Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major

Abstract

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expression.

Figure 1

Analysis of the global transcriptional changes, determined by RNA-seq, during heat shock in L. major promastigotes. (A) PCA plot of the three replicates (r1–r3) derived from each experimental condition (26 or 37 °C). (B) Differential expression analysis of the 10,700 transcripts annotated in the L. major genome. The plot shows the relationship between the mean of the normalized counts and the fold change (log₂) between 26 and 37 °C conditions. Each point represents one transcript. Points colored in red or blue represent transcripts significantly up- or down-expressed at 37 °C, respectively; points colored in black correspond to transcript showing a constant expression in both conditions.

Figure 2

Comparative analysis of the relative expression levels of selected transcripts determined by RNA-seq and validated by quantitative RT-PCR (qPCR). Based on the RNA-seq analysis, two up-regulated transcripts (LmjF.02.T0460 and LmjF.32.T2260), two down-regulated (LmjF.06.T1260 and LmF.36.T3000) and one that did not change (LmjF.16.T1650) after the temperature treatment of the parasites were selected for validation by real-time PCR. In (A) fold-change values in the transcript levels between normal (26 °C) and heat shocked (37 °C) parasites determined by RNA-seq and qPCR. For comparison, and for each one of the transcripts, the lowest values were set arbitrary as 1. The arithmetic mean from triplicate cultures and the standard deviation are represented. In (B) RNA-seq reads derived from promastigotes incubated either at 26 °C (blue) or 37 °C (red), three replicates each, were mapped independently on the genomic region containing the specific transcript (black arrow; the CDS location is represented by a grey arrow). The positions of the oligonucleotides used for qPCR determinations are shown by orange arrowheads. Coverage (Cov.) is expressed as counts per million of reads (CPM).

Figure 3

Gene Ontology (GO) analysis of upregulated (panel A) and downregulated (panel B) transcripts. GO terms associated to annotated genes were extracted from the L. major (Friedlin strain) database and then mapped to the corresponding transcript in the transcriptome (Supplementary Dataset 3). This information was then loaded together with DESeq2 output into goseq R package to carry out GO enrichment analysis. Vertical line marks the negative logarithm for a p-value = 0.05.

Figure 4

Temperature-dependent expression levels for the transcripts derived from the HSP70 locus. Alignment on the L. major HSP70 gene locus of the RNA-seq reads obtained from RNA samples (three biological replicates) isolated from either promastigotes growth at 26 °C (blue) or heat shocked at 37 °C (red). The position of the two transcripts, LmjF.28.T2270 and LmjF.28.T2280 (black arrows), transcribed from the HSP70 locus, is indicated together with the location of their corresponding CDS (grey arrows). Coverage (Cov.) is expressed as counts per million of reads (CPM).

Figure 5

Expression levels for the histone H2B transcripts. There exist three H2B loci in the L. major genome: (A) LmjF.17.1220 gene, (B) LmjF.09.1340 gene, and (C) LmjF.19.0030, LmjF.19.0040 and LmjF.19.0050 genes. Alignment of the RNA-seq reads derived from RNA samples (three biological replicates) isolated from either promastigotes growth at 26 °C (blue) or heat shocked at 37 °C (red). The location of transcripts are indicated by black arrows, whereas the location of CDS is shown by grey arrows. Coverage (Cov.) is expressed as counts per million of reads (CPM).

Figure 6

Temperature-dependent expression of transcript LmjF.28.T3032, which encodes for a previously non-annotated protein. (A) Alignment of the RNA-seq reads derived from RNA samples (three biological replicates) isolated from either promastigotes growth at 26 °C (blue) or heat shocked at 37 °C (red). The location of the transcript is indicated by a black arrow, whereas the location of the putative ORF is shown by a grey arrow. Coverage (Cov.) is expressed as counts per million of reads (CPM). (B) Analysis by Northern blotting of transcript LmjF.28.T3032 in RNA samples derived from parasites incubated at 26 or 37 °C. The sizes (in nucleotides) of the L. major rRNA molecules used as molecular markers are indicated. Bottom, ethidium bromide (EtBr) staining of the gel, before membrane transfer and hybridization. Uncropped images shown in Supplementary Figure 1. (C) Sequence alignment between the protein ABB37_01471 of L. pyrrhocoris and the putative protein encoded in transcript LmjF.28.T3032 (LmjF.28.P3032).

Figure 7

Changes in the selection of SL-addition sites (SASs) associated with the temperature treatment in L. major promastigotes. (A) Schematic representation of the SASs used for trans-splicing of transcripts LmjF.29.T2240, LmjF.34.T1100, LmjF.21.T0891, LmjF.36.T6610 and LmjF.18.T0020 as determined by position of SL-containing RNA-seq reads for each one of the three experimental replicates from either promastigotes grown at 26 °C (blue) or promastigotes incubated for 2 h at 37 °C (red). For each transcript, the relative coverage is shown according to the scale indicated. Below each graph, the position of the CDS (grey arrow), transcript (black arrow) and the size and position of the expected PCR amplification products are shown. (B) PCR amplification of cDNA synthetized from RNA samples of parasites incubated at either 26 °C or 37 °C. For each transcript, a specific oligonucleotide and a common SL-oligonucleotide were used (oligonucleotide sequences are indicated in the Methods section). The negative control, C-(2240), consisted of the amplification from the RNA samples (without retrotranscription step) using the LmjF.29.T2240-specific oligonucleotide and the SL-oligonuclotide. HindIII-digested DNA of bacteriophage Φ29 was used as size marker (lane Φ29), and the size of relevant bands is indicated on the left. (C) Analysis by Northern blotting of transcripts derived from gene LmjF.28.3032 in RNA samples derived from parasites incubated at 26 or 37 °C. Bottom, methylene blue (MeBl) staining of the membrane used for hybridization. Uncropped images shown in Supplementary Figure 2.