Unusual organization of a developmentally regulated mitochondrial RNA polymerase ( TBMTRNAP ) gene in Trypanosoma brucei

Clement, Sandra L.; Koslowsky, Donna J.

doi:10.1016/s0378-1119(01)00538-8

Cited by 19 publications

(17 citation statements)

References 30 publications

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“…Comparison of the predicted amino acid sequence of the putative T. brucei mtRNAP with those of other organisms provides evidence that we have identified an mtRNAP and that it is the same candidate mtRNAP reported earlier (21,22). We demonstrate that the gene is in fact a mitochondrial specific RNA polymerase through functional analyses using RNA interference (RNAi).…”

supporting

confidence: 74%

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A Trypanosome Mitochondrial RNA Polymerase Is Required for Transcription and Replication

Grams¹,

Morris²,

Drew³

et al. 2002

Journal of Biological Chemistry

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Understanding mitochondrial transcription is a requisite first step toward understanding the regulation of mitochondrial gene expression in kinetoplastids. Here we report the identification and functional characterization of a mitochondrial RNA polymerase (mtRNAP) from Trypanosoma brucei, the first trans-acting factor involved in kinetoplast mitochondrial transcription to be identified. Using sequences conserved among the catalytic domains of the single-subunit mtRNAPs, we were able to obtain a full-length sequence for a candidate mtRNAP from T. brucei. Sequence comparison indicates that it shares homology in its catalytic domain with other single-subunit mtRNAPs, including functionally conserved residues that are identical in all single-subunit RNAPs. We used RNA interference to functionally knock out the gene product to determine whether the candidate gene represents an mtRNAP. As predicted for a mitochondrial specific RNA polymerase, reduction of the gene product resulted in a specific decrease of mitochondrial versus nuclear transcripts. Additionally, similar to the mtRNAP of other organisms, the mtRNAP characterized here is involved in replication of the mitochondrial genome. Thus, based on sequence comparison and functional studies, we have cloned an mtRNAP from trypanosomes.The mitochondrial genome of kinetoplastids is unusual in that it is comprised of large and small circular DNAs, maxicircles and minicircles, respectively, catenated into a giant network called kinetoplast DNA (1-3). Little is known of the mechanisms regulating gene expression of either maxicircles or minicircles. In Trypanosoma brucei, the 23-kb maxicircles represent the typical mitochondrial DNA, coding for rRNAs and several proteins involved in mitochondrial respiration. Similar to other organisms, numerous polycistronic transcripts have been detected, indicating that maxicircles are transcribed polycistronically (4 -6). No maxicircle promoter has yet been identified, although a precursor extending at least 1200 nucleotides upstream of the 12 S rRNA has been detected and may represent an initiation site for transcription (7).Many maxicircle genes encode cryptic transcripts that require the post-transcriptional insertion or deletion of uridines to produce a functional mRNA, which is a process known as RNA editing (Ref. 8; for reviews see Ref.9 -11). The information for the editing of maxicircle transcripts is provided by small RNAs termed guide RNAs (gRNAs). 1 With the exception of gMurfII-1 and gMurfII-2 (guide RNAs 1 and 2, respectively, for maxicircle unidentified reading frame II) found on the maxicircle, gRNAs are encoded by the second component of the kinetoplast, the minicircles (12-14). In T. brucei, minicircles are ϳ1-kb circular DNA molecules, and there are several thousand copies/kinetoplast network. Despite some conserved features, they are heterogeneous in sequence and provide the 250 -300 sequence classes necessary to edit maxicircle transcripts (15, 16).Each minicircle of T. brucei contains three or four potential gRNA ...

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confidence: 74%

“…As expected because mtRNAP would be required in active mitochondria, functional knockout of the candidate gene was lethal in procyclic form trypanosomes. Recently, a full-length sequence has been re-ported that represents the same candidate mtRNAP (22). However, there remains no functional demonstration that it is a mtRNAP.…”

mentioning

confidence: 99%

A Trypanosome Mitochondrial RNA Polymerase Is Required for Transcription and Replication

Grams¹,

Morris²,

Drew³

et al. 2002

Journal of Biological Chemistry

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“…However, mitochondrial polymerases are not typically multisubunit RNA polymerases, such as those found in the nucleus. Rather, mitochondrial polymerases thus far identified are singlesubunit polymerases, and such a mitochondrial polymerase for T. brucei has been identified (Wang et al 2000;Clement and Koslowsky 2001;Grams et al 2002). It seems unlikely to us, based on what is known about mitochondrial transcription, that REAP-1 would be acting as a transcription factor.…”

Section: Discussionmentioning

confidence: 99%

RNA-editing-associated protein 1 null mutant reveals link to mitochondrial RNA stability

Hans

Hajduk²,

Madison‐Antenucci³

2007

RNA

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In trypanosomatids, uridylate residues are post-transcriptionally added to or deleted from pre-mRNAs during the complex process of RNA editing. Editing is carried out exclusively in the mitochondrion of these parasites and involves numerous proteins assembled into protein and ribonucleoprotein complexes. Previously we identified RNA-editing-associated protein -1 (REAP-1), an RNA binding protein found in the mitochondrion of Trypanosoma brucei. REAP-1 was shown to specifically recognize and bind to pre-mRNAs that require editing and was proposed to act as a recruitment factor to deliver pre-mRNAs to editing complexes. To help define the role of REAP-1, we have now constructed REAP-1 null mutants. We show that the null mutants, although viable, have a significant growth defect. RNA levels within the mitochondrion were evaluated using reverse transcriptase real-time PCR. Surprisingly, the results show that mitochondrial RNA levels are increased, regardless of the editing status of the RNA. All RNA tested, whether unedited, edited, or never edited were increased in the mutant cell line relative to wild-type levels. This study provides the first evidence for a role of REAP-1 in RNA metabolism.

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“…Total T. brucei genomic DNA was isolated as described previously (7). Mitochondrial RNAs (mtRNAs) were isolated by the acid guanidinium-phenol-chloroform method (6).…”

Section: Methodsmentioning

confidence: 99%

“…The ligations were phenol-chloroform extracted, ethanol precipitated, and resuspended in 1ϫ reverse transcription buffer (50 mM KCl, 20 mM Tris [pH 8.5], 0.5 mM EDTA, 8 mM MgCl 2 ). Poisoned primer extension reactions were performed with each population of ligated RNA (1.0 g of mtRNA or 0.1 g of gRNA), with 100 kcpm (ϳ50 fmol) of primer in the presence of a 1.6 mM concentration (each) of dATP, dCTP, dTTP, and ddGTP, as described previously (7). Reactions were resolved in 8 M urea-8% acrylamide sequencing gels.…”

Section: Methodsmentioning

confidence: 99%

An Intragenic Guide RNA Location Suggests a Complex Mechanism for Mitochondrial Gene Expression in Trypanosoma brucei

Clement¹,

Mingler

Koslowsky³

2004

Eukaryot Cell

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In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events. We demonstrate that the conserved maxicircle gRNA gMURF2-II has an unusual location within the ND4 gene. This is the first report of a completely intragenic gene in kDNA. In addition, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5 end of the ND4 gene. The gCYb(560) gene has an atypical minicircle location in that it is not flanked by the inverted repeat sequences that surround the majority of minicircle gRNA genes. Our data indicate that the mature gCYb(560) gRNA is also a primary transcript and that the 5-end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not of imprecise 5-end processing. Together, these data indicate that gRNA genes represent individual transcription units, regardless of their genomic context, and suggest a complex mechanism for mitochondrial gene expression in T. brucei.

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Unusual organization of a developmentally regulated mitochondrial RNA polymerase ( TBMTRNAP ) gene in Trypanosoma brucei

Cited by 19 publications

References 30 publications

A Trypanosome Mitochondrial RNA Polymerase Is Required for Transcription and Replication

A Trypanosome Mitochondrial RNA Polymerase Is Required for Transcription and Replication

RNA-editing-associated protein 1 null mutant reveals link to mitochondrial RNA stability

An Intragenic Guide RNA Location Suggests a Complex Mechanism for Mitochondrial Gene Expression in Trypanosoma brucei

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