Eukaryotic Cytoplasmic tRNAs Research Articles

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System

The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U(34)]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm(5)) or a 5-carbamoylmethyl (ncm(5)) side chain and sometimes an additional 2-thio (s(2)) or 2'-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm(5) and ncm(5) side chains promote decoding of G-ending codons and that concurrent mcm(5) and s(2) groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm(5)U(34)- and some ncm(5)U(34)-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

Identification of yeast tRNA Um44 2′-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNASer species

A characteristic feature of tRNAs is the numerous modifications found throughout their sequences, which are highly conserved and often have important roles. Um(44) is highly conserved among eukaryotic cytoplasmic tRNAs with a long variable loop and unique to tRNA(Ser) in yeast. We show here that the yeast ORF YPL030w (now named TRM44) encodes tRNA(Ser) Um(44) 2'-O-methyltransferase. Trm44 was identified by screening a yeast genomic library of affinity purified proteins for activity and verified by showing that a trm44-delta strain lacks 2'-O-methyltransferase activity and has undetectable levels of Um(44) in its tRNA(Ser) and by showing that Trm44 purified from Escherichia coli 2'-O-methylates U(44) of tRNA(Ser) in vitro. Trm44 is conserved among metazoans and fungi, consistent with the conservation of Um(44) in eukaryotic tRNAs, but surprisingly, Trm44 is not found in plants. Although trm44-delta mutants have no detectable growth defect, TRM44 is required for survival at 33 degrees C in a tan1-delta mutant strain, which lacks ac(4)C12 in tRNA(Ser) and tRNA(Leu). At nonpermissive temperature, a trm44-delta tan1-delta mutant strain has reduced levels of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), but not other tRNA(Ser) or tRNA(Leu) species. The trm44-delta tan1-delta growth defect is suppressed by addition of multiple copies of tRNA(Ser(CGA)) and tRNA(Ser(UGA)), directly implicating these tRNA(Ser) species in this phenotype. The reduction of specific tRNA(Ser) species in a trm44-delta tan1-delta mutant underscores the importance of tRNA modifications in sustaining tRNA levels and further emphasizes that tRNAs undergo quality control.

Sequence and structure requirements for the biosynthesis of pseudouridine (psi 35) in plant pre-tRNA(Tyr).

All eukaryotic cytoplasmic tRNAs(Tyr) contain pseudouridine in the centre of the anticodon (psi 35). Recently, it has been shown that the formation of psi 35 is dependent on the presence of introns in tRNA(Tyr) genes. Furthermore, we have investigated the structural and sequence requirements for the biosynthesis of psi 35. A number of mutant genes were constructed by oligonucleotide-directed mutagenesis of a cloned Arabidopsis tRNA(Tyr) gene. Nucleotide exchanges were produced in the first and third positions of the anticodon and at positions adjacent to the anticodon. Moreover, insertion and deletion mutations were made in the anticodon stem and in the intron. The mutant genes were transcribed in HeLa cell extract and the pre-tRNAs(Tyr) were used for studying psi 35 biosynthesis in HeLa cell and wheat germ extracts. We have made the following observations about the specificity of plant and vertebrate psi 35 syntheses: (i) insertion or deletion of one base pair in the anticodon stem does not influence the efficiency and accuracy of the psi 35 synthase; (ii) the presence of U35 in a stable double-stranded region prevents its modification to psi 35; and (iii) the consensus sequence U33N34U35A36Pu37 in the anticodon loop is an absolute requirement for psi 35 synthesis. Thus, psi 35 synthases recognize both tRNA tertiary structure and specific sequences surrounding the nucleotide to be modified.

Sequences of two bean mitochondria tRNAsTyr which differ in the level of post-transcriptional modification and have a prokaryotic-like large extra-loop

Two bean mitochondrial tRNAs(Tyr) purified by RPC-5 chromatography and two-dimensional gel electrophoresis have been sequenced using post-labeling techniques. These two tRNAs only differ by three post-transcriptional modifications in the D-loop. They have a large variable loop and therefore resemble prokaryotic tRNAs(Tyr) rather than eukaryotic cytoplasmic tRNAs(Tyr).

TRNA alterations in cancer.

1. 3H-, 125I-, and 32P-labeling methods were developed for base composition and sequence analysis of minute amounts of nonradioactive nucleic acids containing modified constituents. 2. Base composition analysis showed tRNA from two "liver-like" minimal deviation hepatomas, Morris hepatomas 5123D and 7777, to exhibit typical alterations when compared with liver tRNA. Our observations, which were made for different transplant generations of the tumors, indicated a trend toward undermethylation and undermodification of tRNA. 3. Sequence analysis of several cytoplasmic and mitochondrial tRNAs from hepatoma 5123D showed partial lack of m2G and complete lack of Gm and Q. 4. Sequence analysis of mitochondrial tRNAs from hepatoma 5123D indicated several instances of alterations of primary structure, a phenomenon not previously observed for cytoplasmic tRNAs from neoplasms. 5. Biochemical mechanisms underlying these alterations, as well as their functional implications, have yet to be investigated. 6. Modification patterns, but not primary structures, of mitochondrial tRNAs have been highly conserved when compared to prokaryotic and eukaryotic cytoplasmic tRNAs. This implies that (a) post-transcriptional modifications must play a crucial role in tRNA function, and (b) alterations of post-transcriptional modifications in tumor tRNAs have to be regarded as highly significant deviations from the norm.