Messenger RNAs with large numbers of upstream open reading frames are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum

Abstract

The genome of Plasmodium falciparum has one of the most skewed base-pair compositions of any eukaryote, with an AT content of 80–90%. As start and stop codons are AT-rich, the probability of finding upstream open reading frames (uORFs) in messenger RNAs (mRNAs) is high and parasite mRNAs have an average of 11 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream open reading frame (dORF) in P. falciparum, yet the parasite translation machinery is able to bypass these uORFs and reach the dORF to initiate translation. This can happen by leaky scanning and/or reinitiation. In this report, we assessed leaky scanning and reinitiation by studying the effect of uORFs on the translation of a dORF, in this case, the luciferase reporter gene, and showed that both mechanisms are employed in the asexual blood stages of P. falciparum. Furthermore, in addition to the codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and inter-cistronic distance between the uORF and dORF. Based on these features whole-genome data was analysed to uncover classes of genes that might be regulated by uORFs. This study indicates that leaky scanning and reinitiation appear to be widespread in asexual stages of P. falciparum, which may require modifications of existing factors that are involved in translation initiation in addition to novel, parasite-specific proteins.

The role of upstream open reading frames in translation regulation in the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii

“…This number is extremely high when compared to human transcripts where only 49% of the transcripts contain at least one uORF in their 5′ leader (Calvo et al ., 2009 ). With a prediction of an average of 11 uORFs per CDS (Kaur et al ., 2020 ), P. falciparum exhibits the highest ever recorded number of uORFs in a transcript.…”

“…Each uORF has a very different ability to engage the scanning ribosome and experimental approaches have been used to study the features that contribute to the repressive capacity of uORFs. Such features are the Kozak sequence, codon composition, length of the uORF, and the distance between the uORF and the CDS (Kaur et al ., 2020 ). Further, bioinformatics was used to predict translatability of an ORF (therefore, the repressiveness of the uORF) by calculating the probability of translation initiation and elongation of ORFs in the P. falciparum genome (Srinivas et al ., 2016 ).…”

“…However, the use of non-canonical translation mechanisms to circumvent translation repression caused by the uORFs is not limited to this gene. Indeed, it was demonstrated that reinitiation occurs in the case of the hsp70 transcript in the presence of a native uORF and synthetic uORFs suggesting that there is a widespread occurrence of reinitiation in the asexual stages of P. falciparum (Kaur et al ., 2020 ).…”

Evolution of transcriptional control of antigenic variation and virulence in human and ape malaria parasites

“…However, var2csa and its regulatory uORF appear to have evolved relatively late in the Laverania (see below), while PTEF is present in all members of the subgenus, indicating that PTEF must have a function in addition to or beyond regulation of var2csa translation. A recent genome-wide assessment of P. falciparum transcripts noted that the number of potential uORFs is remarkably high [34] and given the substantial degree of synteny and sequence homology within the entire subgenus, this extends to the other Lavarania species as well. The role of PTEF in overcoming the repressive effects of uORFs on mRNA translation therefore could include a broader class of mRNAs beyond var2csa, thus explaining its conservation in parasites that do not possess this particular var gene.…”

In silico identification of novel open reading frames in Plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework

“…Unlike other untranslated regions, slightly more novel peptides were detected to be translated from the 3' and 5′ UTRs. One explanation is that they reflect the incomplete annotation of reference genes, or they could arise from stop-codon read-through [65] and translation of upstream ORFs respectively, where the latter has been shown to regulate the expression of downstream canonical ORFs [66]. However, despite the sparse evidence for translation of these novel transcripts, their regulatory roles on canonical proteins may be at RNA level only, especially antisense transcripts, whose RNA molecules were shown to interact with chromatin and regulate gene expression in P. falciparum [25].…”