Site selection by the tRNA splicing endonuclease of Xenopus laevis

To investigate the mechanism by which the purified Xenopus tRNA splicing endonuclease recognizes its splice sites, we utilized yeast pre-tRNA(3Leu) and pre-tRNA(Phe) variants constructed by in vitro mutagenesis. We found that the endonuclease interacts with conserved features of the mature tRNA domain. In particular, U8 and C56 may be examples of contact points between protein and RNA. Given that there are no conserved sequences at the splice junctions, the specificity of cutting at both splice sites is determined by the length of the anticodon stem. Although in general, the sequence of the intron is unimportant for splicing, there are some structural requirements.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain.

It has been proposed that yeast and Xenopus splicing endonucleases initially recognize features in the mature tRNA domain common to all tRNA species and that the sequence and structure of the intron are only minor determinants of splice-site selection. In accordance with this postulation, we show that yeast endonuclease splices heterologous pre-tRNA(Tyr) species from vertebrates and plants which differ in their mature domains and intron secondary structures. In contrast, wheat germ splicing endonuclease displays a pronounced preference for homologous pre-tRNA species; an extensive study of heterologous substrates revealed that neither yeast pre-tRNA species specific for leucine, serine, phenylalanine and tyrosine nor human and Xenopus pre-tRNA(Tyr) species were spliced. In order to identify the elements essential for pre-tRNA splicing in plants, we constructed chimeric genes coding for tRNA precursors with a plant intron secondary structure and with mature tRNA(Tyr) domains from yeast and Xenopus, respectively. The chimeric pre-tRNA comprising the mature tRNA(Tyr) domain from Xenopus was spliced efficiently in wheat germ extract, whereas the chimeric construct containing the mature tRNA(Tyr) domain from yeast was not spliced at all. These data indicate that intron secondary structure contributes to the specificity of plant splicing endonuclease and that unique features of the mature tRNA domain play a dominant role in enzyme-substrate recognition. We further investigated the influence of specific nucleotides in the mature domain on splicing by generating a number of mutated pre-tRNA species. Our results suggest that nucleotides located in the D stem, i.e. in the center of the pre-tRNA molecule, are recognition points for plant splicing endonuclease.     

Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs.

Intron-containing pre-tRNAs from organisms as different as yeast, Nicotiana, Xenopus and man are efficiently spliced and processed in a HeLa cell extract. They are also correctly processed in a wheat germ extract; however, the intron is removed only from the tobacco pre-tRNA. To determine whether plant pre-tRNA introns have any specific structural and/or sequence feature we have cloned two intron-containing tRNATyr genes from the plant Arabidopsis. Comparison of these genes, of the Nicotiana tRNATyr gene and of a Glycine max tRNAMet gene reveals that plant introns from three different species have no sequence homology and are only 11 to 13 nucleotides long. Thus, short length may be one important feature of plant introns. Furthermore, the 5' and 3' splice sites are separated by 4 bp in the extended anticodon stems of these pre-tRNA structures. In contrast, yeast and vertebrate introns are rather variable in length and the splice sites are separated by 5 or 6 bp. These differences in distance and relative helical orientation of the splice sites in plant pre-tRNAs versus pre-tRNAs from other organisms are obviously tolerated by the vertebrate splicing endonuclease, but not at all by the plant enzyme.     

Site selection by Xenopus laevis RNAase P

Investigation of the mechanism of cleavage site selection by Xenopus RNAase P reveals that the acceptor stem, a 7 bp helix common to all tRNA precursors, is required for cleavage. We propose that Xenopus RNAase P recognizes conserved features of the mature tRNA and that the cleavage site is selected by measuring the length of the acceptor stem. In support of this, we demonstrate that insertion of 2 bp in the acceptor stem of yeast pre-tRNA(3Leu) relocates the cleavage site 2 bases 3' to the original one. In addition, insertion of 1 bp in the acceptor stem of the end-matured yeast pre-tRNA(Phe) generates an RNAase P cleavage site: the enzyme produces a mature tRNA with the characteristic 7 bp stem and releases one 5' flanking nucleotide. Since it has previously been shown that cleavage sites of the splicing endonuclease are determined by the length of the anticodon stem, RNAase P and the splicing endonuclease apparently use different stems to determine their cutting sites.     

Non-enzymatic excision of pre-tRNA introns?

We used human tRNA(Tyr) precursor as a substrate to study self-excision of a pre-tRNA intron. This RNA was synthesized in vitro in a HeLa cell extract. It contains a 5' leader, an intron of 20 nucleotides and a 3' trailer. Self-cleavage of pre-tRNA(Tyr) occurs in 100 mM NH4OAc at a pH ranging from 6 to 8.5 in the presence of spermine, MgCl2 and Triton X-100 under conditions very similar to enzymatic intron excision. The reaction is temperature-dependent, relatively fast as compared to the enzyme-catalysed reaction and leads to fragments which resist further degradation. The detailed structure of all major and minor cleavage products was established by fingerprint analyses. Non-enzymatic cleavage occurs predominantly at the 3' splice site and to a minor extent at the 5' splice site. Other minor cleavage sites are located within the intron and in the 3' trailer. Putative 5' and 3' tRNA halves resulting from pre-tRNA(Tyr) self-cleavage are substrates for wheat germ RNA ligase, suggesting that the cleavage reaction yields 2',3'-cyclic phosphate and 5'-hydroxyl termini. Pre-tRNA splicing endonuclease is believed to cleave both the 5' and the 3' splice site. However, on the basis of our results we propose that this enzyme may support the formation of a pre-tRNA tertiary structure favourable for autocatalytic intron excision and impair unspecific self-cleavage.     

