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

Wong, Sandy; Morales, Tony; Neigel, Joseph E.; Campbell, David A.

doi:10.1128/mcb.13.1.207

Cited by 31 publications

(14 citation statements)

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“…14,1994 on March 21, 2019 by guest http://mcb.asm.org/ Downloaded from Pyrimidine-rich regions followed by the dinucleotide AG function as splice acceptor sites in trypanosomes (10), and it has been shown for the DHFR-TS locus of L. major that one element regulates both polyadenylation and trans splicing (15). This may also be true for a number of transcription units in T. brucei, particularly in cases in which the intergenic regions are small and the first polypyrimidine tract beyond a given gene is the splice acceptor site for the next gene (32,37). In the procyclin and VSG transcription units, however, the (putative) signals for polyadenylation do not correspond to the major splice acceptor sites of downstream genes.…”

Section: Resultsmentioning

confidence: 99%

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Schürch¹,

Hehl²,

Vassella³

et al. 1994

Mol. Cell. Biol.

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Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.The genome of Trypanosoma brucei is organized into clusters of genes which are coordinately transcribed as polycistronic precursor RNAs (reviewed in reference 3). These RNA precursors are subsequently processed into monocistronic mRNAs, each with a 5' miniexon or spliced leader sequence which is acquired by trans splicing (21, 33) and a poly(A) tail. The sequences determining the 3' splice acceptor site for trans splicing in trypanosomes, a polypyrimidine tract followed by the dinucleotide AG, are essentially the same as those used by other organisms for cis splicing (10). In contrast, mRNAs from trypanosomes do not contain the canonical polyadenylation signal AAUAAA, which is found 10 to 30 bases upstream of the poly(A) addition site in most mRNAs from higher eukaryotes (36), and no alternative signals have been defined.The first indication that trans splicing and polyadenylation might be linked in T. brucei stemmed from the observation that 3' processing of ox-tubulin mRNAs was blocked by inhibitors of trans splicing (34). These results were summarized in a model in which addition of the miniexon to the 5' end of a nascent transcript preceded cleavage at the 3' end. More recently, an analysis of the dihydrofolate reductase-thymidylate synthase locus in a related parasite, Leishmania major, has shown that the polyadenylation of transcripts is affected by the 3' splice acceptor site of the next gene in the transcription unit (15). This is still consistent with a coupling of the two processes, but in this case 3' end formation of one mRNA would be linked to trans splicing of the downstream transcript.Some of the most intensively studied transcription units in T. normal housekeeping genes, the procyclin and VSG expression sites are transcribed by an RNA polymerase that is resista...

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Section: Resultsmentioning

confidence: 99%

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Schürch¹,

Hehl²,

Vassella³

et al. 1994

Mol. Cell. Biol.

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“…In an additional example, previously reported by Wong et al (50), transcription of tubulin genes also shows a moderate sensitivity to UV irradiation. Wong et al interpreted this finding to indicate a transcription start site existing proximal to the first tubulin gene in the cluster.…”

Section: Discussionmentioning

confidence: 99%

“…Through the processing events of poly(A) addition and trans splicing, intergenic regions are excised from the precursor RNA, resulting in the generation of the mature mRNAs. As a result of the polycistronic transcription of protein-coding genes, the differential expression of steady-state mRNAs from a single polycistronic transcript requires posttranscriptional control (14,50). The mechanism of posttranscriptional control is not yet completely clear.…”

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confidence: 99%

An RNA Polymerase II Promoter in the hsp70 Locus of Trypanosoma brucei

Lee

1996

Molecular and Cellular Biology

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To study the structure of RNA polymerase (pol) II transcription units and the influence of temperature on the regulation of gene expression in Trypanosoma brucei, an hsp70 intergenic region promoter was characterized. In T. brucei, the hsp70 locus contains, from 5 to 3, a cognate hsp70-related gene (gene 1) which is separated by about 6 kb of DNA from a cluster of five identical hsp70 genes (genes 2 to 6). Transcription proceeds on the entire 23-kb locus, and polycistronic transcription occurs in hsp70 genes 2 to 6. Transcription of hsp70 genes 2 to 6 is only moderately sensitive to UV irradiation, indicating that it cannot be driven by a single far-upstream promoter, which suggests that promoters could be located in the region close to the hsp70 coding region. Transient transformations demonstrated that sequences located upstream of hsp70 gene 2 and in the intergenic region between hsp70 genes 2 and 3 are able to direct transcription of the reporter gene, the chloramphenicol acetyltransferase (CAT) gene. The plasmid DNA driven by the hsp70 intergenic region promoter gave CAT activity ϳ185-fold above the background level. This is equivalent to ϳ1% of that derived from a CAT plasmid driven by the procyclic acidic repetitive protein gene promoter, which is controlled by RNA pol I. The hsp70 intergenic region promoter can drive ␣-amanitin-sensitive transcription at an internal position of the chromosome as well as an episome, suggesting that it is controlled by RNA pol II. However, this hsp70 intergenic region promoter, along with the 3 splice site and the 5 untranslated region of the hsp70 genes that controls the transcription of the reporter gene, cannot up-regulate the expression of the reporter gene during heat shock. This result is consistent with the previous observation that expression of the hsp70 genes in T. brucei is mainly controlled at the posttranscriptional level.Transcription of protein-coding genes and mRNA maturation in trypanosomes and other kinetoplastida involve mechanisms which are different from those in most higher eukaryotes (1,6,37,47). Every mRNA in trypanosomes contains two exons, the 5Ј miniexon and the main coding exon, which are transcribed from two separate genes. Through trans splicing, a capped 39-nucleotide transcript is joined with the main coding exon (1,36,43). Because of the discontinuous synthesis of mRNA, the 5Ј end of the mRNA does not identify the transcription initiation site of the protein-coding genes but identifies that of the miniexon RNA. Therefore, it is still unclear where initiation of RNA polymerase (pol) II-transcribed protein-coding genes occurs.Most genes in trypanosomes are found in multiple copies which are organized in tandem arrays and are separated from each other by short intergenic region sequences of a few hundred nucleotides (19,38,46). These clustered genes are usually polycistronically transcribed. These polycistronically transcribed mRNAs can encode proteins that are differentially expressed or expressed at similar levels. Through the processing eve...

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“…These precursor transcripts are processed into individual mRNAs by cleavages at intergenic sites followed by the addition of a 39-nucleotide spliced leader to the resultant 5' ends and polyadenylation at the 3' ends (6,52,73). In general, the evidence for the existence of polycistronic transcription units has come from Northern (RNA) blots, nuclease protection assays, and nuclear run-on experiments (74). Some polycistronic transcripts are derived from tandem genes of the same family, such as those gene families encoding tubulin (51), actin (3), phosphoglycerate kinase (39), and a flagellar calcium-binding protein (22,29).…”

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confidence: 99%

“…Some polycistronic transcripts are derived from tandem genes of the same family, such as those gene families encoding tubulin (51), actin (3), phosphoglycerate kinase (39), and a flagellar calcium-binding protein (22,29). Other polycistronic transcripts contain the coding regions for different proteins, such as calmodulin, an EF-hand 5 protein, and ubiquitin (10,74), protein phosphatase and a RNA polymerase subunit (23,25), or the VSG and products of the adjacent expression site (ES)-associated genes (ESAGs). The steady-state levels of individual mRNAs derived from a specific polycistronic premRNA can either be similar or differ dramatically (43).…”

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confidence: 99%

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Alarcon¹,

Son²,

Hall³

et al. 1994

Mol. Cell. Biol.

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Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45-to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

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

Abstract: We report genomic linkage of a pair of tandem, identical ubiquitin-extension protein 52 DNA. The genomic library constructed in the cosmid vector c2XB has been described previously (41). The genomic clones p379 (0.4-kb HindIII-EcoRI), pU2Bgl2 207

Cited by 31 publications

References 48 publications

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

An RNA Polymerase II Promoter in the hsp70 Locus of Trypanosoma brucei

A monocistronic transcript for a trypanosome variant surface glycoprotein.

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