Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle.

Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers. In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.

Polyadenylation of telomerase RNA in budding yeast.

Telomerase RNA is a subunit of a stable ribonucleoprotein particle required for telomere replication. We find that, at steady state, 5-10% of the telomerase RNA in Saccharomyces cerevisiae and Kluyveromyces lactis contains a poly(A) tail of about 80 nt. In S. cerevisiae, the poly(A)+ fraction quickly disappeared when a conditional pap1 or rna15 mutant was shifted to the nonpermissive temperature, indicating that polyadenylation is accomplished by the same machinery that polyadenylates mRNAs. Potential cis-acting polyadenylation elements were identified in the telomerase RNA sequence; when they were mutagenized, the polyadenylation pattern shifted, but was not eliminated. The corresponding mutants displayed wild-type growth. By putting the RNA under the control of an inducible promoter, we were able to show that synthesis of the poly(A)+ RNA precedes that of the poly(A)- fraction. This supports, but does not prove, a model in which all telomerase RNA is first polyadenylated and then rapidly processed to give the stable poly(A)-form. Cell cycle arrest experiments showed an increase in the poly(A)+ form between G1 and S phase, consistent with an induction of telomerase RNA transcription at the time of DNA replication.

Relative orientation of RNA helices in a group 1 ribozyme determined by helix extension electron microscopy.

The relative orientation of helical elements in a folded RNA molecule provides key information about its three-dimensional architecture. We have developed a method that involves extending peripheral helices of an RNA, mounting for electron microscopy in the absence of protein and measuring interhelical angles. As a control, extended anticodon and acceptor stems of tRNA(Phe) were found to form a 92 +/- 20 degrees angle, consistent with the X-ray structure. Single, double and triple extensions (50-80 bp) of helical elements P2.1, P6b and P8 of the Tetrahymena group I ribozyme did not alter its catalytic activity. The measured angle between P6b and P8 is consistent with the Michel-Westhof structural model, while the P2.1-P6b and P2.1-P8 angles allow P2.1 to be positioned in the model. The angle distributions of the ribozyme are broader than those of the tRNA, which may reflect the dynamics of the RNA. Helix extension allows low-resolution electron microscopy to provide much higher resolution information about the disposition of helical elements in RNA. It should be applicable to diverse RNAs and ribonucleoprotein complexes.

Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA.

Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association. The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus. Methylation was consistent with the current secondary structure model. Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive. Thus, most telomerase is not base paired to telomeres in vivo. Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions. The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence. For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern. Thus, as expected, comparative analysis gives the structure of the RNA in vivo.

Catalysis of RNA cleavage by a ribozyme derived from the group I intron of Anabaena pre-tRNA(Leu).

In the cyanobacterium Anabaena PCC7120, the precursor to tRNA(Leu) contains a 249-nucleotide group I intron that undergoes efficient self-splicing in vitro. By deleting the 5' and 3' splice sites, this intron has now been converted to an RNA enzyme that uses a guanosine nucleophile to cleave substrate RNAs (S) with multiple turnover. This Anabaena ribozyme has a second-order rate constant for RNA cleavage (kcat/Km)S that is 250-500-fold smaller than that of the Tetrahymena ribozyme, and a multiple-turnover rate constant at saturating S [kcat(mt)] that is approximately 400-fold larger. Several lines of evidence, including kinetic analysis of cleavage of phosphorothioate- and deoxynucleotide-substituted substrates and pH dependence, support the conclusion that both (kcat/Km)S and kcat(mt) are limited by the actual chemical cleavage step. In contrast, for the Tetrahymena ribozyme, it has been shown that neither of these rate constants reflects the chemical step. These kinetic differences are expected from the shorter guide sequence-substrate pairing of the Anabaena ribozyme; for example, weaker binding of RNA speeds product release during multiple turnover and thereby overcomes the rate-limiting product release observed for the Tetrahymena ribozyme. Thus, the large kinetic differences represent superficial rather than fundamental differences between these ribozymes. Furthermore, the strength of the guanosine-binding interaction, the stereospecificity for Rp-phosphorothioate at the cleavage site, and the 10(3)-fold slower cleavage with a deoxyribonucleoside leaving group are properties conserved between the Anabaena and Tetrahymena ribozymes. Finally, log(kcat/Km)S increases linearly with pH in the acid range where chemistry is rate-limiting and becomes pH-independent above pH 7, perhaps because a conformational step becomes rate-limiting; again, these are characteristics shared with the Tetrahymena ribozyme. We conclude that two group I ribozymes, although differing in the identity of many of their active site nucleotides, nevertheless provide functionally similar active sites for sequence-specific RNA cleavage.

Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base-pairing of the exons in the anticodon stem.

