tRNA His Research Articles

Plant mitochondria use two pathways for the biogenesis of tRNA His.

All tRNAHis possess an essential extra G–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G–1 is added by a tRNAHis guanylyl transferase. In prokaryotes, G–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G–1 we find here that both maturation pathways can be used. Indeed, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis precursors at both positions G+1 and G–1. The G–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNAHis without G–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

A possible link between specific transfer RNA methylation and tumorigenic phenotype of breast cancer

The human RNA methyltransferase BCDIN3D is overexpressed in breast cancer cells and involved in cellular invasion and poor prognosis of breast cancer. Several years ago, BCDIN3D was reported to dimethylate the 5'-monophosphate of specific precursor miRNAs (pre-miRNAs), such as the tumor suppressor miR145. Dimethylation of the 5'-monophosphate of the pre-miRNA negatively regulates the subsequent processing by Dicer in vitro , and results in the downregulated expression of the mature form of the miRNA. The depletion of BCDIN3D also reportedly results in the suppression of the tumorigenic phenotype of breast cancer cells. Thus, these findings suggested that BCDIN3D promotes the cellular invasion of breast cancer cells, by downregulating the expression of tumor suppressor miRNAs via the dimethylation of the 5'-monophosphate of the corresponding pre-miRNAs . Recently, we found that cytoplasmic tRNA His is actually the primary target of human BCDIN3D, rather than pre-miR145. BCDIN3D monomethylates the 5'-phosphate of cytoplasmic tRNA His much more efficiently than that of pre-miRNA in vitro , and is responsible for the monomethylation of the 5'-phosphate of cytoplasmic tRNA His in vivo . BCDIN3D recognizes the eight-nucleotide long extended acceptor helix with the G -1 -A 73 mis-pair at the top of the acceptor stem of tRNA His , which are exceptional features among cytoplasmic tRNA species. These results not only reveal the primary target of BCDIN3D, which is overexpressed in breast cancer cells, but also highlight the possible involvement of the 5'-phosphomethylation of tRNA and/or tRNA in the tumorigenesis of breast cancer cells, beyond its established function in protein synthesis.

Abstract 4323: Mitochondrial 22 tRNAs alterations in Iranian breast cancer patients

Abstract Introduction: Mitochondrial dysfunction is related to the genesis of many types of cancers, including breast cancer. Mitochondrial tRNA genes perform several functions including processing and translation in which are essential components of mitochondrial protein synthesis. Until now only few somatic mitochondrial tRNA mutations have been reported in cancer cells. The aim of this study was to find out the relationship between mitochondrial DNA mutations and breast cancer. Patients and Methods: In current this study, 22 mitochondrial tRNAs genes in 30 tumoral tissues and adjacent non-tumoral tissues of patients with breast cancer and 100 blood samples of healthy controls were investigate using PCR-Sequencing methods. Results and conclusions: From 30 breast cancer cases, twelve genetic variations were found in 22 tRNA MtDNA. Six of twelve (50%) were novel and remaining was polymorphism. Among new variants, which incloding C1631A (tRNA Val), T1633A (tRNA Val), A5565G (tRNA Trp), G5849A (tRNA Tyr) were at hetroplasmic state and remaining which including C12187 (tRNA His) and A15948G (tRNA Thr) were at homoplasmic. Among variants, A12308G, a polymorphic mutation in V-loop (tRNALeu(CUN)), was found in 23% breast tumoral tissues (7 of 30) and 3%in healthy blood controls (3 of 100), indicating a statistically significant association of the A12308G alteration between tumoral tissues and controls(p < 0.05).Our finding indicate that , the A12308G alteration may increase the risk of breast cancer through its impact on the tRNA structure result in mitochondrial dysfunction. Therefore, it may consider as a pathogenic predisposition factor for the breast cancer risk. Note: This abstract was not presented at the meeting. Citation Format: Sara Eslamizadeh, Massoud Ghaffarpour, Mohammad Arabzadeh, Massoud Houshmand. Mitochondrial 22 tRNAs alterations in Iranian breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4323. doi:10.1158/1538-7445.AM2014-4323

The complete mitochondrial genome of Macrobrachium nipponense

The complete mitochondrial (mt) genome sequence plays an important role in the accurate determination of phylogenetic relationships among metazoans. Herein, we determined the complete mt genome sequence, structure and organization of Macrobrachium nipponense ( M. nipponense) (GenBank ID: NC_015073.1) and compared it to that of Macrobrachium lanchesteri ( M. lanchesteri) and Macrobrachium rosenbergii ( M. rosenbergii). The 15,806 base pair (bp) M. nipponense mt genome, which is comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs), is slightly larger than that of M. lanchesteri (15,694 bp, GenBank ID: NC_012217.1) and M. rosenbergii (15,772 bp, GenBank ID: NC_006880.1). The M. nipponense genome contains a high AT content (66.0%), which is a common feature among metazoan mt genomes. Compared with M. lanchesteri and M. rosenbergii, we found a peculiar non-coding region of 950 bp with a microsatellite-like (TA) 6 element and many hairpin structures. The 13 PCGs are comprised of a total of 3707 codons, excluding incomplete termination codons, and the most frequently used amino acid is Leu (16.0%). The predicted start codons in the M. nipponense mt genome include ATG, ATC and ATA. Seven PCGs use TAA as a stop codon, whereas two use TAG, three use T and only one uses TA. Twenty-three of the genes are encoded on the L strand, and ND1, ND4, ND5, ND4L, 12S rRNA, 16S rRNA, tRNA His , tRNA Pro , tRNA Phe , tRNA Val , tRNA Gln , tRNA Cys , tRNA Tyr and a tRNA Leu are encoded on the H strand. The two rRNAs of M. nipponense and M. rosenbergii are encoded on the H strand, whereas the M. lanchesteri rRNAs are encoded on the L stand.

New mitochondrial tRNA HIS mutation in a family with lactic acidosis and stroke-like episodes (MELAS)

We report a new mutation in m.12146 A > G in the mt-tRNA His in a family with a remarkable clinical history having different degrees of lactic acidosis and stroke-like episodes. Biochemical measurements of a muscle biopsy established an isolated complex IV deficiency, while similar analysis of fibroblasts showed a combined complex I,III and IV deficiency. Transmitochondrial cybrid analysis proved that this tRNA His mutation causes the enzymatic deficiency. This family illustrates the complexity of the clinical, biochemical and genetic characteristics of a novel mtDNA encoded disorder, as well as the challenge to prove its pathogenicity.

TRNA 5′-end repair activities of tRNA His guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea

The tRNAHis guanylyltransferase (Thg1) family comprises a set of unique 3′–5′ nucleotide addition enzymes found ubiquitously in Eukaryotes, where they function in the critical G−1 addition reaction required for tRNAHis maturation. However, in most Bacteria and Archaea, G−1 is genomically encoded; thus post-transcriptional addition of G−1 to tRNAHis is not necessarily required. The presence of highly conserved Thg1-like proteins (TLPs) in more than 40 bacteria and archaea therefore suggests unappreciated roles for TLP-catalyzed 3′–5′ nucleotide addition. Here, we report that TLPs from Bacillus thuringiensis (BtTLP) and Methanosarcina acetivorans (MaTLP) display biochemical properties consistent with a prominent role in tRNA 5′-end repair. Unlike yeast Thg1, BtTLP strongly prefers addition of missing N+1 nucleotides to 5′-truncated tRNAs over analogous additions to full-length tRNA (kcat/KM enhanced 5–160-fold). Moreover, unlike for −1 addition, BtTLP-catalyzed additions to truncated tRNAs are not biased toward addition of G, and occur with tRNAs other than tRNAHis. Based on these distinct biochemical properties, we propose that rather than functioning solely in tRNAHis maturation, bacterial and archaeal TLPs are well-suited to participate in tRNA quality control pathways. These data support more widespread roles for 3′–5′ nucleotide addition reactions in biology than previously expected.

Plant mitochondria use two pathways for the biogenesis of tRNA His

All tRNAHis possess an essential extra G–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G–1 is added by a tRNAHis guanylyl transferase. In prokaryotes, G–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G–1 we find here that both maturation pathways can be used. Indeed, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis precursors at both positions G+1 and G–1. The G–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNAHis without G–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

3′–5′ tRNA His guanylyltransferase in bacteria

The identity of the histidine specific transfer RNA (tRNA His) is largely determined by a unique guanosine residue at position −1. In eukaryotes and archaea, the tRNA His guanylyltransferase (Thg1) catalyzes 3′–5′ addition of G to the 5′-terminus of tRNA His. Here, we show that Thg1 also occurs in bacteria. We demonstrate in vitro Thg1 activity for recombinant enzymes from the two bacteria Bacillus thuringiensis and Myxococcus xanthus and provide a closer investigation of several archaeal Thg1. The reaction mechanism of prokaryotic Thg1 differs from eukaryotic enzymes, as it does not require ATP. Complementation of a yeast thg1 knockout strain with bacterial Thg1 verified in vivo activity and suggests a relaxed recognition of the discriminator base in bacteria.

