Half Of tRNA Research Articles

Circularly permuted tRNA genes: their expression and implications for their physiological relevance and development.

A number of genome analyses and searches using programs that focus on the RNA-specific bulge-helix-bulge (BHB) motif have uncovered a wide variety of disrupted tRNA genes. The results of these analyses have shown that genetic information encoding functional RNAs is described in the genome cryptically and is retrieved using various strategies. One such strategy is represented by circularly permuted tRNA genes, in which the sequences encoding the 5′-half and 3′-half of the specific tRNA are separated and inverted on the genome. Biochemical analyses have defined a processing pathway in which the termini of tRNA precursors (pre-tRNAs) are ligated to form a characteristic circular RNA intermediate, which is then cleaved at the acceptor-stem to generate the typical cloverleaf structure with functional termini. The sequences adjacent to the processing site located between the 3′-half and the 5′-half of pre-tRNAs potentially form a BHB motif, which is the dominant recognition site for the tRNA-intron splicing endonuclease, suggesting that circularization of pre-tRNAs depends on the splicing machinery. Some permuted tRNAs contain a BHB-mediated intron in their 5′- or 3′-half, meaning that removal of an intron, as well as swapping of the 5′- and 3′-halves, are required during maturation of their pre-tRNAs. To date, 34 permuted tRNA genes have been identified from six species of unicellular algae and one archaeon. Although their physiological significance and mechanism of development remain unclear, the splicing system of BHB motifs seems to have played a key role in the formation of permuted tRNA genes. In this review, current knowledge of circularly permuted tRNA genes is presented and some unanswered questions regarding these species are discussed.

Mechanism of trans-translation revealed by in vitro studies

tmRNA is a bacterial small RNA having a structure resembling the upper half of tRNA and its 3′ end accepts alanine followed by binding to EF-Tu like tRNA. Instead of lacking a lower half of the cloverleaf structure including the anticodon, tmRNA has a short coding sequence for tag-peptide that serves as a target of cellular proteases. An elaborate coordination of two functions as tRNA and mRNA facilitates an irregular translation termed trans-translation: a single polypeptide is synthesized from two mRNA molecules. It allows resumption of translation stalled on a truncated mRNA, producing a chimeric polypeptide comprising the C-terminally truncated polypeptide derived from truncated mRNA and the C-terminal tag-peptide encoded by tmRNA. Trans-translation promotes recycling of the stalled ribosomes in the cell, and the resulting C-terminally tagged polypeptide is preferentially degraded by cellular proteases. Biochemical studies using in vitro trans-translation systems together with structural studies have unveiled the molecular mechanism of trans-translation, during which the upper and lower halves of tRNA are mimicked by the tRNA-like structure of tmRNA and a tmRNA-specific binding protein called SmpB, respectively. They mimic not only the tRNA structure but also its behavior perhaps at every step of the trans-translation process in the ribosome. Furthermore, the C-terminal tail of SmpB, which is unstructured in solution, occupies the mRNA path in the ribosome to play a crucial role in trans-translation, addressing how tmRNA·SmpB recognizes the ribosome stalled on a truncated mRNA.

Trypanosoma cruzi produces a population of tRNA-derived small RNAs which are recruited to specific cytoplasmic granules and secreted to the extracellular medium

