A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2′-phosphotransferase to transfer the splice junction 2′-phosphate from ligated tRNA to NAD, producing ADP ribose 1′′–2′′ cyclic phosphate (Appr>p). We show here that functional 2′-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2′-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2′-phosphate. Analysis of the database shows that likely members of the 2′-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2′-phosphotransferase into Eubacteria, suggesting that the 2′-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2′-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2′-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

tRNA Nuclear Export in Saccharomyces cerevisiae: In Situ Hybridization Analysis

To understand the factors specifically affecting tRNA nuclear export, we adapted in situ hybridization procedures to locate endogenous levels of individual tRNA families in wild-type and mutant yeast cells. Our studies of tRNAs encoded by genes lacking introns show that nucleoporin Nup116p affects both poly(A) RNA and tRNA export, whereas Nup159p affects only poly(A) RNA export. Los1p is similar to exportin-t, which facilitates vertebrate tRNA export. A los1 deletion mutation affects tRNA but not poly(A) RNA export. The data support the notion that Los1p and exportin-t are functional homologues. Because LOS1 is nonessential, tRNA export in vertebrate and yeast cells likely involves factors in addition to exportin-t. Mutation of RNA1, which encodes RanGAP, causes nuclear accumulation of tRNAs and poly(A) RNA. Many yeast mutants, including those with the rna1-1 mutation, affect both pre-tRNA splicing and RNA export. Our studies of the location of intron-containing pre-tRNAs in the rna1-1 mutant rule out the possibility that this results from tRNA export occurring before splicing. Our results also argue against inappropriate subnuclear compartmentalization causing defects in pre-tRNA splicing. Rather, the data support “feedback” of nucleus/cytosol exchange to the pre-tRNA splicing machinery.

Translocation events in the evolution of aminoacyl-tRNA synthetases.

We have characterized hisS, the gene encoding the histidyl-tRNA synthetase (HisRS) from the tetraodontoid fish Fugu rubripes. The hisS gene is about 3.5 kbp long and contains 13 exons and 12 introns of 172 bp, on average. The Fugu hisS gene encodes a putative protein of 519 amino acids with the three motifs identified as signatures of class 2 aminoacyl-tRNA synthetases. A model for the shifting of intron 8 between Fugu and hamster is proposed based on the successive appearance of a cryptic splicing site followed by an insertion mutation that created a new acceptor site. In addition, sequence comparisons suggest that the hisS gene has undergone a translocation through the first intron. As a result, the Fugu HisRS has an N-terminal sequence markedly different from that in the human and hamster enzymes. We propose that similar events have been responsible for variations at the N-terminal end of other aminoacyl-tRNA synthetases. Our analysis suggests that this involves exchanges through introns of two exons encoding an ancestral 32-amino acid motif.

Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny

Nuclear-coded valyl-tRNA synthetase (ValRS) of eukaryotes is regarded of mitochondrial origin. Complete ValRS sequences obtained by us from two amitochondriate protists, the diplomonad, Giardia lamblia and the parabasalid, Trichomonas vaginalis were of the eukaryotic type, strongly suggesting an identical history of ValRS in all eukaryotes studied so far. The findings indicate that diplomonads are secondarily amitochondriate and give further evidence for such conclusion reached recently concerning parabasalids. Together with similar findings on other amitochondriate groups (microsporidia and entamoebids), this work provides critical support for the emerging notion that no representatives of the premitochondrial stage of eukaryotic phylogenesis exist among the species living today.

Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver

Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215–222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5′ or the 3′ adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected Mr are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: The Leptomonas collosoma U5 RNA and its coding gene

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5′ phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016–4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6⋅U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem–loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNACys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5′ splice site region (positions −1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.

A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease

Some group I introns self-splice in vitro, but almost all are thought to be assisted by proteins in vivo. Mutational analysis has shown that the splicing of certain group I introns depends upon a maturase protein encoded by the intron itself. However the effect of a protein on splicing can be indirect. We now provide evidence that a mitochondrial intron-encoded protein from Aspergillus nidulans directly facilitates splicing in vitro. This demonstrates that a maturase is an RNA splicing protein. The protein-assisted reaction is as fast as that of any other known group I intron. Interestingly the protein is also a DNA endonuclease, an activity required for intron mobilization. Mobile elements frequently encode proteins that promote their propagation. Intron-encoded proteins that also assist RNA splicing would facilitate both the transposition and horizontal transmission of introns.

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5′ end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5′ end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3′ end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA2Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA2Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.

Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase

Oligonucleotides that recapitulate the acceptor stems of tRNAs are substrates for aminoacylation by many tRNA synthetases in vitro, even though these substrates are missing the anticodon trinucleotides of the genetic code. In the case of tRNAAla a single acceptor stem G⋅U base pair at position 3·70 is essential, based on experiments where the wobble pair has been replaced by alternatives such as I⋅U, G⋅C, and A⋅U, among others. These experiments led to the conclusion that the minor-groove free 2-amino group (of guanosine) of the G⋅U wobble pair is essential for charging. Moreover, alanine-inserting tRNAs (amber suppressors) that replace G⋅U with mismatches such as G⋅A and C⋅A are partially active in vivo and can support growth of an Escherichia coli tRNAAla knockout strain, leading to the hypothesis that a helix irregularity and nucleotide functionalities are important for recognition. Herein we investigate the charging in vitro of oligonucleotide and full-length tRNA substrates that contain mismatches at the position of the G⋅U pair. Although most of these substrates have undetectable activity, G⋅A and C⋅A variants retain some activity, which is, nevertheless, reduced by at least 100-fold. Thus, the in vivo assays are much less sensitive to large changes in aminoacylation kinetic efficiency of 3·70 variants than is the in vitro assay system. Although these functional data do not clarify all of the details, it is now clear that specific atomic groups are substantially more important in determining kinetic efficiency than is a helical distortion. By implication, the activity of mutant tRNAs measured in the in vivo assays appears to be more dependent on factors other than aminoacylation kinetic efficiency.

Functional association between promoter structure and transcript alternative splicing

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.

Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution

Inteins are protein-splicing elements, most of which contain conserved sequence blocks that define a family of homing endonucleases. Like group I introns that encode such endonucleases, inteins are mobile genetic elements. Recent crystallography and computer modeling studies suggest that inteins consist of two structural domains that correspond to the endonuclease and the protein-splicing elements. To determine whether the bipartite structure of inteins is mirrored by the functional independence of the protein-splicing domain, the entire endonuclease component was deleted from the Mycobacterium tuberculosis recA intein. Guided by computer modeling studies, and taking advantage of genetic systems designed to monitor intein function, the 440-aa Mtu recA intein was reduced to a functional mini-intein of 137 aa. The accuracy of splicing of several mini-inteins was verified. This work not only substantiates structure predictions for intein function but also supports the hypothesis that, like group I introns, mobile inteins arose by an endonuclease gene invading a sequence encoding a small, functional splicing element.

Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are ≈65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

Thermus thermophilus: A link in evolution of the tRNA-dependent amino acid amidation pathways

Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn. Aspartate mischarged on tRNAAsn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems.

The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase

The crystal structure of an enzyme–substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.