Relationships among the Neotropical Candirus (Trichomycteridae, Siluriformes) and the evolution of parasitism based on analysis of mitochondrial and nuclear gene sequences

Phylogenetic relationships among the trichomycterid catfishes are investigated for the first time using molecular sequence data. Data derived from mitochondrial and nuclear DNA sequences for representatives of 17 genera were analyzed to test previous hypotheses of relationships among trichomycterid subfamilies, the monophyly of the subfamily Stegophilinae, and the monophyly and relationships among the genera of parasitic members of the family. We analyzed 2325 aligned base-pairs from mitochondrial 12S, 16S, ND4 (tRNA His tRNA Ser), and the nuclear histone H3 gene for representatives of 10 of 12 stegophiline and 3 of 4 vandelliine genera, plus 10 outgroup taxa selected to represent the range of subfamilial diversity. Maximum parsimony and likelihood approaches resolved a monophyletic semiparasitic Stegophilinae as the sister-group of the obligate hematophagous Vandelliinae. At the level of subfamilies, the pattern of relationships of the parasitic members among the remainder of the family is fully congruent with the most recent hypothesis of relationships for trichomycterids based exclusively on morphological data. Within stegophilines, our results differ from multiple previous morphological studies in recovery of (1) Haemomaster and Ochmacanthus as sister-taxa, (2) the morphologically plesiomorphic Pareidon microps nested within a relatively distal part of the tree topology, (3) Apomatoceros as sister to Henonemus, rather than to the morphologically similar Megalocentor. These result indicate that parasitism arose once and was unreversed within the Trichomycteridae. Survey of diet and feeding morphology among trichomycterids suggests that the semiparasitic lifestyle of the members of the Stegophilinae was retained in the enigmatic Pareiodon microps, despite reversal to the generalized trichomycterid condition of the associated morphological specializations found in all other stegophilines. These results further support the reconstruction of semiparasitism, rather than blood feeding, for the shared common ancestor of the parasitic Trichomycteridae.

Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm 5s 2U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (P S) or 15S rRNA C1409G allele (P R) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNA Lys, tRNA Glu, tRNA Gln, tRNA Leu, tRNA Gly, tRNA Arg and tRNA Phe were decreased significantly while those of tRNA Met and tRNA His were not affected in the mto1 strains carrying the P S allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNA Lys, tRNA Met and tRNA Phe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNA Lys, tRNA Leu, tRNA Arg but almost no effect in those of tRNA His. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.

Complete mitochondrial genome of Chinese big-headed turtle, Platysternon megacephalum, with a novel gene organization in vertebrate mtDNA

The mitochondrial genome of the Chinese big-headed turtle, Platysternon megacephalum, was obtained using polymerase chain reaction (PCR). The entire mtDNA sequence, the longest mitochondrial genome in turtles reported so far, is 19 161 bp. This mitochondrial genome exhibits a novel gene order, which greatly differs from that of any other vertebrates. It is characterized by four distinctive features: 1) the translocation of a gene cluster including three tRNA genes (tRNA His, tRNA Ser, tRNA Leu(CUN)) and ND5 gene, 2) two tRNA Thr pseudogenes, 3) a duplication of pseudo tRNA Thr/tRNA Pro/D-loop region and 4) 3 non-coding spacers. These unique identities represent a new mitogenomic gene order in vertebrates. The TDRL model was proposed to account for the generation of the gene order in P. megacephalum.

