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

Abstract

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.

FIGURE 1.

Preferential translation of Hsp83 is directed by sequences 201–346 in the 3′ UTR. (A) The CAT reporter gene was flanked by complete IRs derived from the Hsp83 genomic cluster, so that the signals for RNA processing were maintained on both sides. The resulting constructs were cloned into the pX transfection vector of Leishmania and used to generate stable cell lines. (B) De novo synthesis of the CAT reporter gene was examined by metabolic labeling of the transgenic parasite cells grown at 26°C, and after their preincubation at 33°C, or 37°C, during 60 min. Protein extracts were separated over 12% polyacrylamide gels. Migration of CAT, Hsp70, Hsp83, and the α- and β-Tubulins are marked with arrowheads.

FIGURE 2.

Structure prediction of the Hsp83 3′ UTR element 1–472 by UNAfold. Color annotation was used to indicate the propensity of individual nucleotides to participate in base-pairing and whether or not a predicted base pair is well determined. Forty colors that range from red (unusually well determined) through orange, yellow, green, blue, purple to black (poorly determined) are used (Zuker and Jacobson 1998). The structure with the lowest ΔG that was obtained using the Mfold program is shown, and the color of each nucleotide indicates its estimated P-value. The deletions which are described in Figure 1 are positioned on the predicted structure. Deletions that abolished preferential translation are colored in green (Δ198–274), purple (Δ249–286), and blue (Δ280–365). Deletions that did not affect preferential translation are marked in gray (Δ346–399, Δ380–441, and Δ428–476).

FIGURE 3.

Sequences 312–341 in the Hsp83 3′ UTR are essential for preferential translation during heat shock. (A) A map of mutations within the region 150–364 in the 3′ UTR of Hsp83. The modified IRs were cloned downstream from the CAT gene to generate 3′ UTRs. The upstream Hsp83 IR was not modified. The chimeric CAT–Hsp83 genes were cloned in the pX vector for transfection of Leishmania, and used to generate transgenic parasite cell lines. (B) Functional fine mapping of sequences that are required for preferential translation of the CAT–Hsp83 chimeric gene. Cells expressing the CAT gene under control of the mutated Hsp83 3′ UTR were grown at 26°C, or transferred to 33°C or 37°C for 1 h, and metabolically labeled for 30 min at the corresponding temperatures with ³⁵[S]-labeled amino acids. Proteins were extracted, separated over 15% SDS-polyacrylamide gels, and autoradiogrammed in a PhosphorImager. Migration of Hsp83. Hsp70, the α- and β-tubulins, and CAT are marked with arrowheads. WT represents the nontransfected negative control cells which do not express CAT. pX-HCH represents transgenic cells expressing CAT under control of nonmodified Hsp83 IRs. Deletion mutations Δ312–331, Δ337–364, Δ332–341, and the C → G exchange mutation at positions 315–327 abrogated preferential translation of the CAT transcript. However, deletion mutations Δ350–364 and Δ342–349 did not interfere with the increased CAT translation at elevated temperatures. (C) Steady-state CAT expression at 26°C in cells transfected with the different 3′ UTR deletions. Cell extracts were separated over 15% SDS-polyacrylamide gels, blotted, and reacted with anti-CAT antibodies. Protein loads were evaluated by control reactions with antibodies against Hsp70. The level of CAT expression was quantified with Multigauge V3.0 and normalized against Hsp70. The values shown at the bottom of the figure represent a mean of two independent experiments.

FIGURE 4.

RNA melting curves of the wild-type and mutated 1–472 RNA fragments. Representative melting profiles were obtained by measuring the optical density of the RNA solutions (0.012 mg/mL) at temperatures that increased by increments of 5°C. The RNA was allowed to equilibrate for 10 min at each temperature, prior to monitoring the absorbance at 260 nm. The change in absorbance for each temperature (ΔOD₂₆₀) was calculated relative to the starting point. The ΔOD₂₆₀ values for each curve are expressed as relative numbers that range from 0% to 100% (the ΔOD₂₆₀ at the highest temperature served as 100%).

FIGURE 5.

RNA melting measured by RNase H cleavage. (A) Location of the antisense oligonucleotides on the predicted RNA structure. The oligonucleotides used for the RNase H assay are positioned on the predicted RNA structure (black lines), and the ratio between the intensity of the two cleavage products is indicated (25/37). Black lettering represents regions that melt at elevated temperatures (25/37 < 1) and gray lettering marks regions that are not affected by temperature elevation (25/37 = 1). (B) RNase H cleavage directed by hybridization of the RNA fragment preincubated at different temperatures. The end-labeled RNA (1–472) was exposed to different temperatures (25°C or 37°C), then incubated with different oligonucleotides and cleaved by RNase H at the corresponding temperature. The RNase H cleavage products were separated over 6% denaturing polyacrylamide gels. Migration of the untreated full-length 1–472 RNA product is shown at the left. Products of the RNase H cleavage reaction following hybridization with oligonucleotides at different temperatures are marked with a star. The oligonucleotide positions in the 3′ UTR are indicated above the lanes. Size markers of 100, 200, and 300 nt are depicted at the left side of each panel (numbers at the far left and short lines between the panels). The ratio between the intensity of the two bands (25/37) is shown at the bottom of each panel.

FIGURE 6.

Preferential translation of Hsp83 occurs via scanning of the 5′ UTR. (A) Introduction of a hairpin structure at the Hsp83 5′ and 3′ UTRs. A scheme of plasmids carrying the CAT gene flanked with Hsp83 IRs, with a foreign hairpin structure introduced either to the 5′ or to the 3′ UTRs, is shown. The CAT constructs were cloned into the pX transfection vector and used to generate transgenic Leishmania lines. (B) A hairpin structure introduced in the 5′ UTR has an inhibitory effect on CAT translation at both temperatures. Cells expressing the CAT gene under control of the Hsp83 IRs carrying a hairpin structure either at the 5′ or at the 3′ UTR were incubated for 1 h at different temperatures, 26°C, 33°C, or 37°C, and metabolically labeled with ³⁵[S]-labeled methionine and cystein during 30 min at the corresponding temperatures. Proteins were extracted and separated over 15% SDS-polyacrylamide gels. The migration distances of Hsp83, Hsp70, tubulin, and the CAT reporter gene are marked by arrows. Introduction of a hairpin structure into the 5′ UTR inhibited the de novo translation of the CAT–Hsp83 chimera at all temperatures. (C) Steady-state CAT expression in cells transfected with a CAT–Hsp83 chimera carrying a hairpin structure at the 5′ (pX-HCH-5′hp) and 3′ (pX-HCH-3′hp) UTRs. Cell extracts were separated over 15% SDS-polyacrylamide gels, blotted, and reacted with anti-CAT antibodies. Protein loads were evaluated by control reactions with antibodies against Hsp70.