Hildburg Beier

UAG readthrough during TMV RNA translation: isolation and sequence of two tRNAsTyr with suppressor activity from tobacco plants

The hypothetical replicase or replicase subunit cistron in the 5′‐proximal part of tobacco mosaic virus (TMV) RNA yields a major 126‐K protein and a minor 183‐K ‘readthrough’ protein in vivo and in vitro. Two natural suppressor tRNAs were purified from uninfected tobacco plants on the basis of their ability to promote readthrough over the corresponding UAG termination codon in vitro. In a reticulocyte lysate the yield of 183‐K readthrough protein increases from ˜10% in the absence of added tobacco plant tRNA up to ˜35% in the case of pure tRNATyr added. Their amino acid acceptance and anticodon sequence (GψA) identifies the two natural suppressor tRNAs as the two normal major cytoplasmic tyrosine‐specific tRNAs. tRNATyr1 has an A:U pair at the base of the TψC stem and an unmodified G10, whereas tRNATyr2 contains a G:C pair in the corresponding location and m2G in position 10. This is the first case that, in a higher eukaryote, the complete structure is known of both the natural suppressor tRNAs and the corresponding viral RNA on which they exert their function. The corresponding codon‐anticodon interaction, which is not in accordance with the wobble hypothesis, and the possible biological significance of the readthrough phenomenon is discussed.

Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAsGln

The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post‐labeling techniques. Two of these tRNAsGln show unusual codon recognition: a previously isolated tRNAGlnUmUA and a second species with CUA in the anticodon (tRNAGlnCUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNAGlnUmUA reads the UAA codon of α‐globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNAGlnCUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAsGln is their unusually strong readthrough efficiency, e.g. purified tRNAGlnCUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNAGln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNAGlnUmUG and tRNAGlnUmUA is 81%, whereas that between tRNAGlnCUA and tRNAGlnUmUA is 95%, indicating that the two unusual tRNAsGln evolved from the normal tRNAGln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.

The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts

Translation of tobacco mosaic virus (TMV) RNA in tobacco protoplasts yields the 17.5‐K coat protein, a 126‐K protein and a 183‐K protein which is generated by an efficient readthrough over the UAG termination codon at the end of the 126‐K cistron. In wheat germ extracts, however, only the 5′‐proximal 126‐K cistron is translated whereas the 183‐K readthrough protein is not synthesized. Purification and sequence analysis of the endogenous tyrosine tRNAs revealed that the uninfected tobacco plant contains two tRNAsTyr, both with GΨA anticodons which stimulate the UAG readthrough in vitro and presumably in vivo. In contrast, ˜85% of the tRNATyr from wheat germ contains a QΨA anticodon and ˜15% has a GΨA anticodon. Otherwise the sequences of tRNAsTyr from wheat germ and tobacco are identical. UAG readthrough and hence synthesis of the 183‐K protein is only stimulated by tRNATyrGΨA and not at all by tRNATyrQΨA. The tRNAsTyr from wheat leaves were also sequenced. This revealed that adult wheat contains tRNATyrGΨA only. This is very much in contrast to the situation in animals, where Q‐containing tRNAs are characteristic for adult tissues whereas Q deficiency is typical for the neoplastic and embryonic state.

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.

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.

Immune-related proteins induced in the hemolymph after aseptic and septic injury differ in honey bee worker larvae and adults.

We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.

A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways

tRNA splicing enzymes had been identified in mammalian and plant cells long before homologous intron‐containing tRNA genes were detected. The tRNATyr gene presented here is the first intron‐containing, human tRNA gene for which transcription and pre‐tRNA maturation has been studied in a homologous system. This gene is disrupted by a 20‐bp long intron and encodes one of the two major human tRNAsTyr which have been purified and sequenced. A tRNATyr gene recently isolated from Nicotiana also contains an intron and codes for a functional, major cytoplasmic tRNATyr. Both tRNA genes are efficiently transcribed in a HeLa cell nuclear extract. Each of them produces two independent primary transcripts because of two initiation and termination sites, respectively. The maturation of the tRNATyr precursors proceeds along different pathways. The intervening sequence of the human pre‐tRNATyr is excised first, followed by ligation of the tRNA halves and maturation of the flanks, as has been shown for all intron‐containing tRNA genes transcribed in HeLa extract. The order of maturation steps is reversed for the plant pre‐tRNATyr: processing of the flanking sequences precedes intron excision. This maturation pathway corresponds to that observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast.

