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

Alberto Rastrojo,Rodrigo Lombraña,Begoña Aguado,Jose M Requena,Jose C Solana,Laura Corvo

doi:10.1038/s41598-019-43354-9

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.

Highlights

Unicellular organisms and cells in multicellular organisms, albeit to different extent, are exposed to sudden changes in the environment that compromise their viability
Total RNA from the six samples was extracted and after poly(A)+ selection, RNA sequencing was carried out using the Illumina methodology
The heat shock response, a universal mechanism aimed to counteract deleterious effects on cellular homeostasis promoted by a sudden increase in the ambient temperature, is classically used as a model system to study gene expression[67]

Summary

Introduction

Unicellular organisms and cells in multicellular organisms, albeit to different extent, are exposed to sudden changes in the environment that compromise their viability. A interesting case is found in digenetic parasites that along their life cycle alternate between poikilothermic and homoeothermic hosts In these organisms, like Leishmania, sudden temperature variations during transmission are natural events in their life cycles[3]. Leishmania and related trypanosomatids regulate the entire gene expression almost exclusively at the post-transcriptional level[8,9,10] This feature has modelled the genome architecture of these parasites: genes are organized into large collinear clusters present on a single strand, and the different gene clusters are separated from each other by short sequences of a few www.nature.com/scientificreports/. Apart from its role in the differentiation program, the heat shock response in Leishmania is being used as a genetic model for deciphering the mechanisms regulating gene expression. Studies on the expression of the two major heat shock proteins (HSPs), i.e. HSP70 and HSP83/90, contributed substantially to demonstrate that control of gene expression in this parasite occurs almost exclusively at the post-transcriptional level, and that HSP synthesis during heat shock depends on regulation of mRNA turnover and translational control[15,16,17,18,19,20]

Methods

Results

Conclusion