Trypanosoma brucei: enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site.

Abstract

The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs) (E. Pays, P. Tebabi, A. Pays, H. Coquelet, P. Revelard, D. Salmon, and M. Steinert, Cell 57:835-845, 1989). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model.In African trypanosomes, antigenic variation is achieved by frequent changes of the variable surface glycoprotein (VSG). Different VSG genes can be expressed, but only one is usually transcribed at a time, and different switching mechanisms have been described (for recent reviews, see references 3, 19, and 26). The transcribed VSG gene is located at the end of a telomeric expression site, which also contains other genes (expression site-associated genes [ESAGs]) and is repeated in 5 to 20 slightly different versions in the genome. This transcription exhibits several characteristics: (i) it resists inhibition by oa-amanitin; (ii) it is preferentially stopped by a lowering of temperature; and (iii) it seems to start upstream from the ESAGs, so that several genes may belong to the same transcription unit (1, 10, 19a, 23). The latter characteristic has also been reported for other genes, such as tubulin, calmodulin, and actin genes and genes of the glycolytic pathway, suggesting that in Trypanosoma brucei, transcription in general might be polycistronic (2,5,8,9,25). However, no direct evidence of the existence of polycistronic transcripts has yet been provided. The trypanosome genes appear devoid of introns, but splicing of the RNA precursors occurs; a 39-nucleotide sequence called the miniexon is cleaved from a short precursor and is added at the 5' termini of all mature mRNAs, irrespective of whether these RNAs are transcribed from the same chromosome as the miniexon (7,11,16,24). This splicing appears necessary for RNA translation (4,27).In an attempt to map the transcription promoter of the AnTat 1.3A VSG gene expression site, we have measured the relative sensitivity of its transcription to inhibition by UV irradiation, as was done for another VSG gene expression site by Johnson et al. (9). As reported previously (19a), we found that transcription of a particular region, located about 45 kilobases (kb) upstream from the VSG gene, appears to be selectively and strongly stimulated by UV light. This apparent stimulation also characterizes the promoter reg...

Genomic and transcriptional linkage of the genes for calmodulin, EF-hand 5 protein, and ubiquitin extension protein 52 in Trypanosoma brucei.

“…7A), we show that transcription of the rRNA gene is inactivated proportionally to its distance from the promoter and that transcription of the 140-bp miniexon (Fig. 7B), which is consistent with the results of Coquelet et al (5,6). Most importantly, synthesis is still observed after 2 min of UV irradiation.…”

A monocistronic transcript for a trypanosome variant surface glycoprotein.

“…Nuclear run-on assays, coupled with UV inactivation of transcription, have been used to demonstrate that for at least some VSG ESs, transcription is initiated 45 to 60 kb upstream of the VSG gene and proceeds through eight or more open translation reading frames, the last of which specifies the VSG (12,30,37,42,56). This transcription occurs in the presence of ox-amanitin, leading to suggestions that it is catalyzed by an RNA polymerase I activity or by a modified RNA polymerase II complex (26,32,59,75).…”

A similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of Trypanosoma brucei.

“…1356 (7). The combination of inhibition of both RNA decay and RNA elongation by UV can account for the characteristic accumulation of promoter-proximal transcripts which has been observed for the VSG (8,23,24,27), procyclin (6,7,23,24), rDNA (6,7,27), and actin gene (3a) transcription units. Such an apparent stimulation of transcription is thus a marker for the proximity of a promoter, while the degree of inhibition of transcription is indicative of the distance to the promoter.…”

“…In order to determine whether all these genes were part of the same transcription unit, the effect of UV irradiation on run-on transcription was analyzed. We have shown before (7,8,23,24,27) Fig. 4); the EcoRI sites at the ends of the cDNA were generated by the cloning procedure (11).…”