Physical and transcriptional analysis of the Trypanosoma brucei genome reveals a typical eukaryotic arrangement with close interspersionof RNA polymerase II- and III-transcribed genes

Marchetti, Maria A.; Tschudi, Christian; Silva, Erika; Ullu, Elisabetta

doi:10.1093/nar/26.15.3591

Cited by 28 publications

(23 citation statements)

References 26 publications

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“…The presence of a termination region between the two convergent polycistronic units on chr3 from LmjF suggests that Leishmania, like S. cerevisiae, might require separation of adjacent Pol II transcription units by proper termination signals to avoid transcriptional collision. It has been previously speculated that Pol III-gene regions might act as Pol II terminators in T. brucei, since Pol III genes have been found at the 3Ј boundary of Pol II transcription units in several cases (17). This also appears to be the case in many (but not all) of the convergent strandswitch regions in LmjF (unpublished data).…”

Section: Vol 3 2004mentioning

confidence: 82%

Transcription Initiation and Termination on Leishmania major Chromosome 3

et al. 2004

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Genome projects involving Leishmania and other trypanosomatids have revealed that most genes in these organisms are organized into large clusters of genes on the same DNA strand. We have previously shown that transcription of the entire Leishmania major Friedlin (LmjF) chromosome 1 (chr1) initiates bidirectionally between two divergent gene clusters. Here, we analyze transcription of LmjF chr3, which contains two convergent clusters of 67 and 30 genes, separated by a tRNA gene, with a single divergent protein-coding gene located close to the "left" telomere. Nuclear run-on analyses indicate that specific transcription of chr3 initiates bidirectionally between the single subtelomeric gene and the adjacent 67-gene cluster, close to the "right" telomere upstream of the 30-gene cluster, and upstream of the tRNA gene. Transcription on both strands terminates within the tRNA-gene region. Transient-transfection studies support the role of the tRNA-gene region as a transcription terminator for RNA polymerase II (Pol II) and Pol III, and also for Pol I.Leishmania is a protozoan parasite (order Kinetoplastida) which alternates life-forms between an intracellular amastigote stage residing in vertebrate macrophages and an extracellular promastigote stage living in the digestive tract of sandflies. The numerous human-infective Leishmania species cause a spectrum of disease ranging from asymptomatic to lethal, resulting in widespread human suffering and death, as well as considerable economic loss (35).Leishmania, as well as other members of the Trypanosomatidae family, possesses unusual mechanisms of gene expression, such as polycistronic transcription (13,19) and RNA editing of the mitochondrial transcripts (37). In these organisms, the mature nuclear mRNAs are generated from primary transcripts by trans-splicing, a process that adds a capped 39-nucleotide (nt) miniexon or splice leader (SL) to the 5Ј termini of the mRNAs (27). The steady-state levels of most of the mature mRNAs appear to be regulated posttranscriptionally by mechanisms that involve their 3Ј untranslated region sequences (23). Promoters for RNA polymerase I (Pol I) have been extensively characterized in trypanosomatids (31,44,46), as have some Pol III promoters (3,26). However, little is known about the sequences that drive the expression of protein-coding genes by Pol II.The Leishmania haploid genome content is ϳ34 Mb, consisting of 36 chromosomes which range in size from 0.3 to 2.5 Mb (40). The Leishmania Genome Network was established with the support of the World Health Organization to map and sequence the genome of Leishmania major Friedlin (LmjF), the reference strain of the project. The sequence of chromosome 1 (chr1), the smallest in the parasite, revealed the presence of 79 putative genes, the first 29 of which are in a cluster on the "bottom" DNA strand, while the remaining 50 are in a cluster on the "top" strand (21). Importantly, nuclear run-on analysis of chr1 showed that specific transcription, leading to the production of stable transcripts, ...

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Section: Vol 3 2004mentioning

confidence: 82%

Transcription Initiation and Termination on Leishmania major Chromosome 3

et al. 2004

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“…A survey of the T. brucei sequence project has resulted in the identification of 50 trypanosomal tRNA genes representing 40 different isoacceptor species (one tRNA with undetermined specificity was also detected [2] but will not further be discussed here) ( Table 2). Several of these genes have been characterized before by different research groups (6,7,15,25,(29)(30)(31)45). It is clear that the trypanosomal genome is not yet completely represented in the sequence database and therefore some tRNA genes may have escaped detection.…”

Section: Resultsmentioning

confidence: 99%

tRNAs inTrypanosoma brucei: Genomic Organization, Expression, and Mitochondrial Import

Tan

Pach

Crausaz

et al. 2002

Molecular and Cellular Biology

116

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The mitochondrial genome of Trypanosoma brucei does not encode tRNAs. Consequently, all mitochondrial tRNAs are imported from the cytosol and originate from nucleus-encoded genes. Analysis of all currently available T. brucei sequences revealed that its genome carries 50 tRNA genes representing 40 different isoacceptors. The identified set is expected to be nearly complete since all but four codons are accounted for. The number of tRNA genes in T. brucei is very low for a eukaryote and lower than those of many prokaryotes. Using quantitative Northern analysis we have determined the absolute abundance in the cell and the mitochondrion of a group of 15 tRNAs specific for 12 amino acids. Except for the initiator type tRNA Met , which is cytosol specific, the cytosolic and the mitochondrial sets of tRNAs were qualitatively identical. However, the extent of mitochondrial localization was variable for the different tRNAs, ranging from 1 to 7.5% per cell. Finally, by using transgenic cell lines in combination with quantitative Northern analysis it was shown that import of tRNA Leu (CAA) is independent of its 5-genomic context, suggesting that the in vivo import substrate corresponds to the mature, fully processed tRNA.

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“…[22][23][24]. Trypanosome U2, U3, and U4 snRNAs have a 2,2,7-trimethylguanosine cap (m 2,2,7 G) derived from cap 0 (25,26). However, these snRNAs appear to be synthesized by pol III (27,28), whereas other kinetoplastid pol III transcripts, such as tRNA, 5S RNA, and 7SL RNA, lack a cap structure.…”

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confidence: 99%

Trypanosoma brucei Encodes a Bifunctional Capping Enzyme Essential for Cap 4 Formation on the Spliced Leader RNA

Takagi

Sindkar

Ekonomidis

et al. 2007

Journal of Biological Chemistry

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The 5 end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m 7 GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.The 5Ј cap is an essential feature of eukaryotic mRNAs and snRNAs, 3 and is required for RNA stability and efficient translation (1). The m 7 GpppN (cap 0) is formed by sequential action of three enzymatic activities. The 5Ј triphosphate of the nacent RNA is hydrolyzed to a diphosphate by RNA triphosphatase, the diphosphate end is capped with GMP by guanylyltransferase, and the GpppN cap is methylated at the N-7 position by (guanine N-7) methyltransferase (2). Whereas the three-step capping reaction is universal to all eukaryotes, organization of individual activities differs between multicellular and unicellular eukaryotes (3). Metazoans and plants have a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase protein and a separate (guanine N-7) methyltransferase protein (4 -9). In contrast, fungi and other unicellular eukaryotes, including Encephalitozoon cuniculi, Giardia lamblia, and Plasmodium falciparum have a three-component system consisting of separate RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase gene products (10 -16). Cap is the earliest modification to the nascent transcripts synthesized by pol II. This specificity is achieved through the direct interaction of one or more components of the capping apparatus with the phosphorylated carboxyl-terminal domain of the large subunit of RNA pol II (7,(17)(18)(19)(20).In kinetoplastid protozoa, such as Trypanosoma brucei, capping is not restricted to pol II transcripts. Kinetoplastid mRNAs possess a unique hypermethylated cap structure called cap 4, which consists of cap 0 with 2Ј-O-methylations on the first four ribose sugars (A m A m C m U m ), and additional base methylations on the first adenine (m 6,6 A) and the fourth uracil (m 3 U) (21). The cap 4 str...

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Physical and transcriptional analysis of the Trypanosoma brucei genome reveals a typical eukaryotic arrangement with close interspersionof RNA polymerase II- and III-transcribed genes

Cited by 28 publications

References 26 publications

Transcription Initiation and Termination on Leishmania major Chromosome 3

Transcription Initiation and Termination on Leishmania major Chromosome 3

tRNAs inTrypanosoma brucei: Genomic Organization, Expression, and Mitochondrial Import

Trypanosoma brucei Encodes a Bifunctional Capping Enzyme Essential for Cap 4 Formation on the Spliced Leader RNA

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