Splicing of yeast tRNA precursors: structure of the reaction intermediates.

The intermediates of the yeast tRNA splicing reaction have been characterized. The intervening sequence is excised as an unique linear molecule. It has 5'-hydroxyl and 3'-phosphate termini. Correspondingly, the half-tRNA molecules are shown to have a 3'-phosphate terminus on the 5' half and 5'-hydroxyl terminus on the 3' half. These isolated halves have been shown to be active in the ligation step of tRNA splicing. Removal of the 3'-phosphate from the 5' half eliminates the ability of the 5' half to participate in ligation.     

Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates.

The primary sequences of all the tRNA precursors which contain intervening sequences and which accumulate in the Saccharomyces cerevisiae rnal mutant are presented. A combination of DNA and RNA sequence analysis has led to elucidation of the primary sequence of four hitherto uncharacterized precursors. The location of the intervening sequence has in all cases been unambiguously determined by analysis of the intermediates in the splicing reaction. Secondary structures based upon the tRNA cloverleaf are shown for all the tRNA precursors and discussed with respect to common recognition by the yeast splicing endonuclease.     

Ribonuclease "XlaI," an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors.

A ribonuclease (RNase) activity, RNase "XlaI," responsible for the excision of intervening sequences from two yeast transfer ribonucleic acid (tRNA) precursors, pre-tRNA(Tyr) and pre-tRNA(3Leu), has been purified 54-fold from nuclear extracts of Xenopus laevis oocytes. The RNase preparation is essentially free of contaminating RNase. A quantitative assay for RNase XlaI was developed, and the reaction products were characterized. RNase XlaI cleavage sites in the yeast tRNA precursors were identical to those made by yeast extracts (including 3'-phosphate and 5'-hydroxyl termini). Cleavage of pre-tRNA(3Leu) by RNase XlaI and subsequent ligation of the half-tRNA molecules do not require removal of the 5' leader or 3' trailer sequences.     

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.     

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.     

Conserved mechanism of tRNA splicing in eukaryotes.

The ligation steps of tRNA splicing in yeast and vertebrate cells have been thought to proceed by fundamentally different mechanisms. Ligation in yeast cells occurs by incorporation of an exogenous phosphate from ATP into the splice junction, with concomitant formation of a 2' phosphate at the 5' junction nucleotide. This phosphate is removed in a subsequent step which, in vitro, is catalyzed by an NAD-dependent dephosphorylating activity. In contrast, tRNA ligation in vertebrates has been reported to occur without incorporation of exogenous phosphate or formation of a 2' phosphate. We demonstrate in this study the existence of a yeast tRNA ligase-like activity in HeLa cells. Furthermore, in extracts from these cells, the entire yeastlike tRNA splicing machinery is intact, including that for cleavage, ligation, and removal of the 2' phosphate in an NAD-dependent fashion to give mature tRNA. These results argue that the mechanism of tRNA splicing is conserved among eukaryotes.     

Splice junction mutations in a yeast tRNA gene which alter the rate and precision of processing

We have introduced mutations into a tRNALeu3 gene which alter the intron boundaries and examined their effects on RNA splicing. Our results show that the 5'-proximal splice junction is not specified by the position of an adjacent base-paired stem present in all naturally occurring tRNA precursors. Also, efficient cleavage of 5'-splice junctions unique to these mutants, -CpU-, -UpA- and -UpG-, indicates the purine found at the 5'-side of this site in all natural precursors is dispensable. Some alterations of the sequence and structure at the 5'-proximal splice site reduce the rate of cleavage therein and result in accumulation of molecules composed of the 5'-half of the tRNA plus the intron. The precise position of the 5'-proximal cleavage site can vary +/- 1 base in these mutants. The 3'-proximal splice junction is rendered inactive by changing the prospective splice junction sequence from -ApC- to -CpC- and reducing the size of an unpaired loop at this site from six to two bases. Very small amounts of RNA composed of the 3'-half of the tRNA plus the intron accumulate from this precursor. We conclude that splice junction sequence and structure affect both the rate and precision of intervening sequence removal.     

A precursor to a minor species of yeast tRNASer contains an intervening sequence.