In the cyanobacterium Anabaena, the precursor to tRNA(Leu) has a 249-nucleotide group I intron inserted between the wobble and second bases of the anticodon; the intron self-splices during transcription in vitro [Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A., & Shub, D. A. (1990) Science 250, 1566-1570]. By studying splicing of isolated pre-tRNA, we confirm that splicing occurs by the two-step transesterification mechanism characteristic of group I introns, resulting in excision of the intron and accurate ligation of the 5' and 3' exons. The first step, guanosine-dependent cleavage of the phosphodiester bond at the 5' splice site, occurs with kcat congruent to 14 min-1 and kcat/Km = 5 x 10(4) M-1 min-1 (32 degrees C, 15 mM MgCl2), unexpectedly efficient for a small group I intron. (kcat/Km is comparable to that of the Tetrahymena pre-rRNA intron, and kcat is an order of magnitude higher than any previously reported for a group I intron). The second step, ligation of the exons, is so slow (k = 0.3 min-1) that it is rate-limiting for splicing in vitro except at very low guanosine concentrations. Disruption of the base pairs that make up the anticodon stem of the tRNA dramatically reduces the rate of the first step of splicing, while compensatory mutations that restore base pairing generally restore activity. We suggest that the very short P1 helix of this pre-tRNA, with only three base pairs preceding the 5' splice site, is unstable without the additional base pairs in the anticodon stem.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoacyl esterase activity of the Tetrahymena ribozyme.

Several classes of ribozymes (catalytic RNA's) catalyze reactions at phosphorus centers, but apparently no reaction at a carbon center has been demonstrated. The active site of the Tetrahymena ribozyme was engineered to bind an oligonucleotide derived from the 3' end of N-formyl-methionyl-tRNA(fMet). This ribozyme catalyzes the hydrolysis of the aminoacyl ester bond to a modest extent, 5 to 15 times greater than the uncatalyzed rate. Catalysis involves binding of the oligonucleotide to the internal guide sequence of the ribozyme and requires Mg2+ and sequence elements of the catalytic core. The ability of RNA to catalyze reactions with aminoacyl esters expands the catalytic versatility of RNA and suggests that the first aminoacyl tRNA synthetase could have been an RNA molecule.

Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena acts as a sequence-specific endoribonuclease. Specificity of cleavage is determined by Watson-Crick base pairing between the active site of the RNA enzyme (ribozyme) and its RNA substrate [Zaug, A. J., Been, M. D., & Cech, T. R. (1986) Nature (London) 324, 429-433]. Surprisingly, single-base changes in the substrate RNA 3 nucleotides preceding the cleavage site, giving a mismatched substrate-ribozyme complex, enhance the rate of cleavage. Mismatched substrates show up to a 100-fold increase in kcat and, in some cases, in kcat/Km. A mismatch introduced by changing a nucleotide in the active site of the ribozyme has a similar effect. Addition of 2.5 M urea or 3.8 M formamide or decreasing the divalent metal ion concentration from 10 to 2 mM reverses the substrate specificity, allowing the ribozyme to discriminate against the mismatched substrate. The effect of urea is to decrease kcat and kcat/Km for cleavage of the mismatched substrate; Km is not significantly affected at 0-2.5 M urea. Thus, progressive destabilization of ribozyme-substrate pairing by mismatches or by addition of a denaturant such as urea first increases the rate of cleavage to an optimum value and then decreases the rate.

Self-splicing RNA and an RNA enzyme in Tetrahymena.

The RNA molecules transcribed from many eukaryotic genes are interrupted by intervening sequences, which are removed by a process called RNA splicing. One structurally related group of intervening sequences, the group I intervening sequences, are found in a variety of microorganisms. Some of these, including the group I intervening sequence from the ribosomal RNA precursor of Tetrahymena thermophila, have been shown to mediate their own splicing in an RNA-catalyzed reaction. Following its excision from the ribosomal RNA precursor, the Tetrahymena intervening sequence acts as an enzyme, cutting and rejoining RNA substrates.

The Tetrahymena ribozyme acts like an RNA restriction endonuclease.

A shortened form of the Tetrahymena self-splicing ribosomal RNA intervening sequence acts as an endoribonuclease, catalysing the cleavage of large RNA molecules by a mechanism involving guanosine transfer. The sequence specificity approaches that of the DNA restriction endonucleases. Site-specific mutagenesis of the enzyme active site alters the substrate sequence specificity in a predictable manner, so that endoribonucleases can be synthesized to cut at a variety of tetranucleotide sequences.

The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase.

A shortened form of the Tetrahymena intervening sequence (IVS) RNA acts as an enzyme, catalyzing nucleotidyl transfer and hydrolysis reactions with oligo(cytidylic acid) substrates [Zaug, A. J., & Cech, T. R. (1986) Science (Washington, D.C.) 231, 470-475]. These reactions involve phosphodiester substrates. We now show that the same enzyme has activity toward phosphate monoesters. The 3'-phosphate of C5p or C6p is transferred to the 3'-terminal guanosine of the enzyme. The pH dependence of the reaction (optimum at pH 5) indicates that the enzyme has activity toward the dianion and much greater activity toward the monoanion form of the 3'-phosphate of the substrate. Phosphorylation of the enzyme is reversible by C5-OH and other oligo(pyrimidines) such as UCU-OH. Thus, the RNA enzyme acts as a phosphotransferase, transferring the 3'-terminal phosphate of C5p to UCU-OH with multiple turnover. At pH 4 and 5, the phosphoenzyme undergoes slow hydrolysis to yield inorganic phosphate. Thus, the enzyme has acid phosphatase activity. The RNA enzyme dephosphorylates oligonucleotide substrates with high sequence specificity, which distinguishes it from known protein enzymes.

Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor.

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.

The intervening sequence RNA of Tetrahymena is an enzyme.

A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro. The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1. The reaction mechanism resembles that of rRNA precursor self-splicing. With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid. Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template. At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis.

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.

Oligomerization of intervening sequence RNA molecules in the absence of proteins.

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA.

The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule can be folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposed mechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated.