- Email: [email protected]
Biological evidence for the world's smallest tRNAs
Biochimie 100 (2014) 151e158
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Biological evidence for the world’s smallest tRNAs Sandra Wende a, Edward G. Platzer b, Frank Jühling d, Joern Pütz c, Catherine Florentz c, Peter F. Stadler d, e, f, g, h, i, Mario Mörl a, * a
University of Leipzig, Institute for Biochemistry, Leipzig, Germany University of California, Riverside, Department of Nematology, Riverside, CA 92521, USA c Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg, France d University of Leipzig, Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig, Germany e Max Planck Institute for Mathematics in the Sciences, Leipzig, Germany f Fraunhofer Institut für Zelltherapie und Immunologie e IZI, Leipzig, Germany g Department of Theoretical Chemistry, University of Vienna, Vienna, Austria h Center for Non-coding RNA in Technology and Health, University of Copenhagen, Frederiksberg C, Denmark i Santa Fe Institute, Santa Fe, NM, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2013 Accepted 24 July 2013 Available online 17 August 2013
Due to their function as adapters in translation, tRNA molecules share a common structural organization in all kingdoms and organelles with ribosomal protein biosynthesis. A typical tRNA has a cloverleaf-like secondary structure, consisting of acceptor stem, D-arm, anticodon arm, a variable region, and T-arm, with an average length of 73 nucleotides. In several mitochondrial genomes, however, tRNA genes encode transcripts that show a considerable deviation of this standard, having reduced D- or T-arms or even completely lack one of these elements, resulting in tRNAs as small as 66 nts. An extreme case of such truncations is found in the mitochondria of Enoplea. Here, several tRNA genes are annotated that lack both the D- and the T-arm, suggesting even shorter transcripts with a length of only 42 nts. However, direct evidence for these exceptional tRNAs, which were predicted by purely computational means, has been lacking so far. Here, we demonstrate that several of these miniaturized armless tRNAs consisting only of acceptor- and anticodon-arms are indeed transcribed and correctly processed by nonencoded CCA addition in the mermithid Romanomermis culicivorax. This is the first direct evidence for the existence and functionality of the smallest tRNAs ever identified so far. It opens new possibilities towards exploration/assessment of minimal structural motifs defining a functional tRNA and their evolution. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Enoplea Mitochondria Armless tRNA Processing tRNA structure CCA addition
1. Introduction To allow an efficient protein biosynthesis in vertebrate mitochondria, the circular genome of these organelles encodes a minimal set of structural RNAs essential for translation. Besides genes for the two ribosomal RNAs, typical animal mitogenomes contain 22 tRNA genes that are required and sufficient for translation in the organelle [1]. In comparison to the nuclear encoded translational machinery, the mitochondrial system is functional not only with such a reduced set of individual tRNAs for 20 different amino acids, but also with smaller tRNA transcripts that have simplified structural properties or lack specific features. Posttranscriptional editing of such tRNA sequences is a frequently observed strategy to obtain a complete and functional set of mitochondrial tRNAs. In these cases,
* Corresponding author. Tel.: þ49 (0) 341 9736 911; fax: þ49 (0) 341 9736 919. E-mail address: [email protected] (M. Mörl). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.07.034
50 - or 30 -terminally truncated tRNA transcripts, resulting from overlapping gene organization, are completed by the addition of the missing residues, ranging from single positions to stretches of several nucleotides [2e4]. Other editing events alter the identity of individual bases by deamination reactions, changing the amino acid identity of the complete tRNA [5]. Due to these facts, the mitochondrially encoded tRNAs frequently show strong deviations from their cytosolic counterparts and their genes are sometimes hard to identify so that specific tRNA search algorithms were developed [6]. In marsupial mitochondria, this is further complicated by processing place holders that are required to ensure a proper release of the neighboring mRNA and rRNAs out of the huge primary transcript that is synthesized. A degenerate tRNA for lysine has lost its original tRNA function but still acts as a processing signal [7]. The most extreme structural deviation and miniaturization of tRNAs, however, is found in the mitochondrial genome of nematodes like Caenorhabditis elegans or Ascaris suum, where either the D- or the T-arm
152
S. Wende et al. / Biochimie 100 (2014) 151e158
is replaced by a simple small loop consisting of five to eight nucleotides [8,9]. Recently, in the genome of nematode and mite mitochondria, the first genes for even smaller tRNA transcripts have been described. In the mite Dermatophagoides farina, the mitochondrial genome harbors genes for tRNAs lacking the D-arm and having only a small and unstable T-arm [10]. In addition, in the nematode group of Enoplea, containing the orders Mermithidia (arthropod parasites), Dorylaimida (plant parasites), Trichocephalida (vertebrate parasites), mitochondrial tRNA gene sets with predicted transcripts lacking both D- or T-domains were detected, leading to further reduced tRNA structures [6]. As an example, the mermithid Romanomermis culicivorax carries two mitochondrial tRNA genes where the D-arm is missing, 11 with a deletion of the T-arm, and nine with a deletion of both arms. These armless tRNAs were inferred from the genomic sequence by purely computational means. Nevertheless, there are several arguments why these transcripts very likely represent functional molecules. First, the gene sequence is highly conserved between the different Enoplea species. Second, the observed base replacements are compensatory and maintain the proposed secondary structure of the transcripts. Third, the observed genome arrangements do not disturb the integrity of the individual tRNA gene sequences [6]. It remains however unclear to what extent they are functional. Here we provide direct experimental evidence for the existence of the corresponding processed transcripts. Intriguingly, these tRNAs carry the highly conserved nucleotide triplet CCA at the 30 -terminus, which is an absolute prerequisite for becoming charged by the cognate aminoacyl tRNA synthetases [11]. As these CCA sequences are not encoded genomically but added in a posttranscriptional reaction by the CCA-adding enzyme, their presence is compelling evidence that these transcripts indeed represent functional tRNAs. 2. Materials and methods 2.1. Rearing and cultivation of R. culicivorax The isogenic female strain of R. culicivorax 3B4 [12] was reared in mass cultures using the procedures of Stirling & Platzer [13]. Autogenous Culex pipiens was employed as the host and the mosquito larvae were fed a mixture of finely ground laboratory rodent diet (LabDiet 5001, meal form; 2 parts) and brewer’s yeast (MP Biochemicals 903312; 1 part). Post-parasitic nematodes were collected in emergence chambers [14] and washed by sedimentation with deionized water and kept at 80 C until use. 2.2. Preparation of RNA Total RNA was isolated from 100 mg R. culicivorax 3B4 cells according to Chomczynski et al. [15]. Worms were homogenized using a 1.4 mm precellys ceramic matrix (Peqlab) for 2 40 s at 6 m/s in a FastPrep-24 homogenizer (MP). Isolated total RNA was DNase I (NEB) digested (2 units) and rRNA was depleted using Ribo-ZeroÔ magnetic gold kit (human/mouse/rat) (Epicentre) according to the manufacturers. 2.3. cDNA synthesis Using T4 RNA ligase, isolated total RNA was fused to a tagging DNA oligonucleotide that carried a ribonucleotide at the phosphorylated 50 -end and a 30 -end blocked by a 30 -30 - linked T [16]. cDNA was synthesized by hybridizing DNA primer P1 to the tagging oligonucleotide and subsequent primer elongation with RevertAid reverse transcriptase according to the manufacturer (Fermentas). Subsequently, the enzyme was inactivated by heat (70 C, 10 min).
2.4. tRNA 30 -end sequence determination cDNA of individual mitochondrial tRNAs was amplified in a 50 ml PCR reaction consisting of 35 cycles with 1 min. 95 C, 1 min. 39 C, 1 min. 60 C. Due to the extreme AT-content of the sequences, such a reduced temperature profile was required for efficient amplification. For polymerization, 1 U Phusion High-Fidelity DNA polymerase (ThermoScientific) was incubated in the presence of the RT primer and an upstream primer specific for the 50 -part of an individual tRNA. PCR products were cloned using the Clone Jet Cloning Kit (Fermentas Molecular Biology Tools; Thermo Scientific) and used for transformation of E. coli. Individual cDNA clones were amplified by colony PCR and sequenced on an ABI Prism 3700 automated sequencer (Amersham Pharmacia Biotech, Freiburg, Germany). Obtained sequences were analyzed using the software SeqMan. 2.5. tRNA 50 -end sequence determination The cDNA preparation described above was used in a tailing reaction with terminal deoxynucleotidyl-transferase and dCTP according to the manufacturer (Invitrogen). The reaction product was amplified by PCR as described above, using a tail-specific primer and a second oligonucleotide annealing to the 30 -part of the individual tRNA. PCR products were cloned and sequenced as described above. 2.6. Oligonucleotides used as primers Tagging oligonucleotide: 50 -pUGG ATC GCG TAG CTC ATA CGA GT(inverse)T-30 . RT primer: 50 -AAC TCG TAT GAG CTA CGC GAT C-30 . C-tail primer (according to invitrogen): 50 -GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-30 . Upstream amplification primer: 50 -GGC CAC GCG TCG ACT AGT AC-30 . tRNAArg primers: 30 -end analysis: 50 -AAA CTT TTA GCA GGA TTT CG-30 . 50 -end analysis: 50 -AAA ACT TAT ATA AAT TAG GAT TC-30 . tRNACys primers: 30 -end analysis: 50 -TAA AGA AGA AAC TTC ATT TG-30 . 50 -end analysis: 50 -CAA AAG AGA TTT TTT TTC ATT TTG-30 . tRNAHis primers: 30 -end analysis: 50 -TAA TAA ATT AAA TAT AAA TTG TG-30 . 50 -end analysis: 50 -ATA ATA AAA TTT TTT ATA AAT TCA C-30 . tRNAIle primers: 30 -end analysis: 50 -TCT TAA TAG AGA ACA ATT TAA ATT G-30 . 50 -end analysis: 50 -ATC TTA AAT TTT AAT TTA AAT TAT C-30 . tRNAPhe primers: 30 -end analysis: 50 -GCT TAC AAA AAA GAT TAA ATTe30 . 50 -end analysis: 50 -ATG CTT ACT TAT TAA TTA AAT TTT C-30 . tRNAThr primers: 30 -end analysis: 50 -TTC CTG CTG ACT ATG TTT TAT TTT G-30 .
S. Wende et al. / Biochimie 100 (2014) 151e158
50 -end analysis: 50 -TTC CTT AGT AAT TTT ATT TAC-30 . 2.7. tRNA structure prediction tRNA secondary structures were predicted using the RNAfold program of the Vienna RNA package [17], output structures were manually curated. Structure presentations were done using VARNA [18]. 3D structure predictions were calculated using RNA Composer [19]. 3. Results Due to several peculiarities of mitochondrial tRNAs in metazoans, including structural deviations and editing, the identification of unusual tRNA genes in Enoplea is not a direct proof for the existence and the functionality of the corresponding transcripts. To investigate whether such unexpected armless tRNAs are indeed expressed in these nematodes, total RNA was isolated from R. culicivorax material and depleted of ribosomal RNA. This total RNA preparation was 30 -terminally ligated to a DNA oligonucleotide
153
and transcribed into cDNA using a primer complementary to the fused oligonucleotide. The resulting cDNA was subjected to 30 -Ctailing and subsequently used as template for the specific amplification of 50 - and 30 -ends of the mitochondrial armless tRNAs for Arg, Cys, Ile, His, Thr, and Phe (Fig. 1A). A tailing reaction with A or T residues was avoided, as the tRNA sequences are extremely AU-rich and would easily lead to frequent mispriming in the subsequent PCR reaction. Due to the high AU content of these transcripts, the PCR elongation step had to be carried out at a reduced temperature of 60 C in order to ensure stable primer binding and efficient polymerization. An example of the resulting amplification products is shown for 50 - and 30 -parts of tRNAIle in Fig. 1B. The PCR products were cloned and individual clones were analyzed by sequencing, yielding between six and 29 sequences for the individual tRNAs. The analysis of the 50 -ends of the tRNAs was successful for three candidates, tRNAArg, tRNAIle and tRNAThr (Fig. 2A, B; Supplementary Table). A possible reason for this inefficient 50 -end amplification might be the considerable difference in the annealing temperatures of the C-tail primer and the tRNA-specific AT-rich oligonucleotide. In addition, most of the sequences show non-encoded heterogeneous extra nucleotides between the G-tail and the 50 -end of the
Fig. 1. Analysis of tRNA 50 - and 30 -ends. A. Strategy: The RNA preparation (black) was ligated to the tagging oligonucleotide (gray). Using the complementary RT primer, cDNA was synthesized. For 50 -RACE, the cDNA was tailed with C residues (gray). Individual tRNA sequences were amplified using a C-tail primer in combination with a primer specific for the 30 -part of the individual tRNA. For 30 -RACE, the PCR was performed with RT primer and a primer specific for the 50 -part of the tRNA. B. The amplification products for tRNAIle are shown as an example. Left: The 50 -part resulted in a PCR product of about 90 bp, depending on the C-tailing and annealing position of the C-tail primer. Right: For the 30 -part of the tRNA, a PCR product of about 70e80 bp could be detected, corresponding to the expected size of 76 bp.
154
S. Wende et al. / Biochimie 100 (2014) 151e158
Fig. 2. Mini tRNAs. A. As an example, the 50 - and 30 -sequences of tRNAIle are shown. Primer binding sites are indicated by black arrows. The predicted sequences are indicated in black characters, identified sequences in color. Lower case characters represent deviations (missing bases) in the predictions, indicated by asterisks. Due to the extreme AU content of the tRNA sequence, both tRNA specific primers ended at the same G position in the tRNA to ensure proper hybridization of the primer 30 -ends. Hence, this individual position is the only one that was not sequenced. B. Presentation of validated tRNA sequences. Black: predicted sequences. Green: identified sequences, gray: missing 50 -parts. Red: deviations from prediction, leading to truncated 50 - and 30 -end positions, or representing CCA-additions that indicate the functionality of these transcripts. Putative acceptor- and anticodonstem pairings are indicated in pink and blue.
tRNA sequence which are probably the result of cDNA synthesis artifact (see discussion). Nevertheless, the 50 -termini of these three tRNAs are a convincing evidence for the correct expression and processing of the transcripts. While tRNAArg and tRNAThr have a 50 end as predicted, tRNAIle is truncated for one position. Hence, this tRNA seems to be even smaller than expected. In contrast to the 50 end determination, the 30 -end analysis was successful for all tRNA candidates. Here, all of the investigated tRNA sequences carried a non-encoded posttranscriptionally added CCA triplet at the 30 -terminus (Fig. 2B) While most of the clones carried a complete version of the triplet, several single sequences also showed partial CCA addition, ending with one or two C-residues. Hence, these products obviously represent either reaction intermediates of the CCA addition process or indicate some degradation during preparation, as it is probably also the case in two 50 -end sequences of tRNAIle,
where 2 nucleotides are missing (Supplementary Table). In addition, in the tRNAs for Ile, Cys, His, and Phe, the actual 30 -end of the encoded tRNA differs from the predicted one, as the expected discriminator position is not present in these transcripts. Instead, the CCA-end is attached to the position upstream of the predicted discriminator nucleotide, indicating that the actual tRNA is at least one nucleotide shorter than predicted originally. In the case of tRNAIle, this truncation corresponds to the situation at the 50 -terminus and generates an acceptor stem with the conventional single-stranded discriminator position upstream of the CCA-end. It is highly likely that similar truncations exist at the 50 -termini of the other identified tRNAs with shorter 30 -ends. As these transcripts show strong deviations of a standard tRNA secondary structure, usual tRNA structure prediction algorithms like tRNAScanSE [20] fail to recognize and fold these sequences,
S. Wende et al. / Biochimie 100 (2014) 151e158
even in a mode optimized for nematode mitochondria. Hence, several of the sequences were analyzed using RNAfold from the Vienna RNA package [17] and the predicted structures were subsequently curated by hand and illustrated using the VARNA tool [18]. For the fully sequenced tRNAArg and tRNAIle, structures with acceptor- and anticodon-like stem elements can be formed (Fig. 3), although the numbers of base pairs in these stems differ from the standard. D- and T-arms, however, are completely missing and are replaced by bulges between 5 and 9 nucleotides. Yet, in the online RNA Composer program [19], these secondary structures can be folded into small L-shaped tertiary structures that show a considerable similarity to conventional tRNA shapes. 4. Discussion The first complete sequence of a tRNA was identified in 1965 and led immediately to the suggestion of several alternative secondary structures [21]. Among these predictions, a cloverleaf-like form of this transcript was proposed which later turned out to be correct. This structure represents the basis for the threedimensional organization of tRNA, resulting in an L-shaped arrangement, where acceptor- and T-stem as well as anticodonand D-stem are stacked to form elongated helices [22,23]. This arrangement turned out to represent the canonical threedimensional structure of tRNAs in all kingdoms as demonstrated by a large number of crystallographic data (reviewed in Giegé et al. [24],), with a conserved distance of about 70 A between the anticodon and the CCA terminus, carrying the amino acid. Surprisingly, metazoan mitochondrial tRNAs show considerable deviations from the cloverleaf secondary structure, with size reductions in loop as well as stem elements, with the most extreme case of lacking the D- or T-stem. Yet, these tRNAs with a size down to 59 nucleotides (human mt tRNASer) still can fold into the canonical L shape with conserved dimensions [24]. Hence, it is very surprising that in several recently analyzed nematode and mite species, even smaller mitochondrial tRNAs were predicted [10,25]. While these annotations are predictions based on DNA sequences, the results presented here are the first experimental proof for the actual existence of these minimalized tRNAs and confirm the correctness of the prediction algorithms. The additional nucleotides located between the 50 -end and the Ctail added during the 50 -RACE analysis do not represent posttranscriptional editing events but are very likely the result of a preparation artifact. First, the added nucleotides differ from clone to clone and are not identical within the sequence analysis of an individual tRNA. For a functional editing reaction, a specific tRNA would have identical nucleotides added. Second, the most likely explanation for the origin of these extra nucleotides is that a remaining excess of dNTPs from the cDNA synthesis was not efficiently removed before the C-tailing reaction was started, resulting in the incorporation of these nucleotides by terminal nucleotidyl transferase. Correspondingly, the extensive use of size exclusion chromatography strongly reduced the amount of the additional nucleotides. Hence, the tRNA 50 -ends are very likely to start at the indicated positions, without carrying extra nucleotides. Nevertheless, in the identified tRNAs for Ile, Cys, His and Phe, the exact 50 - and/or 30 -ends differ for one position compared to the predictions, resulting in even shorter transcripts (Fig. 2B). This correlated recession of the 50 - and 30 -termini restores the typical acceptor stem end in tRNAs, where the 30 -terminal residue (position 73 according to the standard numbering [26]) is unpaired and represents in many transcripts an essential identity element for aminoacylation, referred to as discriminator position [27]. A special case is tRNAHis, where this position 73 (usually a C or an A residue) is basepaired with a G residue at position 1 that e in the case of animal
155
mitochondria e is posttranscriptionally added, leading to an additional G-1/C73 or G-1/A73 base pair [25,28,29]. As it was not possible to identify the 50 -end of the R. culicivorax mitochondrial tRNAHis, the exact situation in this tRNA remains unclear. Nevertheless, the identified 30 -end is one nucleotide shorter than predicted (Fig. 2B). Hence, if the actual 50 -end corresponds to the proposed one, the discriminator position A73 would be base-paired with a U residue at the 50 -end. In other tRNAHis transcripts, the G residue base-paired with the discriminator represents a major identity element for the corresponding synthetase [30e33]. A deviation of this conserved feature was recently identified in the cytosolic tRNAHis of Acanthamoeba castellanii, where the discriminator position remains unpaired [34]. It is possible that in R. culicivorax, a similar situation exists in the corresponding mitochondrial tRNA. The presented data indicate that these armless tRNAs are indeed correctly processed by mitochondrial RNase P and tRNase Z activities and released from the mitochondrial precursor transcripts. In addition, and most significantly, the obtained sequences show that the released tRNAs represent bona-fide substrates for the CCAadding enzyme (Fig. 4). Most of the processing reactions might be carried out by specialized mitochondrial enzyme versions. For the CCA-adding enzyme, however, eukaryotic genomes carry only a single gene, encoding the nuclear/cytosolic as well as the mitochondrial form of this activity [35e37]. Being responsible for CCA incorporation in both cytosolic and mitochondrial sets of tRNAs, this enzyme must have undergone a specific adaptation in order to recognize such a diverse set of tRNA substrates. The fact that all analyzed transcripts carry a complete or (in some rare cases) partial CCA-end at the 30 -termini is a strong evidence that the transcripts do not only represent reduced processing signals for precursor RNA maturation in order to release the adjacent mRNAs according to the mitochondrial punctuation model [38]. Instead, these transcripts seem to be functional tRNAs that are completed by the essential site for aminoacylation, although tRNA charging per se and a direct involvement of the tRNAs in mitochondrial protein synthesis could not be analyzed due to limited material. In agreement with this, the armless tRNAs have the ability to fold into three-dimensional structures that resemble the canonical L-shape as anticipated from folding programs, although the distance between anticodon and aminoacylation site differs strongly from the standard (w63 A for tRNAArg, w46 A for tRNAIle). Such a structure might be further optimized and stabilized by the introduction of modifications, as it was shown to be a requirement for the proper folding of the human mitochondrial tRNALys that adopts an L shape only if an adenosine residue at position 9 is modified to m1A [39]. For the armless tRNAs with such reduced dimensions, two solutions are conceivable. Either the tRNA structure is so flexible that it can extend the distance between the two functional ends in order to be recognized by synthetases and ribosomes, or the mitochondrial translational machinery of R. culicivorax (and of other Enoplea) has a size very different of what is known so far. The sequence data also rule out the possibility that the structural deviations of these minimal transcripts are corrected by massive insertional editing events as they are described for mitochondrial transcripts in trypanosomes [40]. Obviously, the corresponding mitochondrial maturation, aminoacylation and translational machinery co-evolved with the minimization and is adapted to recognize such a peculiar set of tRNAs even in the absence of otherwise highly conserved structural features. Interaction with the synthetase may not be hampered by the size of the tRNA. Indeed, it is well established that synthetases from various origins are capable of aminoacylating much shorter RNAs, such as mini- or micro-helices mimicking the aminoacyl-accepting arm, or only part of it [41]. These findings indicate that the synthetase does
156
S. Wende et al. / Biochimie 100 (2014) 151e158
Fig. 3. Structure predictions for tRNAArg and tRNAIle. In the secondary structure models, the truncations of D- and T-arms are visible, leading to replacement bulges. The anticodon is indicated in red, the CCA-end in green. In the overall structure, these transcripts do not resemble the conventional tRNA cloverleaf that is represented by the human mitochondrial tRNAArg. The predicted three-dimensional structures, however, resemble the standard L-shape of canonical tRNAs, even if acceptor- and anticodon stems show considerable deviations in distance. Although the dimensions of these L-shapes differ from the ones of standard tRNAs, it seem very likely that these transcripts represent functional tRNAs in Romanomermis mitochondria.
S. Wende et al. / Biochimie 100 (2014) 151e158
Fig. 4. tRNA processing reactions. For both conventional as well as bizarre minimized tRNAs, the same set of maturation steps, including aminoacylations, is required. The reactions that were identified in Romanomermis mitochondria are ticked off in green, while the missing reactions are indicated by the red question marks.
not need to interact with both the anticodon and the accepting CCA-end. Additional examples for aminoacylation of unusually structured tRNA substrates include mimics formed by an anticodon hairpin linked to a CCA-end by a single stranded RNA stretch [42]. In mitochondrial ribosomes, a dramatic size reduction in rRNA components was described, leading to ribosomes that obviously have lost the tRNA exit site [43]. Furthermore, the decrease in rRNA size seems to be compensated by an increase in the number of ribosomal proteins, leading to an RNA content of 25e30% in mammalian mitochondria compared to 60e70% in bacteria [44]. In C. elegans, this trend is continued, where the mitochondrial ribosomes have an even higher protein composition [45]. However, as the nematode mitochondrial ribosomes have a size larger than the bacterial counterparts [45], a size reduction compensating the tRNA miniaturization is excluded. It is more likely that specific features of ribosomal RNAs or proteins evolved in order to tolerate such reduced tRNAs as translational adapters. One such co-evolution might concern the A-site finger element that in cytosolic ribosomes interacts with D- and T-arms of an incoming tRNA. In mitochondrial ribosomes, this element is missing so that no sensing for D- and T-loop structures occurs [46]. Despite such likely adaptations in the mitochondrial ribosomes accepting armless tRNAs, it is possible that the efficiency of translation is reduced in comparison to protein synthesis with canonical tRNAs, as it was shown that the bovine mt tRNASerGCU, lacking the D-arm, leads to much lower protein synthesis [47]. In other components of the mitochondrial translation machinery, adaptations to recognize non-canonical tRNA structures were already identified. For the conventional interaction of EF-Tu, recognition of the T-arm of tRNAs is required. In C. elegans, this is not possible for most of the 20 mitochondrial tRNAs, as this structural element is deleted. A gene duplication of EF-Tu then allowed a co-evolution of one copy of the factor with the bizarre mitochondrial tRNA genes [48]. To recognize these transcripts, the new version of EF-Tu obtained an additional C-terminal domain, allowing a specific interaction with the D-loop of these tRNAs [49]. Whether similar adaptations occurred in R. culicivorax in the genes for RNase P, tRNase Z, CCA-adding enzyme and aminoacyl tRNA synthetases currently remains to be clarified. 5. Conclusions Despite the remarkable conservation of tRNAs in most translation systems there is growing evidence for dramatic deviations from the norm in particular in mitochondria. Aberrant genetic codes as well as bizarre tRNA architectures lacking either the T-arm
157
or the D-arm apparently have evolved independently in several clades [6,10,50]. The armless tRNAs of Enoplea emphasize the flexibility of the protein synthesis machinery despite the stringent selection pressures acting on it. We have provided here the first evidence that tRNAs with a size of 42 nts and a structure reduced to two base-paired stems connected by a variable bulge element are processed like conventional tRNAs, leading to transcripts ending with a single unpaired nucleotide at the 30 -end, followed by a posttranscriptionally added CCA-sequence. It will be interesting to see whether there are signs of coevolution of RNA structures and interacting protein partners, as already observed in nematodes for the unique elongation factor EFTu and the T-armless tRNAs in Chromadorea such as C. elegans. There, an extension of the protein recognized the D-loop instead [49]. The drastically reduced structure and the unusual lack of the discriminator position suggests that other steps in tRNA processing, including the cleavage by RNase P RNA and tRNase Z, as well as CCA addition may be adapted to the unusual shape of these tRNAs. The investigation of this co-evolution as well as the interplay of these molecules will expand our understanding of functionality and mechanism of protein synthesis. Acknowledgments We thank Sonja Bonin and Tobias Friedrich for expert technical assistance and Heike Betat for valuable discussion. This work was supported by the Deutsche Forschungsgemeinschaft DFG (Mo 634/ 8-1, Sta 850-3), by CNRS/Université de Strasbourg and the French Excellence Program (Labex MitCross; ANR-10-IDEX-002-02). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2013.07.034. References [1] C.G. Kurland, Problems and paradigns. Evolution of mitochondrial genomes and the genetic code, Bioessays 14 (1992) 709e714. [2] M.G. Abad, Y. Long, A. Willcox, J.M. Gott, M.W. Gray, J.E. Jackman, A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 50 -tRNA editing, RNA 17 (2011) 613e623. [3] J.M. Gott, B.H. Somerlot, M.W. Gray, Two forms of RNA editing are required for tRNA maturation in Physarum mitochondria, RNA 16 (2010) 482e488. [4] A. Reichert, U. Rothbauer, M. Mörl, Processing and editing of overlapping tRNAs in human mitochondria, J. Biol. Chem. 273 (1998) 31977e31984. [5] G.V. Börner, M. Mörl, A. Janke, S. Pääbo, RNA editing changes the identity of a mitochondrial tRNA in marsupials, EMBO J. 15 (1996) 5949e5957. [6] F. Jühling, J. Pütz, C. Florentz, P.F. Stadler, Armless mitochondrial tRNAs in enoplea (nematoda), RNA Biol. 9 (2012) 1161e1166. [7] M. Dörner, M. Altmann, S. Pääbo, M. Mörl, Evidence for import of a lysyl-tRNA into marsupial mitochondria, Mol. Biol. Cell 12 (2001) 2688e2698. [8] R. Okimoto, D.R. Wolstenholme, A set of tRNAs that lack either the T psi C arm or the dihydrouridine arm: towards a minimal tRNA adaptor, EMBO J. 9 (1990) 3405e3411. [9] D.R. Wolstenholme, J.L. Macfarlane, R. Okimoto, D.O. Clary, J.A. Wahleithner, Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms, Proc. Natl. Acad. Sci. USA 84 (1987) 1324e1328. [10] P.B. Klimov, B.M. Oconnor, Improved tRNA prediction in the American house dust mite reveals widespread occurrence of extremely short minimal tRNAs in acariform mites, BMC Genomics 10 (2009) 598. [11] M. Sprinzl, F. Cramer, The -C-C-A end of tRNA and its role in protein biosynthesis, Prog. Nucleic Acid Res. Mol. Biol. 22 (1979) 1e69. [12] T.O. Powers, E.G. Platzer, B.C. Hyman, Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite, Romanomermis culicivorax, Curr. Genet. 11 (1986) 71e77. [13] A.M. Stirling, E.G. Platzer, Catenaria anguillulae in the mermithid nematode Romanomermis culicivorax, J. Invertebr. Pathol. 32 (1978) 348e354. [14] J.J. Petersen, O.R. Willis, Procedures for the mass rearing of a mermithid parasite of mosquitoes, Mosquito News 32 (1972) 226e230. [15] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156e159.
158
S. Wende et al. / Biochimie 100 (2014) 151e158
[16] H. Dickinson, S. Tretbar, H. Betat, M. Mörl, The TRAMP complex shows tRNA editing activity in S. cerevisiae, Mol. Biol. Evol. 29 (2012) 1451e1459. [17] R. Lorenz, S.H. Bernhart, C. Höner Zu Siederdissen, H. Tafer, C. Flamm, P.F. Stadler, I.L. Hofacker, ViennaRNA package 2.0, Algorithms Mol. Biol. 6 (2011) 26. [18] K. Darty, A. Denise, Y. Ponty, VARNA: interactive drawing and editing of the RNA secondary structure, Bioinformatics 25 (2009) 1974e1975. [19] M. Popenda, M. Szachniuk, M. Antczak, K.J. Purzycka, P. Lukasiak, N. Bartol, J. Blazewicz, R.W. Adamiak, Automated 3D structure composition for large RNAs, Nucleic Acids Res. 40 (2012) e112. [20] P. Schattner, A.N. Brooks, T.M. Lowe, The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs, Nucleic Acids Res. 33 (2005) W686eW689. [21] R.W. Holley, J. Apgar, G.A. Everett, J.T. Madison, M. Marquisee, S.H. Merrill, J.R. Penswick, A. Zamir, Structure of a Ribonucleic acid, Science 147 (1965) 1462e1465. [22] S.H. Kim, F.L. Suddath, G.J. Quigley, A. McPherson, J.L. Sussman, A.H. Wang, N.C. Seeman, A. Rich, Three-dimensional tertiary structure of yeast phenylalanine transfer RNA, Science 185 (1974) 435e440. [23] H. Shi, P.B. Moore, The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited, RNA 6 (2000) 1091e1105. [24] R. Giegé, F. Jühling, J. Pütz, P. Stadler, C. Sauter, C. Florentz, Structure of transfer RNAs: similarity and variability, Wiley Interdiscip. Rev. RNA 3 (2012) 37e61. [25] F. Jühling, M. Mörl, R.K. Hartmann, M. Sprinzl, P.F. Stadler, J. Pütz, tRNAdb 2009: compilation of tRNA sequences and tRNA genes, Nucleic Acids Res. 37 (2009) D159eD162. [26] M. Sprinzl, F. Grueter, A. Spelzhaus, D.H. Gauss, Compilation of tRNA sequences, Nucleic Acids Res. 8 (1980) r1er22. [27] D.M. Crothers, T. Seno, G. Söll, Is there a discriminator site in transfer RNA? Proc. Natl. Acad. Sci. USA 69 (1972) 3063e3067. [28] D. L’Abbé, B.F. Lang, P. Desjardins, R. Morais, Histidine tRNA from chicken mitochondria has an uncoded 5’-terminal guanylate residue, J. Biol. Chem. 265 (1990) 2988e2992. [29] C. Francklyn, J. Arnez, Histidyl-tRNA synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), The Aminoacyl-tRNA Synthetases, 2005, pp. 265e277. [30] H. Himeno, T. Hasegawa, T. Ueda, K. Watanabe, K. Miura, M. Shimizu, Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation, Nucleic Acids Res. 17 (1989) 7855e7863. [31] C. Francklyn, P. Schimmel, Enzymatic aminoacylation of an eight-base-pair microhelix with histidine, Proc. Natl. Acad. Sci. USA 87 (1990) 8655e8659. [32] J. Rudinger, C. Florentz, R. Giegé, Histidylation by yeast HisRS of tRNA or tRNAlike structure relies on residues -1 and 73 but is dependent on the RNA context, Nucleic Acids Res. 22 (1994) 5031e5037. [33] I.U. Heinemann, A. Nakamura, P. O’Donoghue, D. Eiler, D. Soll, tRNAHis-guanylyltransferase establishes tRNAHis identity, Nucleic Acids Res. 40 (2011) 333e344.
[34] B.S. Rao, F. Mohammad, M.W. Gray, J.E. Jackman, Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii, Nucleic Acids Res. 41 (2013) 1885e1894. [35] J.Y. Chen, P.B. Joyce, C.L. Wolfe, M.C. Steffen, N.C. Martin, Cytoplasmic and mitochondrial tRNA nucleotidyltransferase activities are derived from the same gene in the yeast Saccharomyces cerevisiae, J. Biol. Chem. 267 (1992) 14879e14883. [36] A.S. Reichert, D.L. Thurlow, M. Mörl, A eubacterial origin for the human tRNA nucleotidyltransferase? Biol. Chem. 382 (2001) 1431e1438. [37] T. Nagaike, T. Suzuki, Y. Tomari, C. Takemoto-Hori, F. Negayama, K. Watanabe, T. Ueda, Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases, J. Biol. Chem. 276 (2001) 40041e40049. [38] D. Ojala, J. Montoya, G. Attardi, tRNA punctuation model for RNA processing in human mitochondria, Nature 290 (1981) 470e474. [39] M. Helm, R. Giegé, C. Florentz, A WatsoneCrick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys, Biochemistry 38 (1999) 13338e13346. [40] A.M. Estévez, L. Simpson, Uridine insertion/deletion RNA editing in trypanosome mitochondria e a review, Gene 240 (1999) 247e260. [41] P. Schimmel, R. Giegé, D. Moras, S. Yokoyama, An operational RNA code for amino acids and possible relationship to genetic code, Proc. Natl. Acad. Sci. USA 90 (1993) 8763e8768. [42] A.D. Wolfson, A.M. Khvorova, C. Sauter, C. Florentz, R. Giegé, Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase, Biochemistry 38 (1999) 11926e11932. [43] R.K. Agrawal, M.R. Sharma, Structural aspects of mitochondrial translational apparatus, Curr. Opin. Struct. Biol. 22 (2012) 797e803. [44] B.E. Christian, L.L. Spremulli, Mechanism of protein biosynthesis in mammalian mitochondria, Biochim. Biophys. Acta 1819 (2012) 1035e1054. [45] F. Zhao, T. Ohtsuki, K. Yamada, S. Yoshinari, K. Kita, Y. Watanabe, K. Watanabe, Isolation and physiochemical properties of protein-rich nematode mitochondrial ribosomes, Biochemistry 44 (2005) 9232e9237. [46] J.A. Mears, M.R. Sharma, R.R. Gutell, A.S. McCook, P.E. Richardson, T.R. Caulfield, R.K. Agrawal, S.C. Harvey, A structural model for the large subunit of the mammalian mitochondrial ribosome, J. Mol. Biol. 358 (2006) 193e212. [47] K. Watanabe, Unique features of animal mitochondrial translation systems. The non-universal genetic code, unusual features of the translational apparatus and their relevance to human mitochondrial diseases, Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86 (2010) 11e39. [48] T. Ohtsuki, Y. Watanabe, T-armless tRNAs and elongated elongation factor Tu, TBMB 59 (2007) 68e75. [49] M. Sakurai, Y. Watanabe, K. Watanabe, T. Ohtsuki, A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of Tarmless tRNAs in nematode mitochondria, Biochem. J. 399 (2006) 249e256. [50] K. Watanabe, S. Yokobori, tRNA modification and genetic code variations in animal mitochondria, J. Nucleic Acids 2011 (2011) 623095.
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Biological evidence for the world’s smallest tRNAs Sandra Wende a, Edward G. Platzer b, Frank Jühling d, Joern Pütz c, Catherine Florentz c, Peter F. Stadler d, e, f, g, h, i, Mario Mörl a, * a
University of Leipzig, Institute for Biochemistry, Leipzig, Germany University of California, Riverside, Department of Nematology, Riverside, CA 92521, USA c Architecture et Réactivité de l’ARN, Université de Strasbourg, CNRS, IBMC, 67084 Strasbourg, France d University of Leipzig, Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig, Germany e Max Planck Institute for Mathematics in the Sciences, Leipzig, Germany f Fraunhofer Institut für Zelltherapie und Immunologie e IZI, Leipzig, Germany g Department of Theoretical Chemistry, University of Vienna, Vienna, Austria h Center for Non-coding RNA in Technology and Health, University of Copenhagen, Frederiksberg C, Denmark i Santa Fe Institute, Santa Fe, NM, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2013 Accepted 24 July 2013 Available online 17 August 2013
Due to their function as adapters in translation, tRNA molecules share a common structural organization in all kingdoms and organelles with ribosomal protein biosynthesis. A typical tRNA has a cloverleaf-like secondary structure, consisting of acceptor stem, D-arm, anticodon arm, a variable region, and T-arm, with an average length of 73 nucleotides. In several mitochondrial genomes, however, tRNA genes encode transcripts that show a considerable deviation of this standard, having reduced D- or T-arms or even completely lack one of these elements, resulting in tRNAs as small as 66 nts. An extreme case of such truncations is found in the mitochondria of Enoplea. Here, several tRNA genes are annotated that lack both the D- and the T-arm, suggesting even shorter transcripts with a length of only 42 nts. However, direct evidence for these exceptional tRNAs, which were predicted by purely computational means, has been lacking so far. Here, we demonstrate that several of these miniaturized armless tRNAs consisting only of acceptor- and anticodon-arms are indeed transcribed and correctly processed by nonencoded CCA addition in the mermithid Romanomermis culicivorax. This is the first direct evidence for the existence and functionality of the smallest tRNAs ever identified so far. It opens new possibilities towards exploration/assessment of minimal structural motifs defining a functional tRNA and their evolution. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Enoplea Mitochondria Armless tRNA Processing tRNA structure CCA addition
1. Introduction To allow an efficient protein biosynthesis in vertebrate mitochondria, the circular genome of these organelles encodes a minimal set of structural RNAs essential for translation. Besides genes for the two ribosomal RNAs, typical animal mitogenomes contain 22 tRNA genes that are required and sufficient for translation in the organelle [1]. In comparison to the nuclear encoded translational machinery, the mitochondrial system is functional not only with such a reduced set of individual tRNAs for 20 different amino acids, but also with smaller tRNA transcripts that have simplified structural properties or lack specific features. Posttranscriptional editing of such tRNA sequences is a frequently observed strategy to obtain a complete and functional set of mitochondrial tRNAs. In these cases,
* Corresponding author. Tel.: þ49 (0) 341 9736 911; fax: þ49 (0) 341 9736 919. E-mail address: [email protected] (M. Mörl). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.07.034
50 - or 30 -terminally truncated tRNA transcripts, resulting from overlapping gene organization, are completed by the addition of the missing residues, ranging from single positions to stretches of several nucleotides [2e4]. Other editing events alter the identity of individual bases by deamination reactions, changing the amino acid identity of the complete tRNA [5]. Due to these facts, the mitochondrially encoded tRNAs frequently show strong deviations from their cytosolic counterparts and their genes are sometimes hard to identify so that specific tRNA search algorithms were developed [6]. In marsupial mitochondria, this is further complicated by processing place holders that are required to ensure a proper release of the neighboring mRNA and rRNAs out of the huge primary transcript that is synthesized. A degenerate tRNA for lysine has lost its original tRNA function but still acts as a processing signal [7]. The most extreme structural deviation and miniaturization of tRNAs, however, is found in the mitochondrial genome of nematodes like Caenorhabditis elegans or Ascaris suum, where either the D- or the T-arm
152
S. Wende et al. / Biochimie 100 (2014) 151e158
is replaced by a simple small loop consisting of five to eight nucleotides [8,9]. Recently, in the genome of nematode and mite mitochondria, the first genes for even smaller tRNA transcripts have been described. In the mite Dermatophagoides farina, the mitochondrial genome harbors genes for tRNAs lacking the D-arm and having only a small and unstable T-arm [10]. In addition, in the nematode group of Enoplea, containing the orders Mermithidia (arthropod parasites), Dorylaimida (plant parasites), Trichocephalida (vertebrate parasites), mitochondrial tRNA gene sets with predicted transcripts lacking both D- or T-domains were detected, leading to further reduced tRNA structures [6]. As an example, the mermithid Romanomermis culicivorax carries two mitochondrial tRNA genes where the D-arm is missing, 11 with a deletion of the T-arm, and nine with a deletion of both arms. These armless tRNAs were inferred from the genomic sequence by purely computational means. Nevertheless, there are several arguments why these transcripts very likely represent functional molecules. First, the gene sequence is highly conserved between the different Enoplea species. Second, the observed base replacements are compensatory and maintain the proposed secondary structure of the transcripts. Third, the observed genome arrangements do not disturb the integrity of the individual tRNA gene sequences [6]. It remains however unclear to what extent they are functional. Here we provide direct experimental evidence for the existence of the corresponding processed transcripts. Intriguingly, these tRNAs carry the highly conserved nucleotide triplet CCA at the 30 -terminus, which is an absolute prerequisite for becoming charged by the cognate aminoacyl tRNA synthetases [11]. As these CCA sequences are not encoded genomically but added in a posttranscriptional reaction by the CCA-adding enzyme, their presence is compelling evidence that these transcripts indeed represent functional tRNAs. 2. Materials and methods 2.1. Rearing and cultivation of R. culicivorax The isogenic female strain of R. culicivorax 3B4 [12] was reared in mass cultures using the procedures of Stirling & Platzer [13]. Autogenous Culex pipiens was employed as the host and the mosquito larvae were fed a mixture of finely ground laboratory rodent diet (LabDiet 5001, meal form; 2 parts) and brewer’s yeast (MP Biochemicals 903312; 1 part). Post-parasitic nematodes were collected in emergence chambers [14] and washed by sedimentation with deionized water and kept at 80 C until use. 2.2. Preparation of RNA Total RNA was isolated from 100 mg R. culicivorax 3B4 cells according to Chomczynski et al. [15]. Worms were homogenized using a 1.4 mm precellys ceramic matrix (Peqlab) for 2 40 s at 6 m/s in a FastPrep-24 homogenizer (MP). Isolated total RNA was DNase I (NEB) digested (2 units) and rRNA was depleted using Ribo-ZeroÔ magnetic gold kit (human/mouse/rat) (Epicentre) according to the manufacturers. 2.3. cDNA synthesis Using T4 RNA ligase, isolated total RNA was fused to a tagging DNA oligonucleotide that carried a ribonucleotide at the phosphorylated 50 -end and a 30 -end blocked by a 30 -30 - linked T [16]. cDNA was synthesized by hybridizing DNA primer P1 to the tagging oligonucleotide and subsequent primer elongation with RevertAid reverse transcriptase according to the manufacturer (Fermentas). Subsequently, the enzyme was inactivated by heat (70 C, 10 min).
2.4. tRNA 30 -end sequence determination cDNA of individual mitochondrial tRNAs was amplified in a 50 ml PCR reaction consisting of 35 cycles with 1 min. 95 C, 1 min. 39 C, 1 min. 60 C. Due to the extreme AT-content of the sequences, such a reduced temperature profile was required for efficient amplification. For polymerization, 1 U Phusion High-Fidelity DNA polymerase (ThermoScientific) was incubated in the presence of the RT primer and an upstream primer specific for the 50 -part of an individual tRNA. PCR products were cloned using the Clone Jet Cloning Kit (Fermentas Molecular Biology Tools; Thermo Scientific) and used for transformation of E. coli. Individual cDNA clones were amplified by colony PCR and sequenced on an ABI Prism 3700 automated sequencer (Amersham Pharmacia Biotech, Freiburg, Germany). Obtained sequences were analyzed using the software SeqMan. 2.5. tRNA 50 -end sequence determination The cDNA preparation described above was used in a tailing reaction with terminal deoxynucleotidyl-transferase and dCTP according to the manufacturer (Invitrogen). The reaction product was amplified by PCR as described above, using a tail-specific primer and a second oligonucleotide annealing to the 30 -part of the individual tRNA. PCR products were cloned and sequenced as described above. 2.6. Oligonucleotides used as primers Tagging oligonucleotide: 50 -pUGG ATC GCG TAG CTC ATA CGA GT(inverse)T-30 . RT primer: 50 -AAC TCG TAT GAG CTA CGC GAT C-30 . C-tail primer (according to invitrogen): 50 -GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-30 . Upstream amplification primer: 50 -GGC CAC GCG TCG ACT AGT AC-30 . tRNAArg primers: 30 -end analysis: 50 -AAA CTT TTA GCA GGA TTT CG-30 . 50 -end analysis: 50 -AAA ACT TAT ATA AAT TAG GAT TC-30 . tRNACys primers: 30 -end analysis: 50 -TAA AGA AGA AAC TTC ATT TG-30 . 50 -end analysis: 50 -CAA AAG AGA TTT TTT TTC ATT TTG-30 . tRNAHis primers: 30 -end analysis: 50 -TAA TAA ATT AAA TAT AAA TTG TG-30 . 50 -end analysis: 50 -ATA ATA AAA TTT TTT ATA AAT TCA C-30 . tRNAIle primers: 30 -end analysis: 50 -TCT TAA TAG AGA ACA ATT TAA ATT G-30 . 50 -end analysis: 50 -ATC TTA AAT TTT AAT TTA AAT TAT C-30 . tRNAPhe primers: 30 -end analysis: 50 -GCT TAC AAA AAA GAT TAA ATTe30 . 50 -end analysis: 50 -ATG CTT ACT TAT TAA TTA AAT TTT C-30 . tRNAThr primers: 30 -end analysis: 50 -TTC CTG CTG ACT ATG TTT TAT TTT G-30 .
