Upstream AUGs and upstream ORFs can regulate the downstream ORF in Plasmodium falciparum

Abstract

BackgroundUpstream open reading frames (uORFs) and upstream AUGs (uAUGs) can regulate the translation of downstream ORFs. The AT rich genome of Plasmodium falciparum, due to the higher AT content of start and stop codons, has the potential to give rise to a large number of uORFs and uAUGs that may affect expression of their flanking ORFs.MethodsA bioinformatics approach was used to detect uATGs associated with different genes in the parasite. To study the effect of some of these uAUGs on the expression of the downstream ORF, promoters and 5′ leaders containing uAUGs and uORFs were cloned upstream of a luciferase reporter gene. Luciferase assays were carried out in transient transfection experiments to assess the effects of uAUGs and mutations on reporter expression.ResultsThe average number of uATGs and uORFs seen in P. falciparum coding sequences (CDS) is expectedly high compared to other less biased genomes. Certain genes, including the var gene family contain the maximum number of uATGs and uORFs in the parasite. They possess ~5 times more uORFs and ~4.5 times more uAUGs within 100 bases upstream of the start codons than other CDS of the parasite. A 60 bp upstream region containing three ORFs and five ATGs from var gene PF3D7_0400100 and a gene of unknown function (PF3D7_0517100) when cloned upstream of the luciferase start codon, driven by the hsp86 promoter, resulted in loss of luciferase activity. This was restored when all the ATGs present in the −60 bp were mutated to TTGs. Point mutations in the ATGs showed that even one AUG was sufficient to repress the luciferase gene.ConclusionsOverall, this work indicates that the P. falciparum genome has a large number of uATGs and uORFs that can repress the expression of flanking ORFs. The role of AUGs in translation initiation suggests that this repression is mediated by preventing the translation initiation complex from reaching the main AUG of the downstream ORF. How the P. falciparum ribosome is able to bypass these uAUGs and uORFs for highly expressed genes remains a question for future research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1040-5) contains supplementary material, which is available to authorized users.

Messenger RNAs with large numbers of upstream ORFs are translated via leaky scanning and reinitiation in the asexual stages ofPlasmodium falciparum

“…falciparum (Kumar et al, 2015). In this report, the effect of a 'T' at the +4 position could not be determined as changing the 'G' at the +4 position to 'T' in the luciferase expression vector (Pf86), altered the wild type codon 'GAA' to a stop codon.…”

“…Another noteworthy distinction between cytosolic translation of the parasite and its host, is a result of the high AT content (80-90%) of the P. falciparum genome. As start and stop codons are AT-rich, this biased sequence leads to the presence of numerous upstream open reading frames (uORFs) in the 5´ leader sequences of messenger RNAs (mRNAs) (Caro et al, 2014;Kumar et al, 2015).…”

“…Unlike human and mouse genomes from which ~50% of the transcribed mRNAs would be expected to contain one or more uORFs (Calvo et al, 2009;Ye et al, 2015), ~98% of P. falciparum mRNAs are predicted to contain an average of ~10 uORFs per coding sequence (CDS) (Caro et al, 2014;Kumar et al, 2015;Srinivas et al, 2016). The presence of such large numbers of uORFs has serious implications for translation.…”

“…falciparum can down-regulate the expression of the dORF (Amulic et al, 2009;Kumar et al, 2015). This post-transcriptional gene regulation (PTGR) mediated by uORFs takes place by engaging or stalling the ribosome at the uORF, subsequently decreasing the probability of the ribosome initiating translation of the dORF (Morris and Geballe, 2000).…”

“…Indeed, it has been reported that the degree of protein expression from the dORF can be regulated by varying the strength of the Kozak sequence of the uORF in mammalian cells (Ferreira et al, 2013). In contrast to other eukaryotic Kozak sequences where the -3 and +4 positions appear to be key, in P. falciparum nucleotides preceding the start codon (-5 to -1) do not play a significant role in determining the strength of the Kozak sequences (Kumar et al, 2015).…”