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. 2015 Dec 21:14:512.
doi: 10.1186/s12936-015-1040-5.

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

Affiliations

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

Mayank Kumar et al. Malar J. .

Abstract

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.

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Figures

Fig. 1
Fig. 1
Distribution of number of uATGS and uORFs across annotated CDS of the genome of P. falciparum. uATGs and uORFs were calculated from 350 bp region upstream of the start codons of 5401 annotated CDS of the parasite. var genes contain highest number of uATGs (highlighted). Number of uORFs has been taken from our previous study [45]
Fig. 2
Fig. 2
Average number of uATGs and uORFs in different protein coding genes. Average number of uATGs (a) and uORFs (b) found in the regions upstream of the start codons of var and other genes. Randomized upstream sequences of var and other genes were used as controls. Mean uATGs and uORFs were calculated in windows of 50 bases, up to 1200 base pairs upstream of the start codons
Fig. 3
Fig. 3
Ratio of uATGs and uORFs. Ratio of average number of uATGs and uORFs associated with var and other genes of the parasite with increasing length of the regions upstream of the start codons. Ratios were calculated in windows of 50 bases, up to 1200 base pairs upstream of the start codons
Fig. 4
Fig. 4
Luciferase assay of the constructs containing var upstream region and upstream region with mutated ATGs. Firefly luciferase readings of the constructs Pf86, Pf86-60var and Pf86-60var(mut 1 to 6) obtained after transiently transfecting them in the parasite. Construct Pf86-stop was used as a negative control where the second codon of the luciferase CDS in the vector Pf86 was converted to a stop codon. Firefly luminescence units were normalized against those of Renilla for each construct. Normalized Firefly readings of each constructs have been expressed with respect to normalized Firefly readings of Pf86. Firefly luciferase readings and standard deviations have been calculated from five replicates. Normalized firefly readings of Pf86 were in the range of 1000–7000 RLUs, measured in a luminometer
Fig