S. Wende et al. / Biochimie 100 (2014) 151e158
50 -end analysis: 50 -TTC CTT AGT AAT TTT ATT TAC-30 . 2.7. tRNA structure prediction tRNA secondary structures were predicted using the RNAfold program of the Vienna RNA package [17], output structures were manually curated. Structure presentations were done using VARNA [18]. 3D structure predictions were calculated using RNA Composer [19]. 3. Results Due to several peculiarities of mitochondrial tRNAs in metazoans, including structural deviations and editing, the identification of unusual tRNA genes in Enoplea is not a direct proof for the existence and the functionality of the corresponding transcripts. To investigate whether such unexpected armless tRNAs are indeed expressed in these nematodes, total RNA was isolated from R. culicivorax material and depleted of ribosomal RNA. This total RNA preparation was 30 -terminally ligated to a DNA oligonucleotide
153
and transcribed into cDNA using a primer complementary to the fused oligonucleotide. The resulting cDNA was subjected to 30 -Ctailing and subsequently used as template for the specific amplification of 50 - and 30 -ends of the mitochondrial armless tRNAs for Arg, Cys, Ile, His, Thr, and Phe (Fig. 1A). A tailing reaction with A or T residues was avoided, as the tRNA sequences are extremely AU-rich and would easily lead to frequent mispriming in the subsequent PCR reaction. Due to the high AU content of these transcripts, the PCR elongation step had to be carried out at a reduced temperature of 60 C in order to ensure stable primer binding and efficient polymerization. An example of the resulting amplification products is shown for 50 - and 30 -parts of tRNAIle in Fig. 1B. The PCR products were cloned and individual clones were analyzed by sequencing, yielding between six and 29 sequences for the individual tRNAs. The analysis of the 50 -ends of the tRNAs was successful for three candidates, tRNAArg, tRNAIle and tRNAThr (Fig. 2A, B; Supplementary Table). A possible reason for this inefficient 50 -end amplification might be the considerable difference in the annealing temperatures of the C-tail primer and the tRNA-specific AT-rich oligonucleotide. In addition, most of the sequences show non-encoded heterogeneous extra nucleotides between the G-tail and the 50 -end of the
Fig. 1. Analysis of tRNA 50 - and 30 -ends. A. Strategy: The RNA preparation (black) was ligated to the tagging oligonucleotide (gray). Using the complementary RT primer, cDNA was synthesized. For 50 -RACE, the cDNA was tailed with C residues (gray). Individual tRNA sequences were amplified using a C-tail primer in combination with a primer specific for the 30 -part of the individual tRNA. For 30 -RACE, the PCR was performed with RT primer and a primer specific for the 50 -part of the tRNA. B. The amplification products for tRNAIle are shown as an example. Left: The 50 -part resulted in a PCR product of about 90 bp, depending on the C-tailing and annealing position of the C-tail primer. Right: For the 30 -part of the tRNA, a PCR product of about 70e80 bp could be detected, corresponding to the expected size of 76 bp.
154
S. Wende et al. / Biochimie 100 (2014) 151e158
Fig. 2. Mini tRNAs. A. As an example, the 50 - and 30 -sequences of tRNAIle are shown. Primer binding sites are indicated by black arrows. The predicted sequences are indicated in black characters, identified sequences in color. Lower case characters represent deviations (missing bases) in the predictions, indicated by asterisks. Due to the extreme AU content of the tRNA sequence, both tRNA specific primers ended at the same G position in the tRNA to ensure proper hybridization of the primer 30 -ends. Hence, this individual position is the only one that was not sequenced. B. Presentation of validated tRNA sequences. Black: predicted sequences. Green: identified sequences, gray: missing 50 -parts. Red: deviations from prediction, leading to truncated 50 - and 30 -end positions, or representing CCA-additions that indicate the functionality of these transcripts. Putative acceptor- and anticodonstem pairings are indicated in pink and blue.
tRNA sequence which are probably the result of cDNA synthesis artifact (see discussion). Nevertheless, the 50 -termini of these three tRNAs are a convincing evidence for the correct expression and processing of the transcripts. While tRNAArg and tRNAThr have a 50 end as predicted, tRNAIle is truncated for one position. Hence, this tRNA seems to be even smaller than expected. In contrast to the 50 end determination, the 30 -end analysis was successful for all tRNA candidates. Here, all of the investigated tRNA sequences carried a non-encoded posttranscriptionally added CCA triplet at the 30 -terminus (Fig. 2B) While most of the clones carried a complete version of the triplet, several single sequences also showed partial CCA addition, ending with one or two C-residues. Hence, these products obviously represent either reaction intermediates of the CCA addition process or indicate some degradation during preparation, as it is probably also the case in two 50 -end sequences of tRNAIle,
where 2 nucleotides are missing (Supplementary Table). In addition, in the tRNAs for Ile, Cys, His, and Phe, the actual 30 -end of the encoded tRNA differs from the predicted one, as the expected discriminator position is not present in these transcripts. Instead, the CCA-end is attached to the position upstream of the predicted discriminator nucleotide, indicating that the actual tRNA is at least one nucleotide shorter than predicted originally. In the case of tRNAIle, this truncation corresponds to the situation at the 50 -terminus and generates an acceptor stem with the conventional single-stranded discriminator position upstream of the CCA-end. It is highly likely that similar truncations exist at the 50 -termini of the other identified tRNAs with shorter 30 -ends. As these transcripts show strong deviations of a standard tRNA secondary structure, usual tRNA structure prediction algorithms like tRNAScanSE [20] fail to recognize and fold these sequences,
S. Wende et al. / Biochimie 100 (2014) 151e158
even in a mode optimized for nematode mitochondria. Hence, several of the sequences were analyzed using RNAfold from the Vienna RNA package [17] and the predicted structures were subsequently curated by hand and illustrated using the VARNA tool [18]. For the fully sequenced tRNAArg and tRNAIle, structures with acceptor- and anticodon-like stem elements can be formed (Fig. 3), although the numbers of base pairs in these stems differ from the standard. D- and T-arms, however, are completely missing and are replaced by bulges between 5 and 9 nucleotides. Yet, in the online RNA Composer program [19], these secondary structures can be folded into small L-shaped tertiary structures that show a considerable similarity to conventional tRNA shapes. 4. Discussion The first complete sequence of a tRNA was identified in 1965 and led immediately to the suggestion of several alternative secondary structures [21]. Among these predictions, a cloverleaf-like form of this transcript was proposed which later turned out to be correct. This structure represents the basis for the threedimensional organization of tRNA, resulting in an L-shaped arrangement, where acceptor- and T-stem as well as anticodonand D-stem are stacked to form elongated helices [22,23]. This arrangement turned out to represent the canonical threedimensional structure of tRNAs in all kingdoms as demonstrated by a large number of crystallographic data (reviewed in Giegé et al. [24],), with a conserved distance of about 70 A between the anticodon and the CCA terminus, carrying the amino acid. Surprisingly, metazoan mitochondrial tRNAs show considerable deviations from the cloverleaf secondary structure, with size reductions in loop as well as stem elements, with the most extreme case of lacking the D- or T-stem. Yet, these tRNAs with a size down to 59 nucleotides (human mt tRNASer) still can fold into the canonical L shape with conserved dimensions [24]. Hence, it is very surprising that in several recently analyzed nematode and mite species, even smaller mitochondrial tRNAs were predicted [10,25]. While these annotations are predictions based on DNA sequences, the results presented here are the first experimental proof for the actual existence of these minimalized tRNAs and confirm the correctness of the prediction algorithms. The additional nucleotides located between the 50 -end and the Ctail added during the 50 -RACE analysis do not represent posttranscriptional editing events but are very likely the result of a preparation artifact. First, the added nucleotides differ from clone to clone and are not identical within the sequence analysis of an individual tRNA. For a functional editing reaction, a specific tRNA would have identical nucleotides added. Second, the most likely explanation for the origin of these extra nucleotides is that a remaining excess of dNTPs from the cDNA synthesis was not efficiently removed before the C-tailing reaction was started, resulting in the incorporation of these nucleotides by terminal nucleotidyl transferase. Correspondingly, the extensive use of size exclusion chromatography strongly reduced the amount of the additional nucleotides. Hence, the tRNA 50 -ends are very likely to start at the indicated positions, without carrying extra nucleotides. Nevertheless, in the identified tRNAs for Ile, Cys, His and Phe, the exact 50 - and/or 30 -ends differ for one position compared to the predictions, resulting in even shorter transcripts (Fig. 2B). This correlated recession of the 50 - and 30 -termini restores the typical acceptor stem end in tRNAs, where the 30 -terminal residue (position 73 according to the standard numbering [26]) is unpaired and represents in many transcripts an essential identity element for aminoacylation, referred to as discriminator position [27]. A special case is tRNAHis, where this position 73 (usually a C or an A residue) is basepaired with a G residue at position 1 that e in the case of animal
155
