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.     

Substrate recognition and splice site determination in yeast tRNA splicing

S. cerevisae tRNA introns interrupt the gene at a constant position in the anticodon loop. Pre-tRNAs are matured by an endonuclease and a ligase. The endonuclease alone can accurately release the intron from the pre-tRNA. Here, we investigate the mechanism of splice site selection by the endonuclease. We propose that it initially recognizes features in the mature domain common to all tRNAs. Once positioned on the enzyme, the splice sites are recognizable because they are a fixed distance from the mature domain. To test this hypothesis, we developed a system for synthesizing pre-tRNA by bacteriophage T7 RNA polymerase. To search for recognition sites, we made several mutations. Mutations of C56 and U8 strongly affect endonuclease recognition of pre-tRNA. With insertion and deletion mutations, we show that the anticodon stem determines splicing specificity. The sequence and structure of the intron are not strong determinants of splice site selection.     

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.     

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.     

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.     

Accumulation of pre-tRNA splicing ''2/3'' intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.     

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.     

Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments

Splicing of precursor tRNAs in plants requires the concerted action of three enzymes: an endonuclease to cleave the intron at the two splice sites, an RNA ligase for joining the resulting tRNA halves and a 2'-phosphotransferase to remove the 2'-phosphate from the splice junction. Pre-tRNA splicing has been demonstrated to occur exclusively in the nucleus of vertebrates and in the cytoplasm of budding yeast cells, respectively. We have investigated the subcellular localization of plant splicing enzymes fused to GFP by their transient expression in Allium epidermal and Vicia guard cells. Our results show that all three classes of splicing enzymes derived from Arabidopsis and Oryza are localized in the nucleus, suggesting that plant pre-tRNA splicing takes place preferentially in the nucleus. Moreover, two of the splicing enzymes, i.e., tRNA ligase and 2'-phosphotransferase, contain chloroplast transit signals at their N-termini and are predominantly targeted to chloroplasts and proplastids, respectively. The putative transit sequences are effective also in the heterologous context fused directly to GFP. Chloroplast genomes do not encode intron-containing tRNA genes of the nuclear type and consequently tRNA ligase and 2'-phosphotransferase are not required for classical pre-tRNA splicing in these organelles but they may play a role in tRNA repair and/or splicing of atypical group II introns. Additionally, 2'-phosphotransferase-GFP fusion protein has been found to be associated with mitochondria, as confirmed by colocalization studies with MitoTracker Red. In vivo analyses with mutated constructs suggest that alternative initiation of translation is one way utilized by tRNA splicing enzymes for differential targeting.     

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.     

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.     

A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α(4), homodimeric: α(2), and heterotetrameric: (αβ)(2)] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε(2)) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε(2) endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αβ)(2) endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)(2) endonuclease. Thus, the discovery of ε(2) endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.     

A cell-free plant extract for accurate pre-tRNA processing, splicing and modification.

An intron-containing tobacco tRNA(Tyr) precursor synthesized in a HeLa cell nuclear extract has been used to develop a cell-free processing and splicing system from wheat germ. Removal of 5' and 3' flanking sequences, accurate excision of the intervening sequence, ligation of the resulting tRNA halves, addition of the 3'-terminal CCA sequence and modification of seven nucleosides were achieved in appropriate wheat germ S23 and S100 extracts. The maturation of pre-tRNA(Tyr) in these extracts resembles the pathway observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast in that processing of the flanks precedes intron excision. Most of the modified nucleosides (m2(2) G, psi 35, psi 55, m7G and m1A) are introduced into the intron-containing pre-tRNA with mature ends, whereas two others (m1G and psi 39) are only found in the mature tRNA(Tyr). Processing and splicing proceed very efficiently in the wheat germ extracts, leading to complete maturation of 5' and 3' ends followed by about 65% conversion to mature tRNA(Tyr) under our standard conditions. The activity of the wheat germ endonuclease is stimulated 3-fold by the non-ionic detergent Triton X-100. All previous attempts to demonstrate the presence of a splicing endonuclease in wheat germ had failed (Gegenheimer et al., 1983). Hence, this is the first cell-free plant extract which supports pre-tRNA processing and splicing in vitro.     

Structural and mutational analysis of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728: catalytic mechanism of tRNA intron-splicing endonucleases

In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728 (EndA_Ta) have been solved to 2.5-Å and 2.7-Å resolution by molecular replacement, using the 2.7-Å resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndA_Ta is a homodimer and that it has overall folding similar to that of other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.     

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.     

Substrate recognition and identification of splice sites by the tRNA-splicing endonuclease and ligase from Saccharomyces cerevisiae.

We have examined the substrate requirements for efficient and accurate splicing of tRNA precursors in Saccharomyces cerevisiae. The effects of Schizosaccharomyces pombe tRNASer gene mutations on the two steps in splicing, intron excision and joining of tRNA halves, were determined independently by using partially purified splicing endonuclease and tRNA ligase from S. cerevisiae. Two mutations (G14 and A46) reduced the efficiency of excision and joining in parallel, whereas two others (U47:7 and C33) produced differential effects on these two steps; U47:7 affected primarily the excision reaction, and C33 had a greater impact on ligation. These data indicate that endonuclease and ligase recognize both common and unique features of their substrates. Another two mutations (Ai26 and A37:13) induced miscutting, although with converse effects on the two splice sites. Thus, the two cutting events appear to be independent. Finally, we suggest that splice sites may be determined largely through their position relative to sites within the tRNA-like domain of the precursors. Several of these important sites were identified, and others are proposed based on the data described here.     

tRNA核酸内切酶的研究进展

tRNA在蛋白质合成过程中起着极其重要的作用。在所有的生物体内,tRNA首先以前体形式转录,然后必需经过一系列的加工后才能成为有功能的tRNA分子。tRNaseZ、RNaseP和tRNA剪接内切酶是参与tRNA前体加工的三种主要的核酸内切酶,分别参与tRNA前体3′末端、tRNA前体5′末端和内含子剪接的加工。这三种酶具有不同的结构特征,并且利用完全不同的催化机制水解磷酸二酯键。tRNaseZ和RNaseP都是金属酶,活性中心分别需要Zn^2＋和Mg^2＋的参与;而tRNA剪接内切酶活性中心不需要金属离子,是一个由不同催化亚基上的关键氨基酸残基构成的组合式活性中心。