Kinetoplast DNA minicircles encode guide RNAs for editing of cytochrome oxidase subunit III mRNA.

Guide RNAs (gRNAs) for the editing of sites 1-8 of COIII mRNA and an "unexpected" partially edited COIII mRNA are encoded in the variable regions of specific kinetoplast DNA minicircles. The gRNAs can form 37 and 44 nucleotide perfect hybrids (allowing for G-U base pairs) with edited mRNAs. The gRNAs were detected on Northern blots and shown to have unique 5' ends situated close to the beginning of the potential base pairing with the edited mRNAs. We suggest that kinetoplast DNA minicircle molecules in general may encode gRNAs for editing of cryptogene mRNAs by a mechanism similar to that previously proposed for editing by maxicircle-encoded gRNAs.     

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.     

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.     

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.     

Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes

Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.     

Protonatable hairpins are conserved in the 5'-untranslated region of tymovirus RNAs.

The secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison. Despite large sequence differences, there are remarkable similarities in the secondary structure. In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches. Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied. UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions. The hairpin found in the 5'-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour. It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5'-UTR. Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding.     

kDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication

The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction.     

Variants within the yeast Ty sequence family encode a class of structurally conserved proteins.

The Ty transposable elements of Saccharomyces cerevisiae form a heterogeneous family within which two broad structural classes (I and II) exist. The two classes differ by two large substitutions and many restriction sites. We show that, like class I elements a class II element, Tyl-17, also appears to contain at least two major protein coding regions, designated TYA and TYB, and the organisational relationship of these regions has been conserved. The TYA genes of both classes encode proteins, designated p1 proteins, with an approximate molecular weight of 50 Kd and, despite considerable variation between the TYA regions at the DNA level, the structures of these proteins are remarkably similar. These observations strongly suggest that the p1 proteins of Ty elements are functionally significant and that they have been subject to selection.     

Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes.

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircles from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircles. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of minicircle replication.     

Strong sequence conservation of a 38 bp region near the center of the extrachromosomal rDNA palindrome in different Tetrahymena species.

The restriction-endonuclease map and the nucleotide sequence of the central region in the extrachromosomal rDNA palindrome of two micronuclear and one a-micronucleate species of Tetrahymena has been determined. The sequence data show that the different species investigated have a 24 or 26 nucleotide sequence region at the very center of the rDNA molecule which is non-palindromic. Comparison of the present sequence data with the published data of another micronucleate species reveal that a segment of 38 base pairs just outside the non-palindromic center is highly conserved in all the different species, while the rest of the central region show little sequence homology. The relevance of this conserved region to the amplification process of the rDNA molecule is discussed.     

The divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA contains a diverse set of repetitive sequences.

A 2.76 kb segment of the 12 kb divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA consists almost entirely of repeated sequences. The repeats can be grouped into six families, some of which are present throughout the remainder of the divergent region. The repeats are oriented in a head-to-tail fashion with the three simplest repeats clustered into large arrays. A 47 bp palindrome and two copies of a "supercluster" of three different types of repeats are also present in the sequenced region. A sequence change in the divergent region is described for a clonal strain of L. tarentolae which was passaged continuously for several years. The repetitive sequences found in the divergent region appear to be appropriate substrates for the presumed deletion/insertion/recombination events occurring in this rapidly evolving portion of the maxicircle.     

Sequence-dependent in vivo importation of tRNAs into the mitochondrion of Leishmania tarentolae.

Sequence determinants for the importation of tRNAs into the mitochondrion of Leishmania tarentolae in vivo were investigated. tRNA(Ile)(UAU) is exclusively localized within the mitochondrion and tRNA(Gln)(CUG) exclusively in the cytosol (Lye LF, Chen DHT, Suyama Y, 1993, Mol Biochem Parasitol 58:233-246; Shi X, Chen DHT, Suyama Y, 1994, Mol Biochem Parasitol 65:23-37). L. tarentolae cells were transfected with plasmids encoding either tRNA(Ile) or tRNA (Gln) that were tagged with altered sequences in the D loop, permitting discrimination from the endogenous tRNAs. Primer extension analysis was used to show that the plasmid-encoded genes were expressed and that the tagged tRNAs showed a similar intracellular localization as the endogenous tRNAs. Exchange or deletion of the 5'-flanking genomic sequences had no effect on the expression or mitochondrial localization of the tagged tRNA(Ile) or on the expression or cytosolic localization of the tagged tRNA(Gln), suggesting that the signals for importation are localized within the tRNA itself. Swapping the D loop+stem from the exclusively cytosolic tRNA(Gln) with that from the tRNA(Ile) produced a partial mitochondrial localization of the plasmid-expressed mutated tRNA(Gln). However, D loop exchange did not eliminate the mitochondrial localization of the plasmid-expressed mutated tRNA(Ile), suggesting that tertiary structure or additional sequence elements may be involved in the importation signal.     

Formation of the 3'' end of U1 snRNA is directed by a conserved sequence located downstream of the coding region.

U1 is a small non-polyadenylated nuclear RNA that is transcribed by RNA polymerase II and is known to play a role in mRNA splicing. The mature 3' end of U1 snRNA is formed in at least two steps. The first step generates precursors of U1 RNA with a few extra nucleotides at the 3' end; in the second step, these precursors are shortened to mature U1 RNA. Here, I have determined the sequences required for the first step. Human U1 genes with various deletions and substitutions near the 3' end of the coding region were constructed and introduced into HeLa cells by DNA transfection. The structure of the RNA synthesized during transient expression of the exogenous U1 gene was analyzed by S1 mapping. The results show that a 13 nucleotide sequence located downstream from the U1 coding region and conserved among U1, U2 and U3 genes of different species is the only sequence required to direct the first step in the formation of the 3' end of U1 snRNA.     

Unusual polypeptide synthesis in the kinetoplast-mitochondria from Leishmania tarentolae. Identification of individual de novo translation products.

The de novo synthesis of cytochrome c oxidase subunits I, II (COI and COII), and apocytochrome b (Cyb) was investigated in kinetoplast-mitochondria of Leishmania. The organelles were isolated after breaking whole cells with nitrogen cavitation. Individual COI, COII, and Cyb polypeptides were identified by fractionation of the kinetoplast membranes, labeled with [(35)S]methionine and cysteine, using two-dimensional (9 versus 14% and 20 versus 11%) denaturing gel electrophoresis. The reaction did not require exogenous energy sources or amino acids. On the contrary, the presence of amino acids other than methionine somewhat inhibited the labeling reaction probably by competing with the uptake of labeled amino acids. The synthesis reaction was insensitive to 100 microg/ml chloramphenicol, gentamycin, paromomycin, lincomycin, hygromycin, and tetracycline, as well as cycloheximide. The process showed a linear increase in the amount of synthesized polypeptides during the first 2 h of incubation, followed by a slower accumulation of products for up to 4 h. The de novo synthesized polypeptides were stable for several additional hours. Their assembly into respiratory complexes, investigated using two-dimensional Blue Native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine-SDS gels, began early during the incubation and continued throughout the course of the synthesis. This work represents the first unequivocal identification of the polypeptide synthesis in kinetoplasts.