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.     

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.     

Lysosomal enzymes produced by immobilized Tetrahymena thermophila

Summary The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 × 10⁷ cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions -glucosidase, -glucosidase, -hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes. Offprint requests to: A. Tiedtke     

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.     

Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex

The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.     

Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila

We have converted the intramolecular cyclization reaction of the self-splicing intervening sequence (IVS) ribonucleic acid (RNA) from Tetrahymena thermophila into an intermolecular guanosine addition reaction. This was accomplished by selectively removing the 3'-terminal nucleotide by oxidation and beta-elimination; the beta-eliminated IVS thereby is no longer capable of reacting with itself. However, under cyclization conditions, a free guanosine molecule can make a nucleophilic attack at the normal cyclization site. We have used this guanosine addition reaction as a model system for a Michaelis-Menten kinetic analysis of the guanosine binding site involved in cyclization. The results indicate that functional groups on the guanine that are used in a G-C Watson-Crick base pair are important for the cyclization reaction. This is the same result that was obtained for the guanosine binding site involved in splicing [Bass, B. L., & Cech, T. R. (1984) Nature (London) 308, 820-826]. Unlike splicing, however, certain additional nucleotides 5' to the guanosine moiety make significant binding contributions. We conclude that the guanosine binding site in cyclization is similar to, but not identical with, the guanosine binding site in splicing. The same binding interactions used in cyclization could help align the 3' splice site of the rRNA precursor for exon ligation. We also report that the phosphodiester bond at the cyclization site is susceptible to a pH-dependent hydrolysis reaction; the phosphodiester bond is somehow activated toward attack by the 3'hydroxyl of a guanosine molecule or by a hydroxyl ion.     

Fate of the junction phosphate in alternating forward and reverse self-splicing reactions of group II intron RNA.

The RNA-catalysed self-splicing reaction of group II intron RNA is assumed to proceed by two consecutive transesterification steps, accompanied by lariat formation. This is effectively analogous to the small nuclear ribonucleoprotein (snRNP)-mediated nuclear pre-mRNA splicing process. Upon excision from pre-RNA, a group II lariat intervening sequence (IVS) has the capacity to re-integrate into its cognate exons, reconstituting the original pre-RNA. The process of reverse self-splicing is presumed to be a true reversion of both transesterification steps used in forward splicing. To investigate the fate of the esterified phosphate groups in splicing we assayed various exon substrates (5'E-*p3'E) containing a unique 32P-labelled phosphodiester at the ligation junction. In combined studies of alternating reverse and forward splicing we have demonstrated that the labelled phosphorus atom is displaced in conjunction with the 3' exon from the ligation junction to the 3' splice site and vice versa. Neither the nature of the 3' exon sequence nor its sequence composition acts as a prominent determinant for both substrate specificity and site-specific transesterification reactions catalysed by bI1 IVS. A cytosine ribonucleotide (pCp; pCOH) or even deoxyoligonucleotides could function as an efficient substitute for the authentic 3' exon in reverse and in forward splicing. Furthermore, the 3' exon can be single monophosphate group. Upon incubation of 3' phosphorylated 5' exon substrate (5'E-*p) with lariat IVS the 3'-terminal phosphate group is transferred in reverse and forward splicing like an authentic 3' exon, but with lower efficiency. In the absence of 3' exon nucleotides, it appears that substrate specificity is provided predominantly by the base-pairing interactions of the intronic exon binding site (EBS) sequences with the intron binding site (IBS) sequences in the 5' exon. These studies substantiate the predicted transesterification pathway in forward and reverse splicing and extend the catalytic repertoire of group II IVS in that they can act as a potential and sequence-specific transferase in vitro.     

Mg2+ effects on a circle opening reaction of the self-splicing intervening sequence from Tetrahymena thermophila

The Mg2+ contribution to the reaction of circular intervening sequence (CIVS) from rRNA precursor of Tetrahymena thermophila with a dinucleotide CU has been investigated. The results indicated that the circle opening of CIVS may involve binding of a weakly held Mg2+ ion.     

The effect of long-range loop-loop interactions on folding of the Tetrahymena self-splicing RNA

Bas?e-pairing between the terminal loops of helices P2.1 and P9.1a (P13) and P2 and P5c (P14) stabilize the folded structure of the Tetrahymena group I intron. Using native gel electrophoresis to analyze the folding kinetics of a natural pre-RNA containing the Tetrahymena intron, we show that P13 and P14 are the only native loop-loop interactions among six possible combinations. Other base-pairing interactions of the loop sequences stabilize misfolded and inactive pre-RNAs. Mismatches in P13 or P14 raised the midpoints and decreased the cooperativity of the Mg(2+)-dependent eqXuilibrium folding transitions. Although some mutations in P13 resulted in slightly higher folding rates, others led to slower folding compared to the wild-type, suggesting that P13 promotes formation of P3 and P7. In contrast, mismatches in P14 increased the rate of folding, suggesting that base-pairing between P5c and P2 stabilizes intermediates in which the catalytic core is misfolded. Although the peripheral helices stabilize the native structure of the catalytic core, our results show that formation of long-range interactions, and competition between correct and incorrect loop-loop base-pairs, decrease the rate at which the active pre-RNA structure is assembled.     

Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila.

We have investigated the occurrence of methylated adenine residues in the macronuclear ribosomal RNA genes of Tetrahymena thermophila. It has been shown previously that macronuclear DNA, including the palindromic ribosomal RNA genes (rDNA), of Tetrahymena thermophila contains the modified base N-6-methyladenine, but no 5-methylcytosine. Purified rDNA was digested with restriction enzymes Sau 3AI, MboI and DpnI to map the positions and levels of N-6-methyladenine in the sequence 5' GATC 3'. A specific pattern of doubly methylated GATC sequences was found; hemimethylated sites were not detected. The patterns and levels of methylation of these sites did not change significantly in different physiological states. A molecular form of the rDNA found in the newly developing macronucleus and for several generations following the sexual process, conjugation, contained no detectably methylated GATC sites. However, both the bulk macronuclear DNA and palindromic rDNA from the same macronuclei were methylated. Possible roles for N-6-methyladenine in macronuclear DNA are discussed in light of these findings.     

The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification

When the circular form of the self-splicing intervening sequence of Tetrahymena thermophila was purified by denaturing polyacrylamide gel electrophoresis by standard methods, the rate of its reaction with tetrauridylate decreased 150-fold at 30 degrees C and at least 1000-fold at 0 degrees C. The activity of the self-splicing RNA was restored by heating it to high temperature and letting it renature in the presence of Mg2+. The rate of reaction of tetrauridylate with the self-splicing RNA flanked by exons was also greatly decreased by gel purification. The difference in activation energies for the reaction of native and denatured intervening sequences suggests that a substantial conformational rearrangement of the gel-purified RNA occurs prior to reaction.     

Structure of the Tetrahymena thermophila telomerase RNA helix II template boundary element

Telomere addition by telomerase requires an internal templating sequence located in the RNA subunit of telomerase. The correct boundary definition of this template sequence is essential for the proper addition of the nucleotide repeats. Incorporation of incorrect telomeric repeats onto the ends of chromosomes has been shown to induce chromosomal instability in ciliate, yeast and human cells. A 5′ template boundary defining element (TBE) has been identified in human, yeast and ciliate telomerase RNAs. Here, we report the solution structure of the TBE element (helix II) from Tetrahymena thermophila telomerase RNA. Our results indicate that helix II and its capping pentaloop form a well-defined structure including unpaired, stacked adenine nucleotides in the stem and an unusual syn adenine nucleotide in the loop. A comparison of the T.thermophila helix II pentaloop with a pentaloop of the same sequence found in the 23S rRNA of the Haloarcula marismortui ribosome suggests possible RNA and/or protein interactions for the helix II loop within the Tetrahymena telomerase holoenzyme.     

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.     

Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway

Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.     

Exon sequences distant from the splice junction are required for efficient self-splicing of the Tetrahymena IVS.

The presence of a natural rRNA secondary structure element immediately preceding the 5' splice site of the Tetrahymena IVS can inhibit self-splicing by competing with base pairing between the 5' exon and the guide sequence of the IVS (P1). Formation of this alternative hairpin is preferred in short precursor RNAs, and results in loss of G-addition to the 5' splice site. Pre-rRNAs which contain longer exons of ribosomal sequence, however, splice rapidly. As many as 146 nucleotides of the 5' exon and 86 nucleotides of the 3' exon are required for efficient self-splicing of Tetrahymena precursors. The presence of nucleotides distant from the 5' splice site apparently alters the equilibrium between the alternative hairpins, and promotes formation of active precursors. This effect is dependent on the specific sequences of the ribosomal pre-RNA, since point mutations within this region reduce the rate of splicing as much as 50-fold. This system provides an opportunity to study the way in which long-range interactions can influence splice site selection in a highly structured RNA.     

Secondary structures of Tetrahymena thermophila rRNA IVS sequence involved in its self-splicing reactions: a new computer analysis

The secondary structures of Tetrahymena thermophila rRNA IVS sequence involved in the self-splicing reactions, are theoretically investigated with a refined computer method previously proposed, able to select a set of the deepest free energy RNA secondary structures under constraints of model hypotheses and experimental evidences. The secondary structures obtained are characterized by the close proximity of self-reactions sites and account for double mutations experiments, and differential digestion data.     

Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila

Conjugating Tetrahymena were irradiated by ultraviolet-B (UV-B) at various stages of conjugation. When the conjugants were exposed to the UV-B at late meiotic prophase (the stage from pachytene to diplotene), abortive conjugation was induced at high frequencies. After completing meiosis, a significant number of the conjugants showed marked anomalies, i.e., failure of nuclear selection after meiosis, and abortion of the subsequent conjugation process such as a postmeiotic division to form gametic nuclei, nuclear exchange, synkaryon formation, and postzygotic development. The conjugating pairs retained the parental macronucleus and separated earlier as compared with a control. The resultant exconjugants degenerated meiotic products and became amicronucleates. These observations strongly suggest the presence of a UV-sensitive molecule that is expressed specifically at the meiotic prophase and that directs the subsequent development after meiosis. Dev. Genet. 23:151–157, 1998. © 1998 Wiley-Liss, Inc.     

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.