RESUMO
Post-transcriptional regulation is a key feature controlling gene expression in the protozoan parasite Leishmania. The nine-nucleotide paraflagellar rod regulatory element (PRE) in the 3'UTR of Leishmania mexicana PFR2 is both necessary and sufficient for the observed 10-fold higher level of PFR2 mRNA in promastigotes compared to amastigotes. It is also found in the 3'UTRs of all known PFR genes. A search of the Leishmania major Friedlin genomic database revealed several genes that share this cis element including a homolog of a heterotrimeric kinesin II subunit, and a gene that shares identity to a homolog of a Plasmodium antigen. In this study, we show that genes that harbor the PRE display promastigote-enriched transcript accumulation ranging from 4- to 15-fold. Northern analysis on episomal block substitution constructs revealed that the regulatory element is necessary for the proper steady-state accumulation of mRNA in L. mexicana paraflagellar rod gene 4 (PFR4). Also we show that the PRE plays a major role in the proper steady-state mRNA accumulation of PFR1, but may not account for the full regulatory mechanism acting on this mRNA. Our evidence suggests that the PRE coordinately regulates the mRNA abundance of not only the PFR family of genes, but also in a larger group of genes that have unrelated functions. Although the PRE alone can regulate some mRNAs, it may also act in concert with additional elements to control other RNA transcripts.
Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , Leishmania mexicana/fisiologia , Proteínas de Protozoários/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Regiões 3' não Traduzidas/fisiologia , Animais , Northern Blotting , DNA de Protozoário/química , DNA de Protozoário/genética , Leishmania major/genética , Leishmania mexicana/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA de Protozoário/genética , Análise de Sequência de DNA , Deleção de SequênciaRESUMO
The Leishmania mexicana PFR2 locus encodes a component of the paraflagellar rod (PFR), a flagellar structure found only in the insect stage of the life cycle. PFR2 mRNA levels are 10-fold lower in the mammalian stage than in the insect stage. Nuclear run-on experiments indicate that the change in PFR2 mRNA abundance is achieved posttranscriptionally. Deletion and block substitution analysis of the entire 1,400-nucleotide 3' untranslated region (UTR) of PFR2C led to the identification of a regulatory element contained within 10 nucleotides of the 3' UTR, termed the PFR regulatory element (PRE), that is necessary for the 10-fold regulation of PFR2 mRNA levels. Comparison of the half-lives of PFR2 transcripts, identical except for the presence or absence of the PRE, revealed that the PRE acts by destabilizing the PFR2 mRNA in amastigotes. The PRE was inserted into a construct which directs the constitutive expression of a chimeric PFR2 transcript. Insertion of the PRE resulted in regulated expression of this transcript, demonstrating that the regulatory element is sufficient for promastigote-specific expression. Since the PRE is present in the 3' UTR of all L. mexicana PFR genes examined so far, we propose that it serves a means of coordinating expression of PFR genes.