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Book Summary:
The protozoan parasite Leishmania undergoes dramatic morphological and biochemical changes as it differentiates between the promastigote and amastigote forms. The molecular mechanisms used to modulate gene expression in this organism are predominately post-transcriptional. We examined the Leishmania mexicana transcriptome using high-density whole-genome oligonucleotide microarrays designed from the genome data of a closely related species, Leishmania major. Statistical analysis on array hybridization data representing 8160 predicted coding regions revealed 182 genes (2.2% of all genes) whose steady-state mRNA levels show at least 2-fold differential regulation between promastigotes and lesion-derived amastigotes. Sample comparison of promastigotes to axenic amastigotes resulted in only 31 genes (0.4%) that meet the same statistical criteria for differential regulation. The reduced number of regulated genes is a consequence of a decrease in the magnitude of the transcript expression in cells under axenic conditions. A nine-nucleotide 3\'UTR regulatory element, termed the paraflagellar rod (PFR) regulatory element (PRE) is both necessary and sufficient for the observed higher levels of PFR2 mRNA in promastigotes compared to amastigotes, and it functions by shortening the half-life of transcripts in the amastigote stage. The PRE is present in the 3\'UTR of all L. mexicana PFR genes known to date, including PFR1 and PFR4 . Northern analyses show that mRNA of PFR1, PFR4, and two additional genes that share this cis element are enriched in the promastigote stage. Block substitution analyses revealed that the presence of the PRE is responsible for the observed regulation of PFR4 and also has a measurable role in the regulation of PFR1. Our studies show that interspecies hybridization on microarrays can be used to analyze closely related protozoan parasites, that axenic culture conditions may alter amastigote transcript abundance, and that there is only a relatively modest change in abundance of a few mRNAs between morphologically distinct promastigote and amastigote cultured cells. Although our microarray data present evidence of an alternative paradigm for eukaryotic differentiation with minimal contributions from changes in mRNA abundance, we show that PRE-dependent mRNA stability is the primary means of regulation in a family of functionally related promastigote-enriched genes in Leishmania mexicana. |
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