Transcriptomic analysis reveals novel downstream regulatory motifs and highly transcribed virulence factor genes of Entamoeba histolytica

Abstract

BackgroundPromoter motifs in Entamoeba histolytica were earlier analysed using microarray data with lower dynamic range of gene expression. Additionally, previous transcriptomic studies did not provide information on the nature of highly transcribed genes, and downstream promoter motifs important for gene expression. To address these issues we generated RNA-Seq data and identified the high and low expressing genes, especially with respect to virulence potential. We analysed sequences both upstream and downstream of start site for important motifs.ResultsWe used RNA-Seq data to classify genes according to expression levels, which ranged six orders of magnitude. Data were validated by reporter gene expression. Virulence-related genes (except AIG1) were amongst the highly expressed, while some kinases and BspA family genes were poorly expressed. We looked for conserved motifs in sequences upstream and downstream of the initiation codon. Following enrichment by AME we found seven motifs significantly enriched in high expression- and three in low expression-classes. Two of these motifs (M4 and M6) were located downstream of AUG, were exclusively enriched in high expression class, and were mostly found in ribosomal protein, and translation-related genes. Motif deletion resulted in drastic down regulation of reporter gene expression, showing functional relevance. Distribution of core promoter motifs (TATA, GAAC, and Inr) in all genes revealed that genes with downstream motifs were not preferentially associated with TATA-less promoters. We looked at gene expression changes in cells subjected to growth stress by serum starvation, and experimentally validated the data. Genes showing maximum up regulation belonged to the low or medium expression class, and included genes in signalling pathways, lipid metabolism, DNA repair, Myb transcription factors, BspA, and heat shock. Genes showing maximum down regulation belonged to the high or medium expression class. They included genes for signalling factors, actin, Ariel family, and ribosome biogenesis factors.ConclusionOur analysis has added important new information about the E. histolytica transcriptome. We report for the first time two downstream motifs required for gene expression, which could be used for over expression of E. histolytica genes. Most of the virulence-related genes in this parasite are highly expressed in culture.Electronic supplementary materialThe online version of this article (10.1186/s12864-019-5570-z) contains supplementary material, which is available to authorized users.

Genomic determinants for initiation and length of natural antisense transcripts in Entamoeba histolytica

“…For example, diverse gene promoters have been identified regulating cell growth 16 , cyst formation 10 or transcription during infection 17 . Recently, the correlative analysis of the genome and of the mRNA revealed several repetitive short nucleotide sequences of DNA which, according to their number of copies, modulate the expression of genes 18 . Splicing sites and polyadenylation sites on mRNA have been also characterized at the genome-wide level 19 .…”

EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica

“…This was also recently validated by RNA-seq analysis where EhP3 was identified as one of the highly transcribed genes of E . histolytica [40]. In this manuscript we have further characterized the important biological functions of the EhP3 isoform.…”

COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

“…This possibility is supported by a recent E. histolytica gene expression study that used RNA-seq technology. Genes were classified according to expression levels with highly expressed genes likely required for E. histolytica growth, which found the parasite Nedd8 amongst the highest expressed [68,69]. In addition, COP9 signalosome inhibitors are being optimized to treat cancer [70], hence one could envision repurposing these drugs for use against parasitic protozoal diseases.…”