mitochondria e is posttranscriptionally added, leading to an additional G-1/C73 or G-1/A73 base pair [25,28,29]. As it was not possible to identify the 50 -end of the R. culicivorax mitochondrial tRNAHis, the exact situation in this tRNA remains unclear. Nevertheless, the identified 30 -end is one nucleotide shorter than predicted (Fig. 2B). Hence, if the actual 50 -end corresponds to the proposed one, the discriminator position A73 would be base-paired with a U residue at the 50 -end. In other tRNAHis transcripts, the G residue base-paired with the discriminator represents a major identity element for the corresponding synthetase [30e33]. A deviation of this conserved feature was recently identified in the cytosolic tRNAHis of Acanthamoeba castellanii, where the discriminator position remains unpaired [34]. It is possible that in R. culicivorax, a similar situation exists in the corresponding mitochondrial tRNA. The presented data indicate that these armless tRNAs are indeed correctly processed by mitochondrial RNase P and tRNase Z activities and released from the mitochondrial precursor transcripts. In addition, and most significantly, the obtained sequences show that the released tRNAs represent bona-fide substrates for the CCAadding enzyme (Fig. 4). Most of the processing reactions might be carried out by specialized mitochondrial enzyme versions. For the CCA-adding enzyme, however, eukaryotic genomes carry only a single gene, encoding the nuclear/cytosolic as well as the mitochondrial form of this activity [35e37]. Being responsible for CCA incorporation in both cytosolic and mitochondrial sets of tRNAs, this enzyme must have undergone a specific adaptation in order to recognize such a diverse set of tRNA substrates. The fact that all analyzed transcripts carry a complete or (in some rare cases) partial CCA-end at the 30 -termini is a strong evidence that the transcripts do not only represent reduced processing signals for precursor RNA maturation in order to release the adjacent mRNAs according to the mitochondrial punctuation model [38]. Instead, these transcripts seem to be functional tRNAs that are completed by the essential site for aminoacylation, although tRNA charging per se and a direct involvement of the tRNAs in mitochondrial protein synthesis could not be analyzed due to limited material. In agreement with this, the armless tRNAs have the ability to fold into three-dimensional structures that resemble the canonical L-shape as anticipated from folding programs, although the distance between anticodon and aminoacylation site differs strongly from the standard (w63 A for tRNAArg, w46 A for tRNAIle). Such a structure might be further optimized and stabilized by the introduction of modifications, as it was shown to be a requirement for the proper folding of the human mitochondrial tRNALys that adopts an L shape only if an adenosine residue at position 9 is modified to m1A [39]. For the armless tRNAs with such reduced dimensions, two solutions are conceivable. Either the tRNA structure is so flexible that it can extend the distance between the two functional ends in order to be recognized by synthetases and ribosomes, or the mitochondrial translational machinery of R. culicivorax (and of other Enoplea) has a size very different of what is known so far. The sequence data also rule out the possibility that the structural deviations of these minimal transcripts are corrected by massive insertional editing events as they are described for mitochondrial transcripts in trypanosomes [40]. Obviously, the corresponding mitochondrial maturation, aminoacylation and translational machinery co-evolved with the minimization and is adapted to recognize such a peculiar set of tRNAs even in the absence of otherwise highly conserved structural features. Interaction with the synthetase may not be hampered by the size of the tRNA. Indeed, it is well established that synthetases from various origins are capable of aminoacylating much shorter RNAs, such as mini- or micro-helices mimicking the aminoacyl-accepting arm, or only part of it [41]. These findings indicate that the synthetase does
156
S. Wende et al. / Biochimie 100 (2014) 151e158
Fig. 3. Structure predictions for tRNAArg and tRNAIle. In the secondary structure models, the truncations of D- and T-arms are visible, leading to replacement bulges. The anticodon is indicated in red, the CCA-end in green. In the overall structure, these transcripts do not resemble the conventional tRNA cloverleaf that is represented by the human mitochondrial tRNAArg. The predicted three-dimensional structures, however, resemble the standard L-shape of canonical tRNAs, even if acceptor- and anticodon stems show considerable deviations in distance. Although the dimensions of these L-shapes differ from the ones of standard tRNAs, it seem very likely that these transcripts represent functional tRNAs in Romanomermis mitochondria.
S. Wende et al. / Biochimie 100 (2014) 151e158
Fig. 4. tRNA processing reactions. For both conventional as well as bizarre minimized tRNAs, the same set of maturation steps, including aminoacylations, is required. The reactions that were identified in Romanomermis mitochondria are ticked off in green, while the missing reactions are indicated by the red question marks.
not need to interact with both the anticodon and the accepting CCA-end. Additional examples for aminoacylation of unusually structured tRNA substrates include mimics formed by an anticodon hairpin linked to a CCA-end by a single stranded RNA stretch [42]. In mitochondrial ribosomes, a dramatic size reduction in rRNA components was described, leading to ribosomes that obviously have lost the tRNA exit site [43]. Furthermore, the decrease in rRNA size seems to be compensated by an increase in the number of ribosomal proteins, leading to an RNA content of 25e30% in mammalian mitochondria compared to 60e70% in bacteria [44]. In C. elegans, this trend is continued, where the mitochondrial ribosomes have an even higher protein composition [45]. However, as the nematode mitochondrial ribosomes have a size larger than the bacterial counterparts [45], a size reduction compensating the tRNA miniaturization is excluded. It is more likely that specific features of ribosomal RNAs or proteins evolved in order to tolerate such reduced tRNAs as translational adapters. One such co-evolution might concern the A-site finger element that in cytosolic ribosomes interacts with D- and T-arms of an incoming tRNA. In mitochondrial ribosomes, this element is missing so that no sensing for D- and T-loop structures occurs [46]. Despite such likely adaptations in the mitochondrial ribosomes accepting armless tRNAs, it is possible that the efficiency of translation is reduced in comparison to protein synthesis with canonical tRNAs, as it was shown that the bovine mt tRNASerGCU, lacking the D-arm, leads to much lower protein synthesis [47]. In other components of the mitochondrial translation machinery, adaptations to recognize non-canonical tRNA structures were already identified. For the conventional interaction of EF-Tu, recognition of the T-arm of tRNAs is required. In C. elegans, this is not possible for most of the 20 mitochondrial tRNAs, as this structural element is deleted. A gene duplication of EF-Tu then allowed a co-evolution of one copy of the factor with the bizarre mitochondrial tRNA genes [48]. To recognize these transcripts, the new version of EF-Tu obtained an additional C-terminal domain, allowing a specific interaction with the D-loop of these tRNAs [49]. Whether similar adaptations occurred in R. culicivorax in the genes for RNase P, tRNase Z, CCA-adding enzyme and aminoacyl tRNA synthetases currently remains to be clarified. 5. Conclusions Despite the remarkable conservation of tRNAs in most translation systems there is growing evidence for dramatic deviations from the norm in particular in mitochondria. Aberrant genetic codes as well as bizarre tRNA architectures lacking either the T-arm
157
or the D-arm apparently have evolved independently in several clades [6,10,50]. The armless tRNAs of Enoplea emphasize the flexibility of the protein synthesis machinery despite the stringent selection pressures acting on it. We have provided here the first evidence that tRNAs with a size of 42 nts and a structure reduced to two base-paired stems connected by a variable bulge element are processed like conventional tRNAs, leading to transcripts ending with a single unpaired nucleotide at the 30 -end, followed by a posttranscriptionally added CCA-sequence. It will be interesting to see whether there are signs of coevolution of RNA structures and interacting protein partners, as already observed in nematodes for the unique elongation factor EFTu and the T-armless tRNAs in Chromadorea such as C. elegans. There, an extension of the protein recognized the D-loop instead [49]. The drastically reduced structure and the unusual lack of the discriminator position suggests that other steps in tRNA processing, including the cleavage by RNase P RNA and tRNase Z, as well as CCA addition may be adapted to the unusual shape of these tRNAs. The investigation of this co-evolution as well as the interplay of these molecules will expand our understanding of functionality and mechanism of protein synthesis. Acknowledgments We thank Sonja Bonin and Tobias Friedrich for expert technical assistance and Heike Betat for valuable discussion. This work was supported by the Deutsche Forschungsgemeinschaft DFG (Mo 634/ 8-1, Sta 850-3), by CNRS/Université de Strasbourg and the French Excellence Program (Labex MitCross; ANR-10-IDEX-002-02). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2013.07.034. References [1] C.G. Kurland, Problems and paradigns. Evolution of mitochondrial genomes and the genetic code, Bioessays 14 (1992) 709e714. [2] M.G. Abad, Y. Long, A. Willcox, J.M. Gott, M.W. Gray, J.E. Jackman, A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 50 -tRNA editing, RNA 17 (2011) 613e623. [3] J.M. Gott, B.H. Somerlot, M.W. Gray, Two forms of RNA editing are required for tRNA maturation in Physarum mitochondria, RNA 16 (2010) 482e488. [4] A. Reichert, U. Rothbauer, M. Mörl, Processing and editing of overlapping tRNAs in human mitochondria, J. Biol. Chem. 273 (1998) 31977e31984. [5] G.V. Börner, M. Mörl, A. Janke, S. Pääbo, RNA editing changes the identity of a mitochondrial tRNA in marsupials, EMBO J. 15 (1996) 5949e5957. [6] F. Jühling, J. Pütz, C. Florentz, P.F. Stadler, Armless mitochondrial tRNAs in enoplea (nematoda), RNA Biol. 9 (2012) 1161e1166. [7] M. Dörner, M. Altmann, S. Pääbo, M. Mörl, Evidence for import of a lysyl-tRNA into marsupial mitochondria, Mol. Biol. Cell 12 (2001) 2688e2698. [8] R. Okimoto, D.R. Wolstenholme, A set of tRNAs that lack either the T psi C arm or the dihydrouridine arm: towards a minimal tRNA adaptor, EMBO J. 9 (1990) 3405e3411. [9] D.R. Wolstenholme, J.L. Macfarlane, R. Okimoto, D.O. Clary, J.A. Wahleithner, Bizarre tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms, Proc. Natl. Acad. Sci. USA 84 (1987) 1324e1328. [10] P.B. Klimov, B.M. Oconnor, Improved tRNA prediction in the American house dust mite reveals widespread occurrence of extremely short minimal tRNAs in acariform mites, BMC Genomics 10 (2009) 598. [11] M. Sprinzl, F. Cramer, The -C-C-A end of tRNA and its role in protein biosynthesis, Prog. Nucleic Acid Res. Mol. Biol. 22 (1979) 1e69. [12] T.O. Powers, E.G. Platzer, B.C. Hyman, Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite, Romanomermis culicivorax, Curr. Genet. 11 (1986) 71e77. [13] A.M. Stirling, E.G. Platzer, Catenaria anguillulae in the mermithid nematode Romanomermis culicivorax, J. Invertebr. Pathol. 32 (1978) 348e354. [14] J.J. Petersen, O.R. Willis, Procedures for the mass rearing of a mermithid parasite of mosquitoes, Mosquito News 32 (1972) 226e230. [15] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156e159.
