Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR. RNA

Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
RNA (Impact Factor: 4.94). 02/2010; 16(2):364-74. DOI: 10.1261/rna.1874710
Source: PubMed


Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3' untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3' UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3' UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5' end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5' UTR, since a hairpin structure abolishes expression of a fused reporter gene.

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Available from: Idan Gabdank, Dec 13, 2013
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    • "However, the steady-state level of various HSP transcripts is regulated by post-transcriptional mechanisms, including RNA stability and translational control. For example, the 3′UTR of Leishmania and Trypanosoma brucei HSP70 confers increased transcript stability under stress conditions (Lee, 1998; Quijada et al., 2000), as does the 3′UTR of Leishmania HSP90 that is also required for preferential translation during stress (Zilka et al., 2001; David et al., 2010). This regulation relies on the interaction of trans-acting RNA-binding proteins that recognize cis-acting elements [reviewed in Kramer and Carrington (2011)]. "
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    ABSTRACT: Across bacterial, archaeal, and eukaryotic kingdoms, heat shock proteins (HSPs) are defined as a class of highly conserved chaperone proteins that are rapidly induced in response to temperature increase through dedicated heat shock transcription factors. While this transcriptional response governs cellular adaptation of fungal, plant, and animal cells to thermic shock and other forms of stress, early branching eukaryotes of the kinetoplastid order, including trypanosomatid parasites, lack classical mechanisms of transcriptional regulation and show largely constitutive expression of HSPs thus raising important questions on the function of HSPs in the absence of stress and the regulation of their chaperone activity in response to environmental adversity. Understanding parasite-specific mechanisms of stress response regulation is especially relevant for protozoan parasites of the genus Leishmania that are adapted for survival inside highly toxic phagolysosomes of host macrophages causing the various immuno-pathologies of leishmaniasis. Here we review recent advances on the function and regulation of chaperone activities in these kinetoplastid pathogens and propose a new model for stress response regulation through a reciprocal regulatory relationship between stress kinases and chaperones that may be relevant for parasite adaptive differentiation and infectivity. This article is protected by copyright. All rights reserved.
    Cellular Microbiology 03/2015; 17(5). DOI:10.1111/cmi.12440 · 4.92 Impact Factor
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    • "Fine deletions finally identified a regulatory element of 30 nucleotides (positions 312–341), containing a stretch of polypyrimidines. This region was shown to be part of an RNA structure that was predicted with high probability [79], using the UNAfold algorithm [80]. A biophysical evaluation of the mRNA melting curves was performed to examine the role of secondary structures in the regulatory region. "
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    ABSTRACT: Trypanosomatids are ancient eukaryotic parasites that migrate between insect vectors and mammalian hosts, causing a range of diseases in humans and domestic animals. Trypanosomatids feature a multitude of unusual molecular features, including polycistronic transcription and subsequent processing by trans-splicing and polyadenylation. Regulation of protein coding genes is posttranscriptional and thus, translation regulation is fundamental for activating the developmental program of gene expression. The spliced-leader RNA is attached to all mRNAs. It contains an unusual hypermethylated cap-4 structure in its 5' end. The cap-binding complex, eIF4F, has gone through evolutionary changes in accordance with the requirement to bind cap-4. The eIF4F components in trypanosomatids are highly diverged from their orthologs in higher eukaryotes, and their potential functions are discussed. The cap-binding activity in all eukaryotes is a target for regulation and plays a similar role in trypanosomatids. Recent studies revealed a novel eIF4E-interacting protein, involved in directing stage-specific and stress-induced translation pathways. Translation regulation during stress also follows unusual regulatory cues, as the increased translation of Hsp83 following heat stress is driven by a defined element in the 3' UTR, unlike higher eukaryotes. Overall, the environmental switches experienced by trypanosomatids during their life cycle seem to affect their translational machinery in unique ways.
    Comparative and Functional Genomics 07/2012; 2012(12):813718. DOI:10.1155/2012/813718 · 2.03 Impact Factor
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    • "Although regulatory sequences have been identified in both 5' and 3' UTRs, most of them have been located in the 3' UTRs [18-23]. For instance, preferential translation of HSP83 in Leishmania requires a thermosensitive polypyrimidine-rich element (PPT) in the 3' UTR [24]. "
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    ABSTRACT: The heat stress suffered by Leishmania sp during its digenetic life-cycle is a key trigger for its stage differentiation. In Leishmania subgenera two classes of HSP70 genes differing in their 3' UTR were described. Although the presence of HSP70-I genes was previously suggested in Leishmania (Viannia) braziliensis, HSP70-II genes had been reluctant to be uncovered. Here, we report the existence of two types of HSP70 genes in L. braziliensis and the genomic organization of the HSP70 locus. RT-PCR experiments were used to map the untranslated regions (UTR) of both types of genes. The 3' UTR-II has a low sequence identity (55-57%) when compared with this region in other Leishmania species. In contrast, the 5' UTR, common to both types of genes, and the 3' UTR-I were found to be highly conserved among all Leishmania species (77-81%). Southern blot assays suggested that L. braziliensis HSP70 gene cluster may contain around 6 tandemly-repeated HSP70-I genes followed by one HSP70-II gene, located at chromosome 28. Northern blot analysis indicated that levels of both types of mRNAs are not affected by heat shock. This study has led to establishing the composition and structure of the HSP70 locus of L. braziliensis, complementing the information available in the GeneDB genome database for this species. L. braziliensis HSP70 gene regulation does not seem to operate by mRNA stabilization as occurs in other Leishmania species.
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