Developmental Regulation of Heat Shock Protein 83 inLeishmania

Abstract

Developmental gene regulation in trypanosomatids proceeds exclusively by post-transcriptional mechanisms. Stability and abundance of heat shock protein (HSP)70 and HSP83 transcripts in Leishmania increase at mammalian-like temperatures, and their translation is enhanced. Here we report that the 3-untranslated region (UTR) of HSP83 (886 nucleotides) confers the temperature-dependent pattern of regulation on a chloramphenicol acetyltransferase (CAT) reporter transcript. We also show that the majority of the 3-UTR sequences are required for increasing mRNA stability during heat shock. Processing of the HSP70 and HSP83 primary transcripts to poly(A)؉ mRNA was more efficient during heat shock; therefore, even when stability at 33°C was reduced by deletions in the 3-UTR, transcripts still accumulated to comparable and even higher levels. Translation of heat shock transcripts in Leishmania increases dramatically upon temperature elevation. Unlike in other eukaryotes in which the 5-UTR confers preferential translation on heat shock transcripts, we show that translational control of HSP83 in Leishmania originates from its 3-UTR. The 5-UTR alone cannot induce translation during heat shock, but it has a minor contribution when combined with the HSP83 3-UTR. We identified an element located between positions 201 and 472 of the 3-UTR which is essential for increasing translation of the CAT-HSP83 reporter RNA at 33-37°C. This region confers preferential translation during heat shock even in transcripts that were less stable. Thus, investigating the traditionally conserved heat shock response reveals that Leishmania parasites use unique pathways for translational control.Unique and unusual features characterize the genomes of trypanosomatids that comprise an ancient group of eukaryotes. Genes transcribed by RNA polymerase II are found in polycistronic transcription units, and gene clusters repeated in tandem frequently encode abundant proteins. There is no evidence for transcriptional activation of developmentally regulated genes, and mRNA abundance is determined exclusively by post-transcriptional mechanisms. These include differential RNA processing (1) and control of mRNA decay (2-4). Polycistronic transcripts mature by trans-splicing that adds a short capped leader (39-mer) to the 5Ј-end of mRNAs and by 3Ј processing that includes cleavage and polyadenylation. Transsplicing and polyadenylation are coupled mechanistically in trypanosomatids and share regulatory signals that consist of polypyrimidine tracks and potential AG splice sites (5, 6). The 3Ј-untranslated sequences lack the consensus eukaryote AAUAAA signal for cleavage and polyadenylation, and no other consensus element of that nature has been identified.Leishmania parasites exist in the alimentary canal of female sand flies as flagellated promastigotes. They develop into virulent metacyclics that are uniquely adapted for transmission by the fly into a mammalian host where they differentiate into amastigotes, an obligate intracellular life form that resides within ...

Distinct 3′-Untranslated Region Elements Regulate Stage-specific mRNA Accumulation and Translation in Leishmania

“…Several 3Ј-UTR-mediated mechanisms of translational control have been reported, including the recruitment of the 40 S ribosomal subunit (45), 60 S ribosomal subunit joining (46), binding to ribosomal protein L13a (47), recruitment of eIF4G (48), transcript circularization through 3Ј-5Ј base-pairing (49), a tRNA-like structure (50), a tRNA-reach-back mechanism (51), and an internal ribosome entry site-like structure at the 3Ј-end of the message (52). In Leishmania, very few 3Ј-UTR elements implicated in translational control have been identified thus far (15,17,53), and the molecular mechanisms are for the most part unknown.…”

“…Due to the absence of transcriptional control in Leishmania, stage-specific regulation of gene expression occurs exclusively at the post-transcriptional level (reviewed in Ref. 11) and involves mainly sequences within the 3Ј-UTR 5 of mRNAs that determine mRNA abundance by modulating RNA stability (12)(13)(14) or translational efficiency (15)(16)(17). However, the molecular mechanisms underlying stagespecific regulation of gene expression in this parasite are not yet understood.…”

A Common Mechanism of Stage-regulated Gene Expression in Leishmania Mediated by a Conserved 3′-Untranslated Region Element

“…We propose that stage-specific regulation of this particular subset of Leishmania mRNAs might be controlled by two distinct mechanisms: one acting via the 450-nt conserved element possibly regulating mRNA translation and the other by an adjacent 3Ј-UTR region that increases mRNA stability. A bipartite mechanism of stage-specific regulation with distinct 3Ј-UTR elements involved either in mRNA stability or translation has also been reported for the PARP mRNA in trypanosomes (31,32) and more recently for the Leishmania amazonensis HSP83 3Ј-UTR (50).…”

“…Few cis-acting regulatory elements within the 3Ј-UTRs of developmentally regulated mRNAs thought to be responsible for either mRNA turnover or translation have previously been identified in both Leishmania (16,50) and Trypanosoma species (30 -32, 51-53). However, none of these regulatory elements share any significant homology to the 450-nt element identified in this study, indicating its implication in a novel mechanism of stage-specific gene regulation in these organisms.…”

Identification of potential detoxification enzyme genes in Leptinotarsa decemlineata (Say) and study of their expression in insects reared on different plants

“…Different 3?-UTRs were found in two transcripts coding for CYP4BN12v1 and two transcripts coding for esterases (Est5320 and Est4512), suggesting that alternative splicing may be a common event in transcription of the detoxification enzyme genes. The use of alternative exons in 3?-UTR has been described in a number of transcripts such as the transcript for hepatic 2Wglobulin in rat (Ichiyoshi et al 1987), the U1 small nuclear ribonucleoprotein particle 70-kDa protein gene transcript in the fruit fly Drosophila melanogaster (Mancebo et al 1990) and the HSP83 protein gene transcript in Leishmania (Zilka et al 2001). The direct correlation has been established between the length of 3?-UTR and the stability of the mRNA species (Sureau and Perbal 1994;Lee 1998).…”