RNase E is required for the maturation of ssrA RNA and normal ssrA RNA peptide-tagging activity

During recent studies of ribonucleolytic “degradosome” complexes of Escherichia coli, we found that degradosomes contain certain RNAs as well as RNase E and other protein components. One of these RNAs is ssrA (for small stable RNA) RNA (also known as tm RNA or 10Sa RNA), which functions as both a tRNA and mRNA to tag the C-terminal ends of truncated proteins with a short peptide and target them for degradation. Here, we show that mature 363-nt ssrA RNA is generated by RNase E cleavage at the CCA-3′ terminus of a 457-nt ssrA RNA precursor and that interference with this cleavage in vivo leads to accumulation of the precursor and blockage of SsrA-mediated proteolysis. These results demonstrate that RNase E is required to produce mature ssrA RNA and for normal ssrA RNA peptide-tagging activity. Our findings indicate that RNase E, an enzyme already known to have a central role in RNA processing and decay in E. coli, also has the previously unsuspected ability to affect protein degradation through its role in maturation of the 3′ end of ssrA RNA.

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.

Glu-tRNAGln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Glutamyl-tRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Posttranscriptional modification of retroviral primers is required for late stages of DNA replication

During reverse transcription of retroviral RNA, synthesis of (−) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5′ long terminal repeat. For (+) strand synthesis using a (−) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3′ terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (−) strand linked to natural modified or synthetic unmodified tRNA3Lys, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA3Lys, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA3Lys. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA3Lys template. Because all retroviral tRNA primers contain the same 1-methyl-A58 modification, our results suggest that 1-methyl-A58 is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A58 may provide a target for HIV chemotherapy is discussed.

Complete mitochondrial genome suggests diapsid affinities of turtles

Despite more than a century of debate, the evolutionary position of turtles (Testudines) relative to other amniotes (reptiles, birds, and mammals) remains uncertain. One of the major impediments to resolving this important evolutionary problem is the highly distinctive and enigmatic morphology of turtles that led to their traditional placement apart from diapsid reptiles as sole descendants of presumably primitive anapsid reptiles. To address this question, the complete (16,787-bp) mitochondrial genome sequence of the African side-necked turtle (Pelomedusa subrufa) was determined. This molecule contains several unusual features: a (TA)n microsatellite in the control region, the absence of an origin of replication for the light strand in the WANCY region of five tRNA genes, an unusually long noncoding region separating the ND5 and ND6 genes, an overlap between ATPase 6 and COIII genes, and the existence of extra nucleotides in ND3 and ND4L putative ORFs. Phylogenetic analyses of the complete mitochondrial genome sequences supported the placement of turtles as the sister group of an alligator and chicken (Archosauria) clade. This result clearly rejects the Haematothermia hypothesis (a sister-group relationship between mammals and birds), as well as rejecting the placement of turtles as the most basal living amniotes. Moreover, evidence from both complete mitochondrial rRNA genes supports a sister-group relationship of turtles to Archosauria to the exclusion of Lepidosauria (tuatara, snakes, and lizards). These results challenge the classic view of turtles as the only survivors of primary anapsid reptiles and imply that turtles might have secondarily lost their skull fenestration.

v-K-ras leads to preferential farnesylation of p21ras in FRTL-5 cells: Multiple interference with the isoprenoid pathway

The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-ras. A dramatic overall reduction of protein prenylation was found in v-K-ras-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [3H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21ras farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-ras oncogene indicated that these variations represent an early and specific marker of active K-ras. Conversely, FRTL-5 cells transformed with Harvey-ras showed a pattern of [3H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-ras oncogene activation also resulted in an overall decrease of [3H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [3H]-MVA and no alteration in [3H]-MVA uptake. The effects of v-K-ras on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [3H]-MVA whereas increasing the [3H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly, v-K-ras expression was found to: (i) down-regulate mevalonate kinase; (ii) induce farnesyl-pyrophosphate synthase expression; and (iii) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-ras oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the ras oncogene product p21ras.

Ribosomes can slide over and beyond “hungry” codons, resuming protein chain elongation many nucleotides downstream

In cells subjected to moderate aminoacyl-tRNA limitation, the peptidyl-tRNA–ribosome complex stalled at the “hungry” codon can slide well beyond it on the messenger RNA and resume translation further downstream. This behavior is proved by unequivocal amino acid sequence data, showing a protein that lacks the bypassed sequence encoded between the hungry codon and specific landing sites. The landing sites are codons cognate to the anticodon of the peptidyl-tRNA. The efficiency of this behavior can be as high as 10–20% but declines with the length of the slide. Interposition of “trap” sites (nonproductive landing sites) in the bypassed region reduces the frequency of successful slides, confirming that the ribosome–peptidyl-tRNA complex passes through the untranslated region of the message. This behavior appears to be quite general: it can occur at the two kinds of hungry codons tested, AUA and AAG; the sliding peptidyl-tRNA can be any of three species tested, phenylalanine, tyrosine, or leucine tRNA; the peptidyl component can be either of two very different peptide sequences; and translation can resume at any of the three codons tested.

Efficient gene tagging in Arabidopsis thaliana using a gene trap approach

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.

The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin β to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin α/β or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

tRNAs and Proteins Are Imported into Mitochondria of Trypanosoma brucei by Two Distinct Mechanisms

Import of tRNA into the mitochondrial matrix of Trypanosoma brucei was reconstituted in vitro. Efficient import required the hydrolysis of externally added ATP and was shown to be a carrier-mediated process depending on proteinaceous receptors on the surface of mitochondria. A partly synthetic tRNATyr as well as a physiological tRNALys were imported along the same pathway. Contrary to import of all matrix-localized proteins, tRNA import does not require a membrane potential. Furthermore, addition of an excess of import-competent tRNA had no effect on import of a mitochondrial matrix protein. In summary, these results show that tRNAs and proteins in T. brucei are imported by fundamentally different mechanisms.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Two Yeast La Motif-containing Proteins Are RNA-binding Proteins that Associate with Polyribosomes

We have characterized two Saccharomyces cerevisiae proteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins. Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts. In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA. We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process. Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic. We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes. Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors. Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.

Dominant negative inhibition by fragments of a monomeric enzyme

Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired. By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway. Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose. We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase. Inhibition is fragment-specific, as not all fragments cause inhibition. An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation. This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive. Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes.

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown.