Spider silkglands contain a tissue-specific alanine tRNA that accumulates in vitro in response to the stimulus for silk protein synthesis

The large ampullate glands of the orb-web spider, Nephila clavipes provide massive amounts of fibroin throughout the lifetime of the adult female. We have developed methods to culture the glands and manipulate their biosynthetic activity. This has allowed us to monitor a series of molecular events that precede silk production in glands excised from appropriately stimulated animals. In this paper, we demonstrate that prior to the transient dramatic production of fibroin, such glands accumulate large amounts of tRNAs cognate to the abundant amino acids in spider silk. One of these, alanine tRNA, appears to consist of two isoaccepting forms--one constitutive, and the other silkgland specific. Moreover, the silkgland-specific form appears to accumulate preferentially in response to stimulation. This phenomenon of tissue-specific tRNA production appears similar to that found in the silkglands of Bombyx mori, but the spider system has the unique property of permitting manipulation in vitro. Thus, it provides an unusual opportunity to study the mechanism of regulated tRNA synthesis.     

Tissue-specific differences in human transfer RNA expression

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms.     

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

A nucleotide that enhances the charging of RNA minihelix sequence variants with alanine.

We showed earlier that a single G3.U70 base pair within the amino acid acceptor helix is a major determinant of the identity of tRNA(Ala). In addition, we demonstrated that an RNA hairpin minihelix that recreates the 12 base pair acceptor-T psi C stem of tRNA(Ala) is also aminoacylated in a G3.U70-dependent manner. Determinants for efficient aminoacylation at pH 7.5 have been further investigated with minihelix substrates that have sequence variations at 3.70 and other locations. Although a U,U mismatch and other 3.70 nucleotide alternatives to G.U were recently proposed by others as also important for alanine acceptance, neither that mismatch nor any of four other 3.70 nucleotide combinations confer aminoacylation in vitro with alanine, even with substrate levels of enzyme. In contrast, permutations of the so-called discriminator nucleotide N73 (at position 73) strongly modulate, but do not block, aminoacylation of those substrates that encode G3.U70. In particular, the efficiency of G3.U70-dependent aminoacylation with alanine is strongly enhanced by having the wild-type A73. The effect of N73 alone can explain most of the difference in aminoacylation efficiency of a G3.U70-containing tRNA and a minihelix substrate whose sequences vary significantly from their tRNA(Ala) counterparts. Comparison with earlier work suggests that the substantial modulating effect of N73 is partly or completely obscured when N73 tRNA variants are expressed as amber suppressors in vivo.     

Specific function of a G.U wobble pair from an adjacent helical site in tRNA(Ala) during recognition by alanyl-tRNA synthetase.

G.U wobble pairs are crucial to many examples of RNA-protein recognition. We previously concluded that the G.U wobble pair in the acceptor helix of Escherichia coli alanine tRNA (tRNA(Ala)) is recognized indirectly by alanyl-tRNA synthetase (AlaRS), although direct recognition may play some role. Our conclusion was based on the finding that amber suppressor tRNA Ala with G.U shifted to an adjacent helical site retained substantial but incomplete Ala acceptor function in vivo. Other researchers concluded that only direct recognition is operative. We report here a repeat of our original experiment using tRNA(Lys) instead of tRNA(Ala). We find, as in the original experiment, that a shifted G.U confers Ala acceptor activity. Moreover, the modified tRNA(Lys) was specific for Ala, corroborating our original conclusion and making it more compelling.     

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.     

A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)

About half of the mammalian genome is composed of retroposons. Long interspersed elements (LINEs) and short interspersed elements (SINEs) are the most abundant repetitive elements and account for about 21% and 13% of the human genome, respectively. SINEs have been detected in all major mammalian lineages, except for the South American order Xenarthra, also termed Edentata (armadillos, anteaters, and sloths). Investigating this order, we discovered a novel high-copy-number family of tRNA derived SINEs in the nine-banded armadillo Dasypus novemcinctus, a species that successfully crossed the Central American land bridge to North America in the Pliocene. A specific computer algorithm was developed, and we detected and extracted 687 specific SINEs from databases. Termed DAS-SINEs, we further divided them into six distinct subfamilies. We extracted tRNA(Ala)-derived monomers, two types of dimers, and three subfamilies of chimeric fusion products of a tRNA(Ala) domain and an approximately 180-nt sequence of thus far unidentified origin. Comparisons of secondary structures of the DAS-SINEs' tRNA domains suggest selective pressure to maintain a tRNA-like D-arm structure in the respective founder RNAs, as shown by compensatory mutations. By analysis of subfamily-specific genetic variability, comparison of the proportion of direct repeats, and analysis of self-integrations as well as key events of dimerization and deletions or insertions, we were able to delineate the evolutionary history of the DAS-SINE subfamilies.     

Functional Analysis of Human tRNA Isodecoders

tRNA isodecoders share the same anticodon but have differences in their body sequence. An unexpected result from genome sequencing projects is the identification of a large number of tRNA isodecoder genes in mammalian genomes. In the reference human genome, more than 270 isodecoder genes are present among the approximately 450 tRNA genes distributed among 49 isoacceptor families. Whether sequence diversity among isodecoder tRNA genes reflects functional variability is an open question. To address this, we developed a method to quantify the efficiency of tRNA isodecoders in stop-codon suppression in human cell lines. First, a green fluorescent protein (GFP) gene that contains a single UAG stop codon at two distinct locations is introduced. GFP is only produced when a tRNA suppressor containing CUA anticodon is co-transfected with the GFP gene. The suppression efficiency is examined for 31 tRNA isodecoders (all contain CUA anticodon), 21 derived from four isoacceptor families of tRNA^Ser genes, 7 from five families of tRNA^Leu genes, and 3 from three families of tRNA^Ala genes. We found that isodecoder tRNAs display a large difference in their suppression efficiency. Among those with above background suppression activity, differences of up to 20-fold were observed. We were able to tune tRNA suppression efficiency by subtly adjusting the tRNA sequence and inter-convert poor suppressors into potent ones. We also demonstrate that isodecoder tRNAs with varying suppression efficiencies have similar stability and exhibit similar levels of aminoacylation in vivo. Our results indicate that naturally occurring tRNA isodecoders can have large functional variations and suggest that some tRNA isodecoders may perform a function distinct from translation.     

Transfer RNA genes of Euglena gracilis chloroplast DNA

Summary Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2–6 genes. None of the known genes contains introns, nor codes for the 3-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.     

Translocation within the acceptor helix of a major tRNA identity determinant

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl-tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific-a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNA(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.     

Genes encoding the 7S RNA and tRNA(Ser) are linked to one of the two rRNA operons in the genome of the extremely thermophilic archaebacterium Methanothermus fervidus

Analysis of gene structure in the extremely thermophilic archaebacterium, Methanothermus fervidus, has revealed the presence of a cluster of stable RNA-encoding genes arranged 5'-7S RNA-tRNA(Ser)-16S rRNA-tRNA(Ala)-23S rRNA-5S rRNA. The genome of M. fervidus contains two rRNA operons but only one operon has the closely linked 7S RNA-encoding gene. The sequences upstream from the two rRNA operons are identical for 206 bp but diverge at the 3' base of the tRNA(Ser) gene. The secondary structures predicted for the M. fervidus 7S, 16S rRNA, tRNA(Ala) and tRNA(Ser) have been compared with those of functionally homologous molecules from moderately thermophilic and mesophilic archaebacteria. A consensus secondary structure for archaebacterial 7S RNAs has been developed which incorporates bases and structural features also conserved in eukaryotic signal-recognition-particle RNAs and eubacterial 4.5S RNAs.