Coordinate regulation of a family of promastigote-enriched mRNAs by the 3′UTR PRE element in Leishmania mexicana☆
Introduction
Protozoan parasites of the genus Leishmania cause widespread diseases whose human manifestations range from self-healing cutaneous lesions to fatal visceral infections [1], [2], [3], [4]. The Phlebotomine sandfly transmits the parasite to human and other mammalian hosts, and the parasites adapt to their environments by cycling between insect-stage flagellated promastigotes and mammalian-stage spherical amastigotes [2], [5], [6]. This dramatic morphological change is accompanied by changes in the parasite's biochemistry and it is likely orchestrated by gene regulatory events [6]. Studying how the parasite accomplishes and controls these changes may lead to the elucidation of novel therapeutic targets [7], [8].
Leishmania and other trypanosomatid protozoans do not share the extensively studied gene expression pathways of bacteria or eukaryotes such as yeast and mammals which regulate primarily at the level of transcription [9]. Although transcription of T. brucei PARP and VSG gene expression can be controlled by specialized RNA polymerase I promoters [10], [11], [12], [13], canonical RNA polymerase II promoters are not known to be intimately associated with protein coding genes in Trypanosomatids. RNA polymerase II promoters may be present in strand-switch regions of the Leishmania chromosomes where they transcribe the genome into long polycistronic pre-mRNAs [14], [15]. Mature mRNAs are chimeric molecules formed by processing polycistronic pre-mRNAs via the trans-splicing of a mini-exon leader sequence to the 5′ end of open reading frames (ORFs) in a process that is coupled with polyadenylation of the adjacent upstream ORF [16], [17]. The parasites are reported to produce pre-mRNAs at a uniform rate across the genome, however these polycistronic units often contain mRNAs whose steady-state levels are significantly different and differentially accumulate in specific life cycle stages. This has led to a paradigm in which post-transcriptional mechanisms for regulation of gene expression predominate [12], [18], including mRNA processing [19], [20], [21], [22], translational efficiency [22], [23], [24], [25], [26], [27], [28], [29] and mRNA stability. Genes whose mRNAs are controlled at the level of mRNA stability include T. brucei procyclin [13], [30], [31] and hexose transporter [32]; T. cruzi amastin [33], mucin [24], and FL169 surface antigen [22]; and Leishmania A2 [34], A600 [35], [36], HSP83 [25], [29], [37], [38], GP63/major surface protease [39], [40], [41], [42], [43], [44], amastin [26], [45], CBP proteases [19], glucose transporters [46], paraflagellar rod genes [47], [48], and S-phase enriched genes [49].
Several studies have begun to illuminate post-transcriptional regulatory mechanisms in Trypanosomatids. Researchers have identified nucleotide sequences that contribute to the regulation of mRNA within the 5′ untranslated region (UTR) [49], [50], the coding sequence [22], intercistronic region [19], [20], [21], [22], but most commonly in the 3′ UTR [22], [24], [26], [30], [32], [33], [34], [35], [42], [47], [51], [52]. These regulatory sequences influence mRNA maturation, translational efficiency, and frequently mRNA abundance. In T. cruzi, AU-rich repeat regions that are structurally and functionally related to mammalian adensoine- and uridine-rich elements (AREs) [53], [54] control the differentially regulated expression of the mucin gene family [24].
In Leishmania, mRNA regulatory sequences have been reported that are greater than 100 nt in length [19], [42], [45] and as short as 8–9 nucleotides. Examples of short regulatory sequences include the putative cycling sequence octamer that directs the accumulation of S-phase enriched mRNAs [49] and a short element that signals for decay of mRNAs which harbor it in amastigotes termed the paraflagellar rod regulatory element (PRE).