In the last decade a new family of small regulatory RNAs (sRNAs) of 20-30 nt in length, were recognized as key players in the regulation of gene expression at the transcriptional and post-transcriptional levels. The enzymatic machinery associated with the biogenesis and effector functions of these sRNAs has been found in the majority of the organisms studied. The exceptions for this are some species of trypanosomatids including Trypanosoma cruzi. This accounts for the absence of RNA interference phenomena in these parasites. In the present work we have cloned and characterized the small RNA population from T. cruzi, in two of its life cycle forms: epimastigotes and metacyclic stages. Our results showed a highly represented population of small RNAs derived from de cleavage of mature tRNAs representing about 30% in epimastigotes and 40% in trypomastigotes of small RNA fraction which we dubbed mini-tRNAs. Surprisingly, more than 98% of mini-tRNAs derived from the 5' half of tRNA for Asp and Glu and localize to particular granular structures in the cytoplasm of T.cruzi at all stages of its life cycle. These tRNA halves seem to be related with an Argonaute protein distinctive of trypanosomatids which was recently cloned and sequenced in our laboratory. This Argonaute protein was differentially expressed through the life cycle of T. cruzi and its expression was accentuated by starvation. Whereas their biological significance is currently unknown this mini-tRNAs population it has been described in the last year in other organisms as Giardia lamblia, Aspergillus fumigatus and many mammalian cancer cells. These mini-tRNAs have also been found in the supernatant of culture of T. cruzi as forming part of the secretome of these organisms, which led us to speculate about a putative role of these molecules in intercellular communication. This could represent a new family of small RNAs with relevance in gene expression regulation in particular for these unicellular parasites where gene regulation is achieved principally by post-transcriptional mechanisms. The complete knowledge of gene expression regulatory pathways in this kind of parasites that concern human health could be a source of study for development of vaccines or anti-parasitic treatment in base new tools as the RNA vaccines or biotechnological drugs.

Molecular mimicry of tRNA / mRNA during trans‐translation

In trans-translation, transfer-messanger RNA (tmRNA) rescues the stalled ribosome, which is caused by truncated or other problemtic mRNAs. When tmRNA enters the stalled ribosome, there is no codon-anticodon interaction, but with a protein factor SmpB. Here, we studied the binding site of SmpB on the ribosome by directed hydroxyl radical probing. It revealed that there are two SmpB binding sites on a ribosome, the A-site and P-site, suggesting that these binding sites reflect pre- and post-translocation states of SmpB during trans-translation. It is also suggested that the N-terminal region (1–133) and the C-terminal tail (134–160) of SmpB mimic the lower half of tRNA and mRNA, respectively. We also examined the role of the C-terminal tail of SmpB using an in vitro trans-translation system. Our data indicate that the residues of the C-terminal tail of SmpB are involved in the early stage of trans-translation. This work was supported by a grant-in-aid for Young Scientists (Start-up) from Japan Society for the Promotion of Science (JSPS) to D.K. (No. 21880009) and a Research Fellowship of Hirosaki University for D.K.

Interaction of SmpB with ribosome from directed hydroxyl radical probing

To add a tag-peptide for degradation to the nascent polypeptide in a stalled ribosome, an unusual translation called trans-translation is facilitated by transfer-messenger RNA (tmRNA) having an upper half of the tRNA structure and the sequence encoding the tag-peptide except the first alanine. During this event, tmRNA enters the vacant A-site of the stalled ribosome without a codon–anticodon interaction, but with a protein factor SmpB. Here, we studied the sites and modes of binding of SmpB to the ribosome by directed hydroxyl radical probing from Fe(II) tethered to SmpB variants. It revealed two SmpB-binding sites, A-site and P-site, on the ribosome. Each SmpB can be superimposed on the lower half of tRNA behaving in translation. The sites of cleavages from Fe(II) tethered to the C-terminal residues of A-site SmpB are aligned along the mRNA path towards the downstream tunnel, while those of P-site SmpB are found almost exclusively around the region of the codon–anticodon interaction in the P-site. We propose a new model of trans-translation in that the C-terminal tail of SmpB initially recognizes the decoding region and the mRNA path free of mRNA by mimicking mRNA.

Was it an ancient gene codifying for a hairpin RNA that, by means of direct duplication, gave rise to the primitive tRNA molecule?

Some sequences of transfer RNAs (tRNAs) which are believed to possess many of the characteristics of the ancestral sequence of this molecule are constructed. Analysis of these ancestral sequences seems to indicate that: (i) self-alignment of these sequences makes it possible for the 3'-half of the reconstructed molecule to display a significant similarity to the 5'-half, thus indicating that the two halves of tRNA might be paralogous, and (ii) the 5'-half of the ancestral tRNA can reconstruct an almost perfect hairpin structure. These observations give weight to the hypothesis suggesting that the primitive tRNA molecule may have originated through direct duplication of an ancient gene codifying for a hairpin structure.