Expression of variant nuclearArabidopsis tRNASer genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts

SummaryWe have recently identified a tRNA gene cluster in theArabidopsis nuclear genome. One tRNASer (AGA) gene and two tRNATyr (GTA) genes occur in tandem arrangement on a 1.5 kb unit that is amplified about 20-fold at a single chromosomal site. Here we have studied the in vitro expression of seven individually cloned tRNASer genes (pAtS1 to pAtS7) derived from this cluster. Five out of the seven tRNASer genes contain point mutations in the coding region which have in part adverse effects on the expression of these genes in different cell-free systems: (i) C10 and A62 in plant tRNASer genes, which correspond to G10 and C62, respectively, in all known vertebrate tRNA genes, result in a reduced transcription efficiency in HeLa but not in yeast extract. This indicates that yeast RNA polymerase III tolerates nucleotide substitutions at positions 10 [5′ internal control region (ICR)] and 62 (3′ ICR), whereas the vertebrate RNA polymerase III requires a more stringent consensus sequence. (ii) Processing of a pre-tRNASer with a mismatch in the aminoacyl stem is impaired in HeLa, yeast and wheat germ extracts, however, a mismatch in the anticodon stem is deleterious for HeLa and wheat germ but not for yeast processing enzymes. The unexpectedly high number of potential tRNASer pseudogenes in the cluster — quite in contrast to the tRNATyr genes which mainly code for functional tRNAs — suggested that tRNASer (AGA) genes also occur elsewhere in the genome. We present evidence that single copies of tRNASer (AGA) genes do indeed exist outside the tRNA gene cluster.

Structure–function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog

Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains—an N-terminal ligase component and a C-terminal 5′-kinase/2′,3′-cyclic phosphodiesterase (CPD) component—that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure–function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2′-PO4 end for tRNA splicing in vivo.

The expression of the TMV-specific 30-kDa protein in tobacco protoplasts is strongly and selectively enhanced by actinomycin

The TMV-encoded 30-kDa protein has been implicated in the cell-to-cell transport of TMV in the infected plant. The polyethylene glycol-mediated inoculation of tobacco protoplasts with TMV particles and TMV RNA was used to compare the time courses of the viral 30-kDa protein synthesis in vivo. Upon infection of protoplasts with TMV RNA, the synthesis of the viral 30-kDa protein starts after 4 to 6 hr, has its maximum after 8 to 10 hr, and decreases. After inoculation of protoplasts with TMV, however, the start of the viral 30-kDa protein synthesis and its maximum are delayed by 2 hr, followed by the same decrease. We show that actinomycin D dramatically stimulates the synthesis of the 30-kDa protein by up to 2 orders of magnitude, whereas the synthesis of the viral 126 kDa, the 183 kDa, and the coat protein is increased only by a factor of 2. Surprisingly, actinomycin V is twice as active as actinomycin D, whereas actinomycin I is nearly inactive. The specific stimulation of the 30-kDa synthesis by actinomycin D in vivo depends neither on the Nicotiana variety nor on the TMV strain used. Final evidence that the 30-kDa protein is truly TMV-derived is provided by the slightly different electrophoretic mobilities of the 30-kDa proteins encoded by TMV strains vulgare, dahlemense, and U2. The identification of the 30-kDa protein in two-dimensional gels was achieved for the first time by a combination of ionic and nonionic detergents for the solubilization of the 30-kDa protein and by the specific stimulation of its synthesis by actinomycin D. The mechanism of the strong and selective actinomycin effect on the viral 30-kDa protein synthesis in vivo is as yet obscure. Actinomycin does not appear to act directly on viral protein biosynthesis, since it neither stimulates the 30-kDa synthesis upon translation of TMV RNA in vitro nor alters the ratio of the products. Actinomycin may rather act by inhibiting selectively the synthesis of a host factor whose synthesis starts at least 4 hr after TMV infection and which strongly inhibits the expression of the viral 30-kDa transport protein.