In vitro genetic analysis of the structural features of the pre-tRNA required for determination of the 3' splice site in the intron excision reaction.

During processing of intron-containing pre-tRNAs, the Xenopus laevis splicing endonuclease binds the precursor and cleaves it at both the 5' and 3' splice sites. In vitro selection was used to determine structural features characteristic of precursor tRNA molecules that are active in this reaction. We performed two types of selection, one for molecules that are not cut, the other for molecules that are cut at only one site. The results shed light on various aspects of the intron excision reaction, including the importance of the three-dimensional structure of the mature domain for recognition and binding of the enzyme, the active role played by the single-stranded region of the intron, and the importance of the cardinal positions which, although not necessarily occupied by the same base in all precursors, nevertheless play a fundamental role in the splicing reaction. A precursor can be cut at the 3' site if a base in the single-stranded loop of the intron is allowed to pair (A-I pair) with the base of the 5' exon situated at the position immediately following the anticodon stem [first cardinal position (CP1)]. The nature of the bases involved in the A-I pair is important, as is the position of the base in the single-stranded loop of the intron. We discuss the role of the cardinal positions in the reaction.     

Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease.

The intron-containing tRNA(Trp) precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. To investigate the role of this structure in cleavage by the halophilic endonuclease, a series of mutant tRNA(Trp) RNAs were prepared and evaluated as substrates. We find that alterations in this structure result in the loss of cleavage at both 5' and 3' sites. Cleavage of a 35-nucleotide model RNA substrate, containing only these features, demonstrates that sequences and structures present at the exon-intron boundaries are sufficient for recognition and cleavage. We have also examined the mechanism used by the halophilic endonuclease to identify the cleavage sites. Addition of a single base, or a base pair in the anticodon stem above the cleavage sites, does not affect the cleavage site selection. The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases.     

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.     

Intron sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae

Predicted single-stranded structure at the 3' splice site is a conserved feature among intervening sequences (IVSs) in eukaryotic nuclear tRNA precursors. The role of 3' splice site structure in splicing was examined through hexanucleotide insertions at a central intron position in the Saccharomyces cerevisiae tRNA gene. These insertions were designed to alter the structure at the splice site without changing its sequence. Endonuclease cleavage of pre-tRNA substrates was then measured in vitro, and suppressor activity was examined in vivo. A precursor with fully double-stranded structure at the 3' splice site was not cleaved by endonuclease. The introduction of one unpaired nucleotide at the 3' splice site was sufficient to restore cleavage, although at a reduced rate. We have also observed that guanosine at the antepenultimate position provides a second consensus feature among IVSs in tRNA precursors. Point mutations at this position were found to affect splicing although there was no specific requirement for guanosine. These and previous results suggest that elements of secondary and/or tertiary structure at the 3' end of IVSs are primary determinants in pre-tRNA splice site utilization whereas specific sequence requirements are limited.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

Mutational Analysis of Pre-mRNA Splicing in Saccharomyces Cerevisiae Using a Sensitive New Reporter Gene, Cup1

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.     

The Eucaryal tRNA Splicing Endonuclease Recognizes a Tripartite Set of RNA Elements

The tRNA splicing endonuclease cleaves intron-containing tRNA precursors on both sides of the intron. The prevailing belief has been that the enzyme binds only to the mature domain through the invariant bases. We show instead that, for recognition, the endonuclease utilizes distinct sets of structural elements, several of which are within the intron. One subset of recognition elements, localized in the mature domain, is needed for recognition of both cleavage sites, while two other subsets, localized at the exon–intron boundaries, are used for recognition of either one or the other cleavage site. The two cleavage sites are essentially independent: neither is required by the other for cleavage to take place. These results support a two-active-site model for the eucaryal endonuclease.     

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.     

Splicing of mRNA mediated by tRNA sequences in mouse cells

tRNA splicing is essential for the formation of tRNAs and therefore for gene expression. A circularly permuted sequence of an amber-suppressor pre-tRNA gene was inserted into the sequence encoding the mouse NEMO protein. We demonstrated that, in mouse cells, the hybrid pre-tRNA/pre-mRNAs can be spliced precisely at the sites of the pre-tRNA intron. This splicing reaction produces functional tRNAs that suppress amber codons as well as translatable mRNAs that sustain the NF-κB activation pathway. The RNA molecules extracted from mouse cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then applied the Archaea-express technology, in which an archaeal RNA endonuclease is expressed in mouse cells. We show that both the endogenous eukaryal endonuclease and the archaeal one cleave the hybrid pre-tRNA/pre-mRNAs in the same manner with an additive effect.