Stage-specific differential polyadenylation of mini-exon derived RNA in African trypanosomes

Abstract

The differentiation of African trypanosomes through several distinct stages in their mammalian host and insect vector correlates with differential expression of some of the parasites' genes. In the search for genes from Trypanosoma brucei brucei involved in switching from the actively dividing long slender to the non-dividing short stumpy bloodstream forms, we have isolated cDNA clones which hybridise specifically to mRNA from short stumpy forms. All clones that were characterised contained similar sequences. Northern blot analysis showed that: (i) RNA transcripts which hybridise to these clones are barely detectable in the poly(A) + fraction of RNA from longslender bloodstream forms and absent in procyclic culture forms, but are abundant in the poly(A) + fraction of RNA from short stumpy forms; (ii) the RNA transcripts are abundant in the poly(A) − fraction of RNA from all life cycle stages of the trypanosomes, without significant differences and, (iii) three transcripts of 160, 280 and 400 nucleotides in size are detected in the poly(A) + fraction of RNA, whereas only a single size-class of transcript of between 140 and 160 nucleotides is detectable in the poly(A) − fraction. Sequence analysis revealed that these clones correspond to mini-exon derived RNA containing a poly(A) tail at their 3′ end. The polyadenylation of the transcript is a post-transcriptional event since sequences from genomic DNA could not be amplified in the polymerase chain reaction when mini-exon and oligo(dT) nucleotide sequences were used as primers. The differences in size of the transcripts detected can be accounted for by variations in the poly(A) tail length.