Certain tRNAs in S. cerevisiae (tRNATyr and tRNAPhe) arise via precursor molecules which are mature at the 5' and 3' termini but contain intervening sequences adjacent to the anticodon (Knapp et al., 1978; O'Farrell et al., 1978). In addition to these molecules, precursors to several other tRNAs accumulate in a temperature-sensitive mutant (ts136) at the nonpermissive temperature. We have analyzed one of these species and shown that it is a precursor to a minor species of tRNASer. This precursor is also mature at both termini and contains an intervening sequence of 19 nucleotides adjacent to the hypermodified A residue 3' to the anticodon. The sequence can be arranged in a secondary structure in which the anticodon stem is extended by additional base-pairing, and contains the sites of excision and ligation within two looped regions. Support for this structure was provided by analysis of the products of limited digestion with RNAase T1. recently Piper (1978) reported the isolation of a minor species of tRNASer which decodes UCG. He found this species to be structurally heterogeneous and determined that the less abundant form corresponds to the tRNA which is altered in the recessive lethal SUP-RL1 amber suppressor. Our data now suggest that the more abundant form may be restricted to reading UCA in vivo; thus mutation of the minor species would result in complete loss of UCG-decoding ability and explain the recessive lethality of SUP-RL1. We have shown that the precursor which accumulates in ts136 corresponds exclusively to this minor tRNASerUCG species. Our results suggest that this may be the only gene for tRNASer in yeast which contains an intervening sequence.     

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.     

Domain structure in yeast tRNA ligase

Yeast tRNA ligase is one of two proteins required for the splicing of precursor tRNA molecules containing introns. The 95-kDa tRNA ligase has been purified to homogeneity from a strain of Escherichia coli which overexpresses the protein. The ligation reaction requires three enzymatic activities: phosphodiesterase, polynucleotide kinase, and ligase. By partial proteolytic digestion, we have produced fragments of tRNA ligase which contain the constituent activities. These results provide evidence for a model in which the three constituent activities of ligase are located in three distinct domains separated by protease-sensitive regions. We have also located the active adenylylated site in the ligase domains. It is lysine-114. The tRNA ligase sequence in this region has limited homology to the active-site region of T4 RNA ligase.     

Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates.

We show here that yeast tRNA ligase protein is essential in the cell and participates in joining together tRNA half-molecules resulting from excision of the intron by the splicing endonuclease. A haploid yeast strain carrying a chromosomal deletion of the ligase gene is viable only if ligase protein can be supplied from a plasmid copy of the gene. When synthesis of the plasmid-borne ligase gene is repressed, cells eventually die and accumulate endonuclease cut but unligated half-molecules and intervening sequences. Half-molecules that accumulate appear to be fully end-processed. Two temperature-sensitive ligase mutant strains have been isolated; these strains accumulate a similar set of unligated half-molecules at the nonpermissive temperature.     

Frameshift suppressor mutations outside the anticodon in yeast proline tRNAs containing an intervening sequence.

Extragenic suppressors of +1 frameshift mutations in proline codons map in genes encoding two major proline tRNA isoacceptors. We have shown previously that one isoacceptor encoded by the SUF2 gene (chromosome 3) contains no intervening sequence. SUF2 suppressor mutations result from the base insertion of a G within a 3'-GGA-5' anticodon, allowing the tRNA to read a 4-base code word. In this communication we describe suppressor mutations in genes encoding a second proline tRNA isoacceptor (wild-type anticodon 3'-GGU-5') that result in a novel mechanism for translation of a 4-base genetic code word. The genes that encode this isoacceptor include SUF7 (chromosome 13), SUF8 (chromosome 8), trn1 (chromosome 1), and at least two additional unmapped genes, all of which contain an intervening sequence. We show that suppressor mutations in the SUF7 and SUF8 genes result in G-to-U base substitutions at position 39 that disrupted the normal G . C base pairing in the last base pair of the anticodon stem adjacent to the anticodon loop. These anticodon stem mutations might alter the size of the anticodon loop and permit the use of a 3'-GGGU-5' sequence within the loop to read 4-base proline codons. Uncertainty regarding the exact structure of the mature suppressor tRNAs results from the possibility that anticodon stem mutations might affect sites of intervening sequence removal. The possible role of the intervening sequence in the generation of mature suppressor tRNA is discussed. Besides an analysis of suppressor tRNA genes, we have extended previous observations of the apparent relationship between tRNA genes and repetitive delta sequences found as solo elements or in association with the transposable element TY1. Hybridization studies and a computer analysis of the DNA sequence surrounding the SUF7 gene revealed two incomplete, inverted delta sequences that form a stem and loop structure located 165 base pairs from the 5' end of the tRNA gene. In addition, sequences beginning 164 base pairs from the 5' end of the trn1 gene also exhibit partial homology to delta. These observations provide further evidence for a nonrandom association between tRNA genes and delta sequences.     

Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.