Messenger RNAs with large numbers of upstream open reading frames are translated via leaky scanning and reinitiation in the asexual stages of Plasmodium falciparum Chhaminder Kaur, Mayank Kumar, Swati Patankar Parasitology, 2020 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.
ORFpred: A machine learning program to identify translatable small open reading frames in intergenic regions of the plasmodium falciparum genome Vivek Srinivas, Mayank Kumar, Santosh Noronha, Swati Patankar Current Bioinformatics, 2016 Motivation: Small Open Reading Frames (smORFs) are involved in a variety of cellular processes varying from metabolism to gene regulation and eukaryotic genomes have been predicted to contain a large number of smORFs. Only a meager 174 smORFs have been annotated in the genome of the human malaria parasite Plasmodium falciparum. Although millions of smORFs can be extracted from the parasite genome, the identification of translatable smORFs from the P. falciparum genome is a challenging task due to low accuracy of existing smORF predictors when applied to an AT biased genome. Result: We developed ORFpred, a machine learning algorithm which calculates the probability of translation initiation and elongation of ORFs in the P. falciparum genome. ORFpred identified 2204 translatable smORFs and when compared to available predictors, showed higher accuracy. We believe that ORFpred will help in identification of probable protein coding smORFs in other eukaryotic genomes. Availability and Implementation: Database used for training and testing the algorithm and source codes are freely available at http://www.bio.iitb.ac.in/~patankar/software/ORFpred. Keywords: Small open reading frames, upstream open reading frames, translatability, low molecular weight proteins, post transcriptional gene regulation, Plasmodium falciparum, AT rich genome.
Upstream AUGs and upstream ORFs can regulate the downstream ORF in Plasmodium falciparum Mayank Kumar, Vivek Srinivas, Swati Patankar Malaria Journal, 2015 BACKGROUND: Upstream 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: Overall, 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.
Regulation of gene expression in Plasmodium falciparum Current Science, 2012