Previously, we reported that expression of the Leishmania mexicana paraflagellar rod 2 (PFR2) genes is regulated post-transcriptionally by modulation of mRNA decay rates by a negative regulatory mechanism [47]. PFR2 encodes a structural component of the paraflagellar rod (PFR), a cytoskeletal structure essential for motility in promastigotes [55], [56]. The steady-state level of PFR2 mRNA is 10-fold greater in flagellated promastigotes than in amastigotes [57]. Differential accumulation of PFR2 mRNA depends on the PRE. This novel AU-rich RNA element, is a short sequence (AUGUAnAGU) contained within the 3′ UTR of the PFR2 mRNA that accelerates its decay in amastigotes. Mutation of the PRE results in an increase in amastigote mRNA half-life and steady-state abundance to levels that coincide with PFR2 mRNA levels in promastigotes [47]. Insertion of the PRE into an unregulated transcript that harbors the PFR2 ORF flanked by a 3′UTR consisting solely of vector-derived sequences confers regulation on the chimeric transcript by decreasing mRNA levels in amastigotes. Thus, the PRE appears to be both necessary and sufficient for post-transcriptional regulation of PFR2 mRNA accumulation.
The PRE is present in the 3′ UTR of all known L. mexicana PFR gene family members, including all three copies of PFR2, the two sequenced copies of PFR1, PFR4, and in other genes that have a putative flagellar function [47]. The recent completion of sequencing of the Leishmania major genome [58] has made possible a genome-wide search for the PRE in L. major. This, in addition to several reports of Leishmania transcript profiling [59], [60], [61], [62], [63], [64], [65], [66], has allowed candidate genes that display PRE-dependent regulation to be identified.
In this report, we show that L. mexicana mRNAs that harbor the PRE display promastigote-enriched transcript accumulation, including genes not related to the PFR. We provide evidence that the PRE is necessary for proper mRNA regulation of PFR4 and plays a major role in the proper steady-state mRNA accumulation of PFR1. This evidence suggests that the PRE coordinately regulates mRNA abundance not only in the PFR family, but also in a larger group of genes with diverse or unknown function.
Section snippets
Parasite strains and culture
Promastigotes of L. mexicana (WHO strain MNYC/BZ/62/m379) were cultured in M199 medium containing 5% (v/v) fetal bovine serum, and 5% (v/v) bovine embryonic fluid at 26 °C as described previously [67]. All amastigotes of L. mexicana used in this study were from axenic cultures and were obtained by shifting the incubation conditions of promastigotes from 26 to 33 °C and pH 5.5 in a modified UM 54 medium as described previously [48]. Promastigote and amastigote cultures were maintained by serial
Identification of PRE-harboring mRNAs
An initial interspecies analysis of sequences of the PFR family of genes showed that the PRE consensus sequence (ATGTAnAGT) is conserved among L. mexicana and L. major orthologs [47]. We reasoned that we could identify PRE-harboring L. mexicana mRNAs by identifying instances of the PRE within or adjacent to predicted L. major open reading frames. We identified 343 occurrences of the element in the entire genome. Because consensus sequences for neither a 5′ spliced leader addition signal nor a
PRE family: coordinate regulation of a family of promastigote-enriched genes by a common regulatory element
The PRE signals for amastigote-specific decay when present in the 3′UTR of Leishmania mRNAs. It was originally studied in the paraflagellar rod 2 gene, is both necessary and sufficient for the accelerated decay of PFR2 in amastigotes, and is present in the 3′ UTRs of all PFR genes known to date. In this report, we demonstrate by mutational analysis that the PRE is also necessary for proper regulation of PFR4 and PFR1, thus showing that this element is befitting of its given name, the
Conclusion
We have identified a family of genes that harbor the PRE instability sequence and have shown that it is functional in several contexts, suggesting coordinate expression of genes by the presence of this element. The identification of the PRE in the 3′ UTRs of Leishmania genes may be a rapid way of identifying developmentally regulated transcripts which can hasten the discovery of additional novel therapeutic targets [7].
Acknowledgements
The authors thank Zane Bergman and Jane Kinney (Department of Biochemistry, Purdue University) for their contributions to this work, and the Forney and Broyles labs for their helpful input. This work was supported by National Institute of Health grant A147909. J.H.L. was a Burroughs Welcome Fund new investigator in Molecular Parasitology. This is journal paper 2007-18165 from Purdue Agriculture Research Programs.
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Work performed at Purdue University, Department of Biochemistry, 175 S. University St., West Lafayette, IN 47907-2063, United States.