RNA helices that recapitulate sequences of the tRNA acceptor stem, including the 3′ NCCA nucleotides, can be substrates for aminoacyl–tRNA synthetases (Frugier et al., 1994; Hamann & Hou, 1995; Martinis & Schimmel, 1995; Quinn et al., 1995). Although the catalytic efficiency of aminoacylation of RNA helices is reduced from that of the full-length parent tRNA, the specificity is maintained. The specific aminoacylation lies in the ability of aminoacyl–tRNA synthetases to recognize functional groups within the RNA helices. Analysis of tRNA–synthetase structures has suggested a general principle (Rould et al., 1989; Ruff et al., 1991; Arnez & Moras, 1997). The class I synthetases, which attach an amino acid initially to the 2′-OH of the terminal ribose, approach the acceptor and NCCA end from the minor groove side. The class II synthetases, which attach an amino acid to the terminal 3′-OH, approach from the major groove side (Arnez & Moras, 1997). The class-specific approach leads to tRNA–synthetase complexes that are near mirror images of each other and provides a structural rationale for the stereochemistries of aminoacylation. We report here the identification of a functional group in the acceptor end of Escherichia coli tRNACys that is important for the class I cysteine–tRNA synthetase. This functional group makes one of the largest energetic contributions to aminoacylation. However, it is located on the major groove side of the acceptor stem. Kinetic analysis of the contribution of this functional group to aminoacylation suggests new features that are not anticipated from the class-specific approach of synthetases.

Molybdenum is an essential cofactor in many enzymes, but must first be complexed by molybdopterin, whose synthesis requires four enzymatic activities. The first two enzymes of this pathway are encoded by the MOCS1 locus in humans. We describe here a remarkably well-conserved novel mRNA splicing phenomenon that produces both an apparently bicistronic MOCS1AM-OCS1B transcript, as well as a distinct class of monocistronic transcript. The latter are created by a variety of splicing mechanisms (alternative splice donors, alternative splice acceptors, and exon-skipping) to bypass the normal termination nonsense codon of MOCS1A resulting in fusion of the MOCS1A and MOCS1B open reading frames. Therefore, these “no-nonsense” transcripts encode a single bifunctional protein embodying both MOCS1A and MOCS1B activities. This coexpression profile was observed in vertebrates (human, mouse, cow, rabbit, opossum, and chicken) and invertebrates (fruit fly and nematode) spanning at least 700 million years of evolution. Our phylogenetic data also provide evidence that the bicistronic form of MOCS1 mRNA is likely to only produce MOCS1A protein and, combined with Northern analyses, suggests that MOCS1B is translated only as a fusion with MOCS1A. Taken together, the data presented here demonstrate a very highly conserved and physiologically relevant dynamic splicing scheme that profoundly influences the protein-coding potential of the MOCS1 locus.

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

In higher eukaryotes, the regulation of pre-mRNA processing is still poorly known. The accumulation of various mature mRNAs, which can be observed in the nuclei of mammalian cells, is suggestive of a regulatory role of transport. However, the significance of these nuclear mRNA is presently unknown. We have used a tetracycline-regulated promoter to investigate the dynamics of these pools of mRNAs upon arrest of transcription. We observed, for β-globin and LT-α genes, a slow disappearance of these mRNA from the nucleus, with an apparent half-life that is similar to their cytoplasmic half-life. In view of these dynamics, these mRNA cannot simply be mature mRNAs in transit to the cytoplasm. They could be mRNAs retained in the nucleus, provided that the regulation of mRNA stability is comparable in the nucleus and the cytoplasm. But, because of their limited stability, these nuclear mRNAs cannot constitute a significant stock for gene expression. Alternatively, they could reflect a bidirectional transport of mRNA, that is, to and from the cytoplasm, which would provide a direct explanation for the similarity in both compartments of their half-life and poly(A) tail shortening over time.

The coxsackie B3 virus oriR is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientation-dependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNAIle(UAU) and tRNAGln(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNAAsp was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than ∼17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNAIle as compared to the level of importation of the mainly cytosol-localized tRNAGln. Furthermore, exchanging the D-arm between the tRNAIle and the tRNAGln resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6–7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.

RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2′,3′ cyclic phosphate and 5′-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNAMet has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2′,3′ cyclic phosphate at the 3′ end of the 5′ exon is converted into the splice junction phosphate forming a 3′,5′-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.

The tRNA 3′ end contains the conserved CCA sequence at the 74–76 positions. The CCA sequence is synthesized and maintained by the CCA-adding enzymes. The specificity of the Escherichia coli enzyme at each of the 74–76 positions was investigated using synthetic minihelix substrates that contain permuted 3′ ends. Results here indicate that the enzyme has the ability to synthesize unusual 3′ ends. When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs. Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not. In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme. However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA. The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs. Thus, the E. coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP. These features led to a model of CCA synthesis that is independent of a nucleic acid template. The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5′-to-3′ polarity. The DNA helicase activity of the enzyme preferentially unwound 5′-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting α-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the α-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.