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PubMed1987

In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

Base Sequence↗

PubMed1988

Group II intron domain 5 facilitates a trans-splicing reaction.

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

Electrophoresis, Polyacrylamide Gel↗

PubMed1989

Miniribozymes, small derivatives of the sunY intron, are catalytically active.

The self-splicing sunY intron from bacteriophage T4 has the smallest conserved core secondary structure of any of the active group I introns. Here we show that several nonconserved regions can be deleted from this intron without complete loss of catalytic activity. The 3' stems P9, P9.1, and P9.2 can be deleted while retaining 5' cleaving activity. Two base-paired stems (P7.1 and P7.2) that are peculiar to the group IA introns can also be deleted; however, the activities of the resulting derivatives depend greatly on the choice of replacement sequences and their lengths. The smallest active derivative is less than 180 nucleotides long. These experiments help to define the minimum structural requirements for catalysis.

Base Sequence↗

PubMed1992

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.

Amino Acid Sequence↗

PubMedDate not supplied

[Design of ribozymes].

A review of the data on the synthesis of RNA-fragments with endonuclease activity. The Haseloff and Gerlach model for design of ribozymes and the results of the synthesis of the three new ribozymes targeted against the chloramphenicol acetyl transferase gene transcript are considered. The possibility of the use of a shortened form of Tetrahymena self-splicing ribosomal RNA intervening sequence for the ribozyme design also are discussed.

Base Sequence↗

PubMedDate not supplied

[Splicing. I. Splicing of tRNA, rRNA and mRNA in organelles].

Mosaic structure of genes is shown for three out of four known types of RNA: transfer, ribosomal and messenger. At least three different mechanisms are involved in maturation of transcripts from these genes. The peculiarity of tRNA splicing is connected with the possibility of existence of tRNA molecules free of ribonucleoprotein complexes. The mode of Tetrahymena pre-tRNA self-splicing may also take place during maturation of other RNAs (rRNA of protozoa, ribosomal and messenger rRNA of lower fungi, plant messenger rRNA), which share the similar structure. The third type of mechanism is involved in splicing messenger RNA in eukaryotic cell nuclei.

Animals↗

PubMed1989

RNA as an enzyme.

The catalytic activity of ribonucleic acid is reviewed, with the intervening sequence (IVS) of the ribosomal RNA precursor of Tetrahymena serving as a major example. The IVS catalyzes its own excision from the precursor RNA and at the same time ligation of the flanking sequences, a reaction termed self-splicing. The excised IVS RNA can act as an enzyme to catalyze sequence-specific cleavage and ligation reactions on substrate RNA molecules. The RNA polymerization activity of the IVS supports the possibility that RNA catalysis could have been important in establishing a prebiotic self-replicating system. Other systems in which RNA catalysis has been found include related group I IVSs, group II IVSs, ribonuclease P, and certain plant infectious RNAs.

Animals↗

PubMed1983

RNA splicing in Neurospora mitochondria. The large rRNA intron contains a noncoded, 5'-terminal guanosine residue.

The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5' end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.

Base Sequence↗

PubMed1995

Self-assembly of a group I intron active site from its component tertiary structural domains.

The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro.

The group II intron bl1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bl1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component in this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bl1.

Adenosine Triphosphate↗

PubMed1995

Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro.

The group II intron bI1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bI1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component is this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS-116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bI1.

Adenosine Triphosphate↗

PubMed1994

Intron-containing T4 bacteriophage gene sunY encodes an anaerobic ribonucleotide reductase.

The function of the SunY protein, encoded by an intron-containing gene of bacteriophage T4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the SunY intron. Here we show that anaerobic T4 infections of Escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme. Inactivation of the T4 sunY gene (in this communication renamed nrdD) significantly decreased both the induced activity and the anaerobic production of phage, confirming the role of the T4 NrdD (SunY) protein as a phage-specific anaerobic ribonucleotide reductase. With the identification of the T4 nrdD (sunY) gene product as an anaerobic ribonucleotide reductase, all known bacteriophage introns are found to share the common and as yet unexplained property of residing within genes of DNA metabolism.

Amino Acid Sequence↗

PubMed1995

Generation of nuclease resistant circular RNA decoys for HIV-Tat and HIV-Rev by autocatalytic splicing.

Circular exon sequences can be generated by splicing permuted intron-exon (PIE) sequences. The Anabaena pre-tRNA group I self-splicing PIE sequence was modified to generate circular forms of the HIV-TAR and the high affinity region of the HIV-RRE (RBE). RNA products containing TAR and the RBE were purified from splicing reactions and demonstrated to be circular. The circular form of these sequences was shown to be resistant to nuclease degradation in cellular extracts. Gel shift assays demonstrate that the circular form of the RBE is specifically bound by a Rev derived peptide. These data suggest that PIE-circularization of RNA may be an effective way to express small stable RNAs designed for therapeutics (eg-decoys).

Base Sequence↗

PubMed1995

Generation of nuclease resistant circular RNA decoys for HIV-Tat and HIV-Rev by autocatalytic splicing.

Anabaena↗

PubMed1996

Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.

Animals↗

PubMed1995

Horizontal transmission of group-I ribozymes: viruses as a carrier of the introns.

The nucleotide sequences of two self-splicing group I introns found in viruses that infect the eukaryotic green alga Chlorella were compared with those of 19 nuclear group I introns of 11 green algae, 1 red alga, 2 yeasts and 1 protist. The similarity ranging from 69.5% to 89.0% in any comparison strongly suggested a close relatedness of these introns. Since this kind of viruses infect algal cells residing in protists such as Paramecium, they may serve as a mediator to spread group I introns horizontally among organisms of different phylogenetic kingdoms.

Animals↗

PubMed1996

X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering.

For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.

Animals↗