Mitochondrial DNA of kinetoplastids.
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Results for “RNA, Guide, Kinetoplastida”
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The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions.
The guide RNA (gRNA) paradigm states that the uridine (U) insertion/deletion type of RNA editing is mediated by short 3' uridylylated gRNAs that are complementary to specific blocks of mature edited sequence. These gRNAs contain the edited sequence information in the form of guiding purine residues that can base pair with the inserted U's and do not base pair with encoded U's that are to be deleted. The minicircle gRNA genes in trypanosomatids are localized at specific sites within the variable region, with the number and the precise localization of genes also being species-specific. The total number of minicircle sequence classes and thereby minicircle-encoded gRNAs varies greatly between species and even between different strains of the same species, with the greatest number being in the trypanosome species. Several conundrums which appeared to raise problems for the gRNA paradigm arose during comparative analysis of minicircle gRNA gene organization. The solution of these conundrums has led to a better understanding of the function and evolution of this RNA modification phenomenon.
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Guide RNAs and guide RNA genes in the cryptobiid kinetoplastid protozoan, Trypanoplasma borreli.
Trypanoplasma borreli belongs to the bodonid/cryptobiid group of kinetoplastid protozoa, which represents a sister group to the trypanosomatids. RNA transcripts from several mitochondrial genes in this organism undergo the trypanosomatid type of uridine addition/deletion RNA editing. A guide RNA (gRNA) cDNA library was constructed and five gRNAs were identified, one for editing the ribosomal protein S12 mRNA, three for editing the cytochrome oxidase subunit I mRNA, and one for editing the cytochrome b mRNA. All of the gRNAs contained nonencoded oligo[U] sequences at the 3' end, as is common with gRNAs in trypanosomatids, but also contained nonencoded oligo[U] sequences at the 5' end. The mechanism for addition of the 5' nonencoded oligo[U] sequence and the function of this sequence are unknown. The T. borreli gRNAs were shorter (25-35 nt, excluding the 5' oligo[U]) than gRNAs in trypanosomatids (45-50 nt), indicating a smaller size of editing blocks in this organism. Genomic sequences for two gRNAs were cloned and sequenced. These two gRNA-encoding sequences were shown to originate from the 180-kb Component I molecules, which represent a possible homologue of minicircle DNA in trypanosomatids, and not from the 80-kb Component II molecules, which contain the structural genes and cryptogenes.
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The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.
Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.
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RNA editing in kinetoplastid protozoa.
RNA editing produces mature mRNAs by adding and removing uridines within the mitochondrial transcripts. The edited sequence appears to be specified by small complementary RNAs using a non-templated process that may have features resembling RNA splicing. The accumulation of edited mRNAs is developmentally regulated.
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Native mRNA editing complexes from Trypanosoma brucei mitochondria.
The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.
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Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata.
We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.
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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.
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Disruption of RNA editing in Leishmania tarentolae by the loss of minicircle-encoded guide RNA genes.
RNA editing in kinetoplastids appears to be a labile genetic trait that is affected by prolonged cell culture. The transcripts of the G1-G5 cryptogenes are pan-edited in the recently isolated LEM125 strain of Leishmania tarentolae, but not in the UC strain which has been in culture for 55 years. At least 32 minicircle-encoded guide RNAs (gRNAs) for the editing of G1-G5 transcripts are present in LEM125 and absent in UC. We hypothesize that specific minicircle sequence classes encoding gRNAs for the editing of these transcripts were lost during the long culture history of the UC strain. The protein products, which include components of complex I of the respiratory chain, are probably not required during the culture stage of the Leishmania life cycle.
Amino Acid Sequence↗
Investigating the mechanism and machinery of RNA editing.
RNA editing in trypanosomes produces mature mRNAs by posttranscriptional guide RNA (gRNA)-directed uridylate insertion and deletion. This article describes methods for the study of RNA editing with an emphasis on an in vitro editing system that was used to explore the general mechanism of editing and that can be adapted for more in-depth studies of this intriguing and important process. Methods used to investigate the macromolecular complex that catalyzes RNA editing are also described. This complex is composed of multiple proteins and contains several catalytic activities. It is in the early stages of characterization. The methods described here are intended to assist in its further analysis.
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In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing.
A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.
Base Sequence↗
RNA editing in trypanosomes. The us(e) of guide RNAs.
Guide RNAs are encoded in maxicircle and minicircle DNA of trypanosome mitochondria. They play a pivotal role in RNA editing, a process during which the nucleotide sequence of mitochondrial RNAs is altered by U-insertion and deletion. Guide RNAs vary in length from 35 to 78 nucleotides, which correlates with the variation in length of the three functionally important regions of which they are composed: (i) a 4-14 nucleotide 'anchor' sequence embedded in the 5' region, which is complementary to a target sequence on the pre-edited RNA downstream of an editing domain, (ii) a middle part containing the editing information, which ranges from guiding the insertion of just one U into one site to that of the insertion of 32 Us into 10 sites, and (iii) a 5-24 nucleotide 3' terminal oligo [U] extension. Moreover, a variable uridylation site creates gRNAs containing a varying segment of editing information for the same domain. Comparison of different guide RNAs demonstrates that, besides the U-tail, they have no obvious common primary and secondary sequence motifs, each particular sequence being unique. The occurrence in vivo and the synthesis in vitro of chimeric molecules, in which a guide RNA is covalently linked through its 3' U-tail to an editing site of a pre-edited RNA, suggests that RNA editing occurs by consecutive transesterification reactions and is evidence that the guide RNAs not only provide the genetic information, but also the Us themselves.
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The absence of genes for cytochrome c oxidase and reductase subunits in maxicircle kinetoplast DNA of the respiration-deficient plant trypanosomatid Phytomonas serpens.
By completing the sequencing of the maxicircle conserved region in the kinetoplast DNA of Phytomonas serpens, we showed that the genes for subunits I and II (COI and COII) of cytochrome c oxidase in this organism were missing. We had previously shown that the genes for cytochrome c oxidase subunit III and apocytochrome b were also missing. These deletions did not affect the structure or expression of the remaining genes. Partial editing of the mRNA for NADH dehydrogenase subunit 8, previously found in strain IG from insects, was complete in two other strains isolated from plants. The appearance of a novel maxicircle gene for MURF2 block I gRNA, which substitutes for the gene missing due to the COII gene deletion, may illustrate a general mechanism for the origin of gRNAs.
Amino Acid Sequence↗
Organization of minicircle genes for guide RNAs in Trypanosoma brucei.
We have identified four T. brucei minicircle sequences that are complementary to cytochrome oxidase III (COIII) edited mRNA sequence and have shown the existence of transcripts from three of these minicircle sequences. These minicircle transcripts potentially serve as guide RNAs (gRNAs) for RNA editing of the COIII transcript. These gRNAs range in size from 55 to 70 nucleotides, are heterogeneous in sequence, and have a 5' terminal triphosphate. The genes for these gRNAs are flanked by imperfect 18 bp repeats separated by approximately 110 bp. Transcription initiates at the first purine within a conserved sequence, 5'-RYA-YA-3', 31 or 32 bp from the upstream inverted repeat. We propose that these 18 bp inverted repeats are important for minicircle gRNA expression in T. brucei.
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Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: misediting produced by misguiding.
We have analyzed the generation of unexpected patterns of RNA editing, i.e., those not following a strict 3' to 5' progression, which occur in junction regions between fully edited and preedited sequences. Evidence is presented that these patterns are generated by misediting due to specific events of misguiding. Misediting can occur through the interaction of inappropriate gRNAs with mRNAs or appropriate gRNAs in an incorrect fashion. Four possible mechanisms for the generation of misedited sequences are presented. Chimeric molecules have been detected in steady-state mitochondrial RNAs that are composed of misguiding gRNAs covalently linked to mRNAs at misediting sites by the 3' oligo(U) tail. We propose that misediting within junction regions can be corrected by appropriately acting gRNAs.
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Conservation of gRNA gene cassette structure in African trypanosomes despite divergence in the defining flanking repeats.
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Editing and misediting of transcripts of the kinetoplast maxicircle G5 (ND3) cryptogene in an old laboratory strain of Leishmania tarentolae.
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Leishmania gymnodactyli and Leishmania infantum minicircles contain the same guide RNA genes as do minicircles of Leishmania tarentolae.
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