RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.

Fantoni, A; Daré, Amos O.; Tschudi, Christian

doi:10.1128/mcb.14.3.2021

Cited by 68 publications

(59 citation statements)

References 45 publications

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“…In most eukaryotes, a single set of capping enzymes is sufficient to cap mRNA and snRNA as they are both transcribed by pol II and the cap is added co-transcriptionally to all pol II transcripts. In trypanosomes, while the SL RNA is transcribed by pol II and capped co-transcriptionally, the trimethylguanosine-capped U2, U3, and U4 snRNAs appear to be transcribed by pol III (Tschudi et al 1986;Mottram et al 1989;Fantoni et al 1994;Nakaar et al 1994). It is not clear whether U snRNAs are capped co-transcriptionally, as other pol III transcripts, such as tRNA, 5S RNA, and 7SL RNA, lack a cap structure.…”

Section: Discussionmentioning

confidence: 91%

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

Hall

Ho²

2006

RNA

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The m 7 GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m 7 GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324-amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from Sadenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m 7 GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predictedon the basis of the crystal structure of Ecm1-to be located at or near the active site identified six conserved residues in the putative AdoMet-or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050-amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain, which is homologous with TbCmt1, and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily.

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Section: Discussionmentioning

confidence: 91%

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

Hall

Ho²

2006

RNA

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“…There is no obvious sequence homology between the upstream sequences or the 5' end of the coding region between the 87U6 gene and the 7SL gene. Another recent report describes a trypanosome U2 gene that is transcribed by RNA polymerase III and requires upstream sequences as well as coding sequences near the 5' end of the gene for efficient transcription (10). Both of these promoters require 5' flanking sequences to be efficiently expressed.…”

Section: Discussionmentioning

confidence: 99%

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Tichelaar¹,

Knerer²,

Vrabel³

et al. 1994

Mol. Cell. Biol.

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Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.The transcription of the small nuclear RNA U6 has been well characterized in both vertebrate (2,8,17,21,22,28) and yeast (6, 7, 9) species. In the three vertebrate species in which a U6 gene has been cloned (humans, mice, and Xenopus tropicalis), transcription has been shown to be entirely dependent on 5' flanking sequences, including a classical TATA box and the U small nuclear RNA specific proximal sequence element (for review, see references 19 and 38). Both of these elements are normally found in RNA polymerase II promoters but have been shown to function in the RNA polymerase III promoter of U6. Indeed, the TATA box has been shown to be the element responsible for conferring RNA polymerase III specificity to the U6 promoter (23,24). In yeast species, the promoter structure is more complex, consisting of both external and internal elements. The U6 gene in Saccharomyces cerevisiae requires a 5' flanking TATA box, but transcription in vivo requires the presence of an internal A block and a B block located 120 nucleotides downstream (4, 6, 7, 9). The fission yeast Schizosaccharomyces pombe also contains a potentially functional B block homology within the intron of its U6 gene (11,32). However, in all cases studied thus far, the transcription of U6 relies wholly or in part on sequences upstream of the coding region.The human genome contains approximately 200 copies of U6-related sequences (14), only five of which have been sequenced and only three of which have been stud...

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“…Bloodstream-form parasites (BSF-SM) were grown in HMI-9 medium at 37°C supplemented with 10% Tet -serum and 3 μg/ml G418 to maintain the RNAi machinery. Cells were grown and transfected as described previously (Fantoni et al, 1994).…”

Section: Cell Culture and Transgenic Expressionmentioning

confidence: 99%

TbG63, a golgin involved in Golgi architecture inTrypanosoma brucei

Ramirez

Graffenried

Ebersberger

et al. 2008

Journal of Cell Science

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Golgins are coiled-coil proteins that have been implicated in the structure and function of the Golgi complex. Here, we identify and characterize a trypanosomal golgin, TbG63, showing that it has a C-terminal membrane anchor and an N-terminus that projects into the cytoplasm. TbG63 in procyclic parasites is localized to the Golgi and interacts with the active, GTP-form of TbRab1A. Overexpression of TbG63 has dramatic effects on Golgi architecture – effects that require the N-terminus – whereas depletion has little, if any, effect on the growth rate. By contrast, in the bloodstream form of the parasite, depletion of TbG63 slows growth, although it has no obvious effect on the transport of a variant surface glycoprotein (VSG) or on Golgi structure. TbG63 might be a useful tool to study the structure and functioning of the Golgi complex.

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RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.

Cited by 68 publications

References 45 publications

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

TbG63, a golgin involved in Golgi architecture inTrypanosoma brucei

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