Poly(A)-binding protein I of Leishmania:: functional analysis and localisation in trypanosomatid parasites

Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional. Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript stability is a major factor controlling stage-specific gene expression. Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (LmPAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression. Impab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids. LmPAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo. It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus. LmPAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae, These properties are indicative of functional divergence in vivo.