Inhibition of human immunodeficiency virus type 1 replication by siRNA targeted to the highly conserved primer binding site

The initiation of HIV-1 reverse transcription occurs at an 18-nucleotide sequence in the viral genome designated as the primer binding site (PBS), which is complementary to the 3′ terminal nucleotides of tRNA Lys,3. Since the PBS is highly conserved among all infectious HIV-1, it represents an attractive target for the development of new therapeutics to inhibit viral replication. In this study, we have evaluated three approaches using small interfering RNA (siRNAs) targeted to the PBS for the capacity to inhibit HIV-1 replication. In the first, transfection of a 21-nucleotide siRNA complementary to the PBS into cells inhibited production of HIV-1 following infection. Control siRNAs of the same length complementary to HIV-1 gag mRNA or to gfp mRNA decreased the production of virus or had no effect on virus replication, respectively. Analysis of the PBS of integrated proviruses derived from viruses that ultimately grew in cultures transfected with siRNA all contained wild-type PBS sequence, demonstrating that HIV-1 did not mutate to escape inhibition by siRNA. In the second approach, hairpin siRNA targeted to the wild-type PBS were expressed using an adeno-associated virus (AAV) vector. HIV-1 replication was inhibited in cells infected with AAV encoding the siRNA to the wild-type PBS, but not in cells infected with AAV encoding an siRNA of the same length targeted to an irrelevant PBS. Finally, studies from this laboratory have shown that alteration of the PBS to be complementary to tRNA His results in the production of infectious virus that rapidly reverts to utilize tRNA Lys,3 following in vitro culture. A proviral genome containing a PBS complementary to tRNA His that encodes an siRNA molecule complementary to the wild-type PBS under control of a U6 promoter within the nef gene was as infectious as the parent HIV-1 genome containing no insert in nef. The virus with the PBS only complementary to tRNA His reverted to use tRNA Lys,3, coincident with rapid virus growth, while the virus encoding siRNA grew slower than the virus without siRNA and maintained the PBS complementary to tRNA His longer in culture. At later times of infection, viruses with the PBS complementary to tRNA His and the siRNA exhibited a rapid increase in p24 antigen in the culture. Analysis of the PBS revealed that it was now complementary to tRNA Lys,3. Analysis of the gene encoding the siRNA revealed that the reversion of the PBS coincided with the deletion of the gene encoding siRNA. The results of these studies show that siRNA targeted to the PBS of HIV-1 can inhibit virus replication, supporting the concept that HIV-1 has evolved a strong preference to select tRNA Lys,3 for high-level replication and establishing the PBS and primer selection as a potential target for new therapeutics.

EEF1A binding to aminoacylated viral RNA represses minus strand synthesis by TYMV RNA-dependent RNA polymerase

The genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3′-end that can be valylated and then form a stable complex with translation elongation factor eEF1A·GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3′-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1A·GTP to the valylated viral RNA. eEF1A·GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1A·GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1A·GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1A·GTP was also observed with a methionylated variant of TYMV RNA and with aminoacylated tRNA His, tRNA Ala, and tRNA Phe transcripts. It is proposed that minus strand repression by eEF1A·GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA.

Molecular phylogeny of the armored catfish family Callichthyidae (Ostariophysi, Siluriformes)

The family Callichthyidae comprises eight genera of fishes widely distributed across the Neotropical region. In the present study, sequences of the mitochondrial genes 12S rRNA, 16S rRNA, ND4, tRNA His, and tRNA Ser were obtained from 28 callichthyid specimens. The sample included 12 species of Corydoras, three species of Aspidoras, two species of Brochis, Dianema, Lepthoplosternum, and Megalechis, and two local populations of Callichthys and Hoplosternum. Sequences of Nematogenys inermis (Nematogenyidae), Trichomycterus areolatus, and Henonemus punctatus (Trichomycteridae), Astroblepus sp. (Astroblepidae), and Neoplecostomus paranensis, Delturus parahybae, and Hemipsilichthys nimius (Loricariidae) were included as the outgroup. Phylogenetic analyses were performed by using the methods of maximum parsimony and maximum likelihood. The results of almost all analyses were very similar. The family Callichthyidae is monophyletic and comprises two natural groups: the subfamilies Corydoradinae ( Aspidoras, Brochis, and Corydoras) and Callichthyinae ( Callichthys, Dianema, Hoplosternum, Lepthoplosternum, and Megalechis), as previously demonstrated by morphological studies. The relationships observed within these subfamilies are in several ways different from those previously proposed on the basis of morphological data. Molecular results were compared with the morphologic and cytogenetic data available on the family.

Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells

Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA Asn and tRNA His, mannosyl-queuosine (manQ) in tRNA Asp, and galactosyl-queuosine (galQ) in tRNA Tyr. An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA Asp is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.

