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.     

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.     

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.     

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.     

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.     

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.     

The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena

The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing. We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei. We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA. The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping. A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event. This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions. The EV of the IVS is a circular RNA. This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels. When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed. Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion. The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.     

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.     

One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity

The specificity of reactions catalyzed by the Tetrahymena pre-rRNA intervening sequence (IVS) was studied using site-specific mutagenesis. Two sequences required for 5' splice-site selection during self-splicing were defined. Single-base changes in either a 5' exon sequence or a 5' exon-binding site within the IVS disrupt their ability to pair and result in inefficient or inaccurate splicing. Combinations that restore complementarity suppress the effect of the single-base changes. Sequence alterations in the 5' exon-binding site also change the specificity of two other reactions: intermolecular exon ligation (trans-splicing) and the enzymatic nucleotidyltransferase activity of the IVS RNA. Thus the substrate specificity of an RNA enzyme can be changed in a manner predictable by the rules of Watson-Crick base-pairing.     

The intervening sequence of the ribosomal RNA gene is highly conserved between two Tetrahymena species.

The entire intervening sequence of Tetrahymena thermophila ribosomal DNA has been determined. It is 413 nucleotides long and has the same splice junctions as those in T. pigmentosa. There is 93% homology between the intervening sequences in the two species, and 100% homology between their adjacent 26S RNA coding regions.     

5' exon replacement and repair by spliceosome-mediated RNA trans-splicing

Spliceosome-mediated RNA trans-splicing (SMaRT) has been used previously to reprogram mutant endogenous CFTR and factor VIII mRNAs in human epithelial cell and tissue models and knockout mice, respectively. Those studies used 3' exon replacement (3'ER); a process in which the distal portion of RNA is reprogrammed. Here, we also show that the 5' end of mRNA can be completely rewritten by 5'ER. For proof-of-concept, and to test whether 5'ER could generate functional CFTR, we generated a mutant minigene target containing CFTR exons 10-24 (deltaF508) and a mini-intron 10, and a pretrans-splicing molecule (targeted to intron 10) containing CFTR exons 1-10 (+F508), and tested these two constructs in 293T cells for anion efflux transport. Cells cotransfected with target and PTM showed a consistent increase in anion efflux, but there was no response in control cells that received PTM or target alone. Using a LacZ reporter system to accurately quantify trans-splicing efficiency, we tested several unique PTM designs. These studies provided two important findings as follows: (1) efficient trans-splicing can be achieved by binding the PTM to different locations in the target, and (2) relatively few changes in PTM design can have a profound impact on trans-splicing activity. Tethering the PTM close to the target 3' splice site (as opposed to the donor site) and inserting an intron in the PTM coding resulted in a 65-fold enhancement of LacZ activity. These studies demonstrate that (1) SMaRT can be used to reprogram the 5' end of mRNA, and (2) efficiency can be improved substantially.     

Alternative promoter and 5' exon generate a novel Gs alpha mRNA

Several species of mRNA have been shown to encode the alpha subunit of the stimulatory GTP-binding regulatory protein, Gs alpha. The various Gs alpha mRNAs are generated through alternative splicing of a single precursor RNA and through the use of alternative acceptor splice sites. We now report the existence of a Gs alpha mRNA that uses a previously unidentified promoter and leading exon (termed exon 1'). In both the canine and human Gs alpha genes, exon 1' is located 2.5 kilobases 5' of exon 1. Exon 1' does not contribute an in-frame ATG, and thus its mRNA encodes a truncated form of Gs alpha. Initiation of translation is predicted to begin at an AUG in exon 2, as demonstrated both by in vitro translation and COS cell expression studies.     

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.     

Three-dimensional model and molecular dynamics simulation of the active site of the self-splicing intervening sequence of the bacteriophage T4 nrdB messenger RNA

The secondary and 3D structure of the active site of the self-splicing T4 nrdB RNA has been modeled on a graphics workstation by use of the suggested 3D arrangement of the active site of the Tetrahymena IVS [Kim, S.H., & Cech, T.R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8788-8792] as a guideline. The initially obtained crude structure was then subjected to molecular mechanics energy minimization and molecular dynamics simulation to relax tensions. In this process the energy decreased considerably and gave a final structure that deviated by 3 A [root mean square (rms)] from the initial structure. The cofactor guanosine (and the competitive inhibitor arginine) was docked to a proposed [Michel, F., Hanna, M., Green, R., Bartel, D.P., & Szostak, J.W. (1989) Nature 342, 391-395] binding site, where it was found to fit rather well. A minor modification of the binding mode easily brought the O3' end of the guanosine within 2 A of the phosphodiester bond where the primary cleavage occurs.     

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.