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

Abstract

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...

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

“…Thus, elements within the 3′ UTR of the TBMTRNAP mRNA are likely to be important for the developmental regulation of its expression during the T. brucei life cycle. Polyadenylation in T. brucei is dependent upon a downstream polypyrimidine track followed by a trans-splice site (Matthews et al, 1994;Schurch et al, 1994). Trans-splicing is rapid and seems to occur as soon as the splice site is available to the processing machinery (Ullu et al, 1993).…”

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei

“…A clear shift towards a shorter poly(A) tail, or no tail, was observed in samples after 6 h incubation with ActD. We also identified three alternative poly(A) sites, located in close proximity to the major polyadenylation site [41] (Fig. 5B).…”

“…A possible explanation might be that processing of GPEET transcripts is altered in late PF, thus preventing the generation of mature mRNA. Interestingly, the genomic organisation of GPEET and the adjacent EP3 gene is unusual, as the intergenic region contains two distinct polypyrimidine stretches followed by a downstream AG dinucleotide, which both can function as polyadenylation/splice acceptor sites [41,49]. Trans-splicing of EP3 mRNA can thus be uncoupled from 3 end formation of the upstream GPEET mRNA, permitting alternative processing of GPEET transcripts in late PF without interfering with EP3 maturation.…”

Control of gene expression in Trypanosomatidae

“…Several experiments using deletion mutants have tested this hypothesis and have shown the importance of intergenic regions in governing trans-splicing and polyadenylation in T. brucei. In addition to the correct distance, the presence of a polypyrimidine-rich motif is also crucial, since only AG dinucleotides situated downstream from a polypyrimidine tract are used as splice acceptor site ( Figure 1) (4)(5)(6)8).…”