Evaluation of the Use of Antisense tRNAmetas an Inhibitor for Eukaryotic Protein Synthesis

We attempted to explore the use of antisense RNAs against tRNA as an inhibitor of eukaryotic protein synthesis. For this purpose, antisense RNA against the 5'-end half of the initiator tRNA of wheat germ was synthesized, and its effect on translation of the Brome mosaic virus mRNA was investigated in a wheat germ cell-free system. When the antisense RNA against the 5'-end half of the initiator tRNA including the anticodon sequence was added at the concentration of 8 microM to the cell-free system, protein synthesis was completely inhibited. This inhibitory effect could be suppressed by the addition of wheat germ tRNA. In contrast, sense and control RNA showed slight inhibitory effects, which were not, however, suppressed by wheat germ tRNA. The antisense tRNA formed a double-stranded RNA duplex with the target methionine tRNA in the wheat germ extract which became resistant to ribonuclease treatment. These experiments suggest that antisense tRNA could be utilized for control of tRNA functions and to block protein synthesis.

Catalytic RNA reactions of yeast tRNA(Phe) fragments.

We describe 12 new catalytic RNA reactions which are intermolecular variants of the well-known intramolecular Pb(2+)-promoted hydrolysis of yeast tRNA(Phe). Fragments derived from the native yeast tRNA(Phe) which possess the T stem-loop can function as catalysts for the site-specific hydrolysis at p18 of D stem-loop-containing fragments. An initial report described the catalytic cleavage of an unmodified T7 transcript corresponding to the 5' half of tRNA(Phe) by a 3' half-molecule derived from the native tRNA. [Sampson, J. R., Sullivan, F. X., Behlen, L. S., DiRenzo, A. B., & Uhlenbeck, O. C. (1987) Cold Spring Harbor Symp. Quant. Biol. 52, 267-275]. We have investigated the trans reaction further by creating a family of substrate and catalyst RNA molecules by dissection of the native tRNA(Phe) using a combination of chemical and enzymatic methods. A search for cleavage activity in trans was conducted using a combinatorial approach with the available T and D stem-loop-containing fragments. Twelve combinations were found to be catalytic, and initial rates, kcat's, and Km's are reported for each. The kcat's for the reactions differ by approximately 20-fold, whereas Km's vary by only approximately 2-fold. Differences in some of the cleavage rates argue that tertiary interactions present in the intact molecule can be reconstituted in the fragment combinations. Secondary structural features remote from the cleavage site can also affect the apparent cleavage rates. A minimum catalytic complex consisting of a substrate fragment corresponding to nucleotides 1-24 of the native molecule and a catalytic RNA corresponding to 46-76 is identified. This complex is of interest since the transition state for cleavage involves only three helices, with no elements of the anticodon required for cleavage. This is reminiscent of the proposed secondary structure of the hammerhead catalytic RNA cleavage motif.

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.

The Distribution of Transfer Ribonucleic Acid in Rabbit Reticulocytes: LEVELS OF AMINOACYLATION AND RIBOSOMAL ATTACHMENT DURING HEMOGLOBIN SYNTHESIS

Abstract This study is a consideration of the condition and distribution of tRNA in rabbit reticulocytes. Levels of tRNA aminoacylation by 18 different amino acids were determined by assaying tRNA resistance to periodate oxidation. The levels range from 71% aminoacylation for tRNA accepting glycine to 97% for tRNAs accepting leucine and phenylalanine, with most values being close to 90%. Amounts of tRNA accepting the 18 amino acids which are attached to ribosomes were determined. Yields of ribosomal material from known numbers of reticulocytes are equivalent to 100,000 80 S ribosomal monomers per cell. There are approximately 100,000 tRNA molecules per cell attached to these ribosomes, this being approximately one-quarter of the total reticulocyte tRNA content. The numbers of ribosomal tRNA molecules for the different amino acids vary widely and have no demonstrable relationship to rabbit hemoglobin amino acid composition or to the content of tRNA for these amino acids in whole reticulocytes. More than half of the total reticulocyte tRNA for leucine and phenylalanine is attached to ribosomes while only 10% of serine and alanine tRNA is ribosomal. The results are discussed in relation to the cycling of different kinds of tRNA during hemoglobin synthesis and to the possibility that the availability of certain tRNA species may limit the rate of hemoglobin synthesis in reticulocytes.