Complete mitochondrial DNA sequence of the swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura)

We determined the complete mitochondrial DNA sequence for a swimming crab Portunus trituberculatus (Miers, 1876) (Crustacea: Decapoda: Brachyura). The entire genome was amplified using a long PCR technique, and the products were subsequently used as templates for direct sequencing using a primer walking strategy. The genome (16,026 bp) contained the same 37 genes (two rRNAs, 22 tRNAs, and 13 proteins) plus the putative control region as found in other arthropods. The gene order of P. trituberculatus was largely identical to those so far obtained for other arthropods such as Drosophila yakuba, although the relative position of the tRNA His gene differed from all other arthropods studied so far. While the tRNA His gene in other arthropod mitochondrial genomes is located between the ND4 and ND5 genes, that of P. trituberculatus was found between the tRNA Glu and the tRNA Phe genes, which were located downstream of the ND5 gene. The present gene order could have resulted from tandem duplication occurring in the tRNA Phe - ND5- tRNA His region (typical gene order in other arthropods) and following deletions of redundant genes. This is the first report of a mitochondrial genome of a brachyuran crab, one of the most diversified and economically important groups of crustaceans.

Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome

We provide phylogenetic analyses for primary Reptilia lineages including, for the first time, Sphenodon punctatus (tuatara) using data from whole mitochondrial genomes. Our analyses firmly support a sister relationship between Sphenodon and Squamata, which includes lizards and snakes. Using Sphenodon as an outgroup for select squamates, we found evidence indicating a sister relationship, among our study taxa, between Serpentes (represented by Dinodon) and Varanidae. Our analyses support monophyly of Archosauria, and a sister relationship between turtles and archosaurs. This latter relationship is congruent with a growing set of morphological and molecular analyses placing turtles within crown Diapsida and recognizing them as secondarily anapsid (lacking a skull fenestration). Inclusion of Sphenodon, as the only surviving member of Sphenodontia (with fossils from the mid-Triassic), helps to fill a sampling gap within previous analyses of reptilian phylogeny. We also report a unique configuration for the mitochondrial genome of Sphenodon, including two tRNA Lys copies and an absence of ND5, tRNA His, and tRNA Thr genes.

Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome- c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA Leu(CUN), tRNA Ser(AGY), and tRNA His were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO. In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.

The residue immediately upstream of the RNase P cleavage site is a positive determinant

We have studied the importance of the residue at the position immediately upstream of the RNase P RNA cleavage site using model substrates that mimic the structure at and near the cleavage site of the tRNA His precursor. The various model substrates were studied with respect to cleavage site recognition as well as the kinetics of cleavage using M1 RNA, the catalytic subunit of Escherichia coli RNase P. Our studies showed that the identity of the residue immediately upstream of the cleavage site critically influences both these aspects. Among the ones tested, U is the preferred nucleotide at this position. Hence, these findings rationalize why most bacterial tRNA His genes/transcripts harbor a U immediately upstream of the RNase P cleavage site and extend our understanding of the cleavage site recognition process in general and the unusual cleavage of the tRNA His precursor in particular. Based on our as well as the data of others, we suggest that the nucleotide immediately upstream of the cleavage site is a positive determinant for cleavage by RNase P in general and the expression of tRNA genes is influenced by structural elements localized outside the promoter region i.e. in the leader and spacer regions of tRNA transcripts.

Regulated tRNA import in Leishmania mitochondria

The genes for three new tRNA and a 5S RNA were identified from a genomic DNA clone of 917 nucleotide pairs from the protozoon Leishmania tarentolae. They were encoded in the following order. The transcriptional directions and anticodons are in parentheses: tRNA Val (CAC→)–5SRNA (→)–tRNA His (←GUG)–tRNA Phe (GAA→). The tRNA His and tRNA Phe sequences have not been reported previously in trypanosomatid organisms. By northern analysis, tRNA Val and tRNA Phe were equally distributed between the cytosol and mitochondria, while tRNA His was less abundant in mitochondria than in the cytosol. Accordingly, the latter tRNA is classified as Import restricted (Imp r). As shown before, 5S RNA was not imported. Recently, Mahapatra and Adhya [S. Mahapatra, T. Ghosh, S. Adhya, Nucl. Acids Res. 22 (1994) 3381–3386; S. Mahapatra, S. Adhya, J. Biol. Chem. 271 (1996) 20432–20437] have developed an in vitro import system in Leishmania and suggested that the D-loop sequence could serve as the import determinant. We examined all available tRNA gene sequences in trypanosomatids but found no apparent consensus sequences within the D-loop that might account for tRNA-import regulation.