158
S. Wende et al. / Biochimie 100 (2014) 151e158
[16] H. Dickinson, S. Tretbar, H. Betat, M. Mörl, The TRAMP complex shows tRNA editing activity in S. cerevisiae, Mol. Biol. Evol. 29 (2012) 1451e1459. [17] R. Lorenz, S.H. Bernhart, C. Höner Zu Siederdissen, H. Tafer, C. Flamm, P.F. Stadler, I.L. Hofacker, ViennaRNA package 2.0, Algorithms Mol. Biol. 6 (2011) 26. [18] K. Darty, A. Denise, Y. Ponty, VARNA: interactive drawing and editing of the RNA secondary structure, Bioinformatics 25 (2009) 1974e1975. [19] M. Popenda, M. Szachniuk, M. Antczak, K.J. Purzycka, P. Lukasiak, N. Bartol, J. Blazewicz, R.W. Adamiak, Automated 3D structure composition for large RNAs, Nucleic Acids Res. 40 (2012) e112. [20] P. Schattner, A.N. Brooks, T.M. Lowe, The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs, Nucleic Acids Res. 33 (2005) W686eW689. [21] R.W. Holley, J. Apgar, G.A. Everett, J.T. Madison, M. Marquisee, S.H. Merrill, J.R. Penswick, A. Zamir, Structure of a Ribonucleic acid, Science 147 (1965) 1462e1465. [22] S.H. Kim, F.L. Suddath, G.J. Quigley, A. McPherson, J.L. Sussman, A.H. Wang, N.C. Seeman, A. Rich, Three-dimensional tertiary structure of yeast phenylalanine transfer RNA, Science 185 (1974) 435e440. [23] H. Shi, P.B. Moore, The crystal structure of yeast phenylalanine tRNA at 1.93 A resolution: a classic structure revisited, RNA 6 (2000) 1091e1105. [24] R. Giegé, F. Jühling, J. Pütz, P. Stadler, C. Sauter, C. Florentz, Structure of transfer RNAs: similarity and variability, Wiley Interdiscip. Rev. RNA 3 (2012) 37e61. [25] F. Jühling, M. Mörl, R.K. Hartmann, M. Sprinzl, P.F. Stadler, J. Pütz, tRNAdb 2009: compilation of tRNA sequences and tRNA genes, Nucleic Acids Res. 37 (2009) D159eD162. [26] M. Sprinzl, F. Grueter, A. Spelzhaus, D.H. Gauss, Compilation of tRNA sequences, Nucleic Acids Res. 8 (1980) r1er22. [27] D.M. Crothers, T. Seno, G. Söll, Is there a discriminator site in transfer RNA? Proc. Natl. Acad. Sci. USA 69 (1972) 3063e3067. [28] D. L’Abbé, B.F. Lang, P. Desjardins, R. Morais, Histidine tRNA from chicken mitochondria has an uncoded 5’-terminal guanylate residue, J. Biol. Chem. 265 (1990) 2988e2992. [29] C. Francklyn, J. Arnez, Histidyl-tRNA synthetases, in: M. Ibba, C. Francklyn, S. Cusack (Eds.), The Aminoacyl-tRNA Synthetases, 2005, pp. 265e277. [30] H. Himeno, T. Hasegawa, T. Ueda, K. Watanabe, K. Miura, M. Shimizu, Role of the extra G-C pair at the end of the acceptor stem of tRNA(His) in aminoacylation, Nucleic Acids Res. 17 (1989) 7855e7863. [31] C. Francklyn, P. Schimmel, Enzymatic aminoacylation of an eight-base-pair microhelix with histidine, Proc. Natl. Acad. Sci. USA 87 (1990) 8655e8659. [32] J. Rudinger, C. Florentz, R. Giegé, Histidylation by yeast HisRS of tRNA or tRNAlike structure relies on residues -1 and 73 but is dependent on the RNA context, Nucleic Acids Res. 22 (1994) 5031e5037. [33] I.U. Heinemann, A. Nakamura, P. O’Donoghue, D. Eiler, D. Soll, tRNAHis-guanylyltransferase establishes tRNAHis identity, Nucleic Acids Res. 40 (2011) 333e344.
[34] B.S. Rao, F. Mohammad, M.W. Gray, J.E. Jackman, Absence of a universal element for tRNAHis identity in Acanthamoeba castellanii, Nucleic Acids Res. 41 (2013) 1885e1894. [35] J.Y. Chen, P.B. Joyce, C.L. Wolfe, M.C. Steffen, N.C. Martin, Cytoplasmic and mitochondrial tRNA nucleotidyltransferase activities are derived from the same gene in the yeast Saccharomyces cerevisiae, J. Biol. Chem. 267 (1992) 14879e14883. [36] A.S. Reichert, D.L. Thurlow, M. Mörl, A eubacterial origin for the human tRNA nucleotidyltransferase? Biol. Chem. 382 (2001) 1431e1438. [37] T. Nagaike, T. Suzuki, Y. Tomari, C. Takemoto-Hori, F. Negayama, K. Watanabe, T. Ueda, Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases, J. Biol. Chem. 276 (2001) 40041e40049. [38] D. Ojala, J. Montoya, G. Attardi, tRNA punctuation model for RNA processing in human mitochondria, Nature 290 (1981) 470e474. [39] M. Helm, R. Giegé, C. Florentz, A WatsoneCrick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys, Biochemistry 38 (1999) 13338e13346. [40] A.M. Estévez, L. Simpson, Uridine insertion/deletion RNA editing in trypanosome mitochondria e a review, Gene 240 (1999) 247e260. [41] P. Schimmel, R. Giegé, D. Moras, S. Yokoyama, An operational RNA code for amino acids and possible relationship to genetic code, Proc. Natl. Acad. Sci. USA 90 (1993) 8763e8768. [42] A.D. Wolfson, A.M. Khvorova, C. Sauter, C. Florentz, R. Giegé, Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase, Biochemistry 38 (1999) 11926e11932. [43] R.K. Agrawal, M.R. Sharma, Structural aspects of mitochondrial translational apparatus, Curr. Opin. Struct. Biol. 22 (2012) 797e803. [44] B.E. Christian, L.L. Spremulli, Mechanism of protein biosynthesis in mammalian mitochondria, Biochim. Biophys. Acta 1819 (2012) 1035e1054. [45] F. Zhao, T. Ohtsuki, K. Yamada, S. Yoshinari, K. Kita, Y. Watanabe, K. Watanabe, Isolation and physiochemical properties of protein-rich nematode mitochondrial ribosomes, Biochemistry 44 (2005) 9232e9237. [46] J.A. Mears, M.R. Sharma, R.R. Gutell, A.S. McCook, P.E. Richardson, T.R. Caulfield, R.K. Agrawal, S.C. Harvey, A structural model for the large subunit of the mammalian mitochondrial ribosome, J. Mol. Biol. 358 (2006) 193e212. [47] K. Watanabe, Unique features of animal mitochondrial translation systems. The non-universal genetic code, unusual features of the translational apparatus and their relevance to human mitochondrial diseases, Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86 (2010) 11e39. [48] T. Ohtsuki, Y. Watanabe, T-armless tRNAs and elongated elongation factor Tu, TBMB 59 (2007) 68e75. [49] M. Sakurai, Y. Watanabe, K. Watanabe, T. Ohtsuki, A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of Tarmless tRNAs in nematode mitochondria, Biochem. J. 399 (2006) 249e256. [50] K. Watanabe, S. Yokobori, tRNA modification and genetic code variations in animal mitochondria, J. Nucleic Acids 2011 (2011) 623095.