Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence

Circularization and reverse circularization of the Tetrahymena thermophila rRNA intervening sequence resemble the first and second steps in splicing, respectively. However, site-specific base substitutions show that different nucleotides are involved in selection of the 5' splice site and the circularization sites. Furthermore, a substitution at the major circularization site that prevents circularization can be suppressed by second substitutions at two different nucleotide positions. A model is proposed in which adjacent and overlapping sequences can function as a binding site, forming a short duplex with the sequence at the circularization site and thus directing circularization and reverse circularization. Because the 5' exon-binding site and three potential circularization binding sites fall within a contiguous eight nucleotide region, this sequence may translocate relative to the catalytic core of the ribozyme in a template-like manner.     

Isolation and characterization of the human apolipoprotein A-II gene. Electron microscopic analysis of RNA:DNA hybrids, nucleotide sequence, identification of a polymorphic MspI site, and general structural organization of apolipoprotein genes

Three cloned apolipoprotein A-II genes were isolated from a human genomic cosmid library constructed in our laboratory. An approximately 3-kilobase HindIII insert containing the entire gene was analyzed by RNA:DNA hybridization and electron microscopy. The apo-A-II gene was found to consist of 4 exons and 3 intervening sequences (IVS), and the lengths of each exon and IVS were estimated by direct observation of the hybrids. The entire approximately 3-kilobase HindIII insert was sequenced. The 5' end of the gene was determined by primer extension. The DNA sequence confirms the presence of 4 exons and 3 IVS: exon 1, 34 nucleotides; exon 2, 76 nucleotides; exon 3, 133 nucleotides; exon 4, 230 nucleotides; IVS-I, 169 nucleotides; IVS-II, 299 nucleotides; and IVS-III, 396 nucleotides. A "TATA box" is located at position -29 from the CAP site. A "CAT box" is present at position -78. A "TG" element consisting of (TG)19 is identified at the 3' end of IVS-III. Furthermore, an enhancer core sequence, CTTTCCA, is identified at position -355 in the 5' flanking sequence. At positions -497 to -471 upstream from the CAP site is a stretch of 27 nucleotides that show high homology to stretches of 5' flanking sequences in the apo-C-II, apo-A-I, apo-E, and apo-C-III genes. An Alu dimer sequence is located approximately 300 nucleotides from the 3' end of the gene. Within this Alu sequence, we have identified a polymorphic MspI site. Restriction fragment length polymorphism involving this site has been previously shown to correlate with apo-A-II levels and high density lipoprotein structure. Analysis of conformation by Chou-Fasman analysis and by the helical hydrophobic moment of Eisenberg et al. (Eisenberg, D., Weiss, R. M., and Tergwillager, T. C. (1982). Nature (Lond.) 299, 371-374) indicates that in all of the 5 apolipoproteins characterized at the nucleotide level to date, i.e. apo-C-II, apo-A-II, apo-E, apo-A-I, and apo-C-III, the 2 IVS within the peptide coding regions of the gene tend to occur at regions corresponding to the surface of the polypeptide chain and divide the protein into distinct functional domains.     

Structure of the catalytic core of the Tetrahymena ribozyme as indicated by reactive abbreviated forms of the molecule.

The precursor rRNA of Tetrahymena thermophila contains a group I intervening sequence (IVS) that catalyzes its own excision to yield mature rRNA. The excised IVS catalyzes a number of cleavage/ligation reactions that are analogous to the transesterification reactions of splicing. We examined the behavior of a variety of 3'-truncated forms of the IVS and found several abbreviated molecules that retained catalytic activity. The reactivity of these molecules indicates that the site at which cleavage/ligation occurs lies in close proximity to all of the conserved sequence elements within the catalytic core of the IVS.     

Characterization of an authentic intermediate in the self-splicing process of ribosomal precursor RNA in macronuclei of Tetrahymena thermophila.

We have characterized a 1.5 kb RNA species in T. thermophila macronuclei previously found in vivo and including intron sequences linked to the 3' exon. This IVS-3' exon RNA could be detected in gels as a discrete molecule only after denaturation of nuclear RNA. After addition of 32P-GTP, as splicing cofactor in a nuclear in vitro system, the IVS-3' exon RNA was labeled at its 5' terminus, as was the by-product of splicing, the excised IVS RNA. The time course of labeling indicates that the IVS-3' exon RNA acts like a reaction intermediate and specifically a kinetic precursor to IVS RNA. Partial nuclease digestions showed that the IVS-3' exon RNA and the IVS RNA have the same 5' terminal sequence. In addition the IVS-3' exon RNA can release the 15-mer oligonucleotide cleaved off during circularization of IVS RNA under conditions of high temperature. Taken together, the structural, functional, and kinetic properties of the IVS-3' exon RNA strongly suggest that it represents a previously postulated in vivo intermediate in the splicing pathway.     

Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient.

A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing.     

Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron.

A thalassemic beta-globin gene cloned from a haplotype I chromosome contains a T to G transversion at position 116 of IVS1 which results in the generation of an abnormal alternative acceptor splice site. Transient expression studies revealed a 4-fold decrease in the amount of RNA produced with greater than 99% of it being abnormally spliced despite preservation of the normal acceptor splice site at position 130. These results suggest that the mutation at IVS1 position 116 results in beta zero thalassemia. A closely related mutation at position 110 of IVS1 also generates a novel acceptor site and results in a similar decrease in total mRNA produced, but approximately 20% of the mRNA produced is normally spliced and thus the phenotype is that of beta + thalassemia. These observations suggest that short range position effects may play a dramatic role in the choice of potential splice acceptor sites. We demonstrate the presence of abnormally spliced mRNA in reticulocytes of affected individuals and show the mutation at IVS1 position 116 segregating from the mutation at IVS1 position 110 in a three generation pedigree. The mutation results in the creation of a MaeI restriction site, as do a number of other thalassemic mutations, and we demonstrate some difficulties that may arise in the differential diagnosis of these mutations.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

Identification and characterization of an intervening sequence within the 23S ribosomal RNA genes of Campylobacter jejuni

Campylobacter jejuni is a significant cause of bacterial enteritis in humans. Three of seven C. jejuni isolates examined were found to contain fragmented 23S rRNA. The occurrence of fragmented 23S rRNA correlated with the presence of an intervening sequence (IVS) within the 23S rRNA genes. The IVS is 157 nucleotides in length and replaces an eight nucleotide sequence in the 23S rRNA genes of C. jejuni isolates that contain intact 23S rRNA. The two ends of the IVS share 31 bases of complementarity that could form a stem-loop structure. Fragmentation of the 23S ribosomal RNA results from the excision of the IVS from the transcribed RNA; the 3’ cleavage site maps within the putative stem-loop formed by the IVS. Southern hybridization analysis revealed that the IVS is not present in the genomes of isolates that contain intact 23S rRNA, suggesting that the IVS is not derived from Campylobacter chromosomal sequences. The C. jejuni IVS is located at a position analogous to that of the IVSs found in both Salmonella and Yersinia spp.     

Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA.

The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS). The altered genes were transcribed and the RNA tested for self-splicing in vitro. A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity. Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site. Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS. Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro. Three other regions were found to be necessary. The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences. The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing.     

Reactivity of modified ribose moieties of guanosine: new cleavage reactions mediated by the IVS of Tetrahymena precursor rRNA.

An RNA molecule consisting of the 5' exon and intervening sequence (IVS) of Tetrahymena precursor rRNA was oxidized with sodium periodate to convert the ribose moiety of the 3' terminal guanosine into a dialdehyde form. The modified RNA undergoes a specific cleavage reaction at the 5' splice site, but has no apparent cyclization activity. This novel reaction mediated by the IVS RNA is pH dependent over the range 6.5-8.5 and leaves a 5' phosphate and a 3'-OH at the newly created termini. The dialdehyde form of monomer guanosine is also capable of causing a specific cleavage reaction at the 5' splice site although the nucleotide is not covalently attached to the IVS RNA in the final product. These and other findings described in this report suggest that the cis diol of the intact ribose moiety of guanosine is not an absolute requirement for the IVS-mediated reactions.     

Circulation of oligonucleotides by disulfide bridge formation.

An effective, convenient method for the circularization of oligonucleotides has been developed. This procedure involved preparation of an oligonucleotide with backbone-linked 5'- and 3'-terminal hexamethylenethiol groups, followed by oxidation of the thiol groups with air of oxygen to produce the corresponding circular sequence bridged via a bis(hexamethylene)-disulfide moiety. The method has been applied to the circularization of oligodeoxynucleotide sequences of varying lengths (5, 10, 15, 20, 30 and 40 bases), and the circularization process was highly efficient as shown by HPLC or gel electrophoresis of the crude reaction mixtures. Competing reactions such as dimerization were not significant except for the longer sequences (30 and 40 bases). The circularization of an eight base RNA sequence was also accomplished, as well as hexa-ethylene glycol bridged poly-T sequences capable of triplex formation.     

The complete nucleotide sequence of the chick a-actin gene and its evolutionary relationship to the actin gene family

The nucleotide sequence of the chick a-actin gene reveals that the gene is comprised of 7 exons separated by six very short intervening sequences (IVS). The first IVS interrupts the 73 nucleotide 5' untranslated segment between nucleotides 61 and 62. The remaining IVS interrupt the translated region at codons 41/42, 150, 204, 267, and 327/328. The 272 nucleotide 3' untranslated segment is not interrupted by IVS. The amino acid sequence derived from the nucleotide sequence is identical to the published sequence for chick a-actin except for the presence of a met-cys dipeptide at the amino-terminus. The IVS positions in the chick a-actin gene are identical to those of the rat a-actin gene. While there is partial coincidence of the IVS in the a-actin genes with the vertebrate b-actin genes and 2 sea urchin actin genes, there is no coincidence with actin genes from any other source except soybean where one IVS position is shared. This discordance in IVS positions makes the actin gene family unique among the eucaryotic genes analyzed to date.     

Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs

The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.     

Reversibility of cyclization of the Tetrahymena rRNA intervening sequence: implication for the mechanism of splice site choice

The Tetrahymena rRNA intervening sequence (IVS) excises itself from the pre-rRNA and then mediates its own cyclization. We now find that certain di- and trinucleotides with free 3' hydroxyl groups reopen the circular IVS at the cyclization junction, producing a linear molecule with the oligonucleotide covalently attached to its 5' end. This linear molecule recyclizes with release of the added oligonucleotide. Thus the IVS RNA, like an enzyme, lowers the activation energy for both forward and reverse cleavage-ligation reactions. Certain combinations of pyrimidines are required for circle reopening. The most reactive oligonucleotide is UCU. This sequence resembles those preceding the major and minor cyclization sites in the linear IVS RNA (UUU and CCU) and the 5' splice site in the pre-rRNA (UCU). We propose that an oligopyrimidine binding site within the IVS binds the sequences upstream of each of these target sites for cleavage-ligation.