate. Lead ions cut RNA at specific residues; these sites are very sensitive to RNA tertiary structure. Repressed and derepressed RNAs show entirely different cleavage patterns after incubation with lead acetate. Remarkably, hybridizing a derepressed RNA with an RNA comprising the ascending arm of stem III not only re-establishes repression, but also converts the pattern of susceptibility to attack by lead ions over the whole molecule. We suggest that RNA conformation plays a role in keeping exon 6B from being spliced into non-muscle cell mRNA.     

In vivo facilitation of Tetrahymena group I intron splicing in Escherichia coli pre-ribosomal RNA.

The observation that the large ribosomal RNA intron of Tetrahymena is spliced 20-50-fold more rapidly in vivo than in vitro (Brehm SL, Cech TR, 1983, Biochemistry 22:2390-2397; Bass BL, Cech TR, 1984, Nature 308:820-826) suggests facilitation of RNA folding in vivo. To determine whether a specific group I splicing factor is required in Tetrahymena, the intron was inserted into the analogous position of the Escherichia coli 23S rRNA. We report that the intron is rapidly excised from pre-rRNA in bacteria and that the magnitude of the in vivo rate enhancement is similar to that in Tetrahymena. These results demonstrate that a species-specific protein is not required. Instead, a common mechanism of assisting RNA folding is sufficient to accelerate the removal of self-splicing introns from ribosomal RNA.     

Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA

The yeast mitochondrial group II intron bI1 is self-splicing in vitro. We have introduced a deletion of hairpin C1 within the structural domain 1 that abolishes catalytic activity of the intron in the normal splicing reaction in cis, but does less severely affect a reaction in trans, the reopening of ligated exons. Since exon reopening is supposed to correspond to a reverse 3' cleavage this suggests that the deletion specifically blocks the first reaction step. The intron regains its activity to self-splice in cis by intermolecular complementation with a small RNA harbouring sequences lacking in the mutant intron. These results demonstrate the feasibility to reconstitute a functionally active structure of the truncated intron by intermolecular complementation in vitro. Furthermore, the data support the hypothesis that group II introns are predecessors of nuclear pre-mRNA introns and that the small nuclear RNAs of the spliceosome arose by segregation from the original intron.     

Chemical probing of RNA with the hydroxyl radical at single-atom resolution

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5′-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5′-aldehyde. We conclude that hydroxyl radical abstracts a 5′-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5′ deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2′-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.     

A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA.

We report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components.     

Fine structure of ribosomal RNA: I. Conservation of homologous regions within ribosomal RNA of eukaryotes

By molecular hybridization experiments the homologies between ribosomal RNAs from a unicellular organism (Gyrodinium cohnii), three invertebrates (Drosophila hydei, Chironomus thummi, Sciara coprophila), an amphibian (Xenopus laevis), and a mammal (mouse) were determined. Competition hybridization experiments demonstrated that portions of these homologous regions are the same in all the ribosomal RNAs tested, regardless of animal species. This conclusion based on hybridization data was confirmed by comparative fingerprint analysis. The ribosomal RNA sequences involved in heterologous hybridization have a higher A + T composition than the bulk ribosomal RNA. It appears from competition experiments of a heterologous hybridization that two thirds of the conserved similar regions are present in 18 S ribosomal RNA, and the remaining one third in 28 S ribosomal RNA. It is argued that these similar regions have been conserved during evolution due to their structural and/or functional role in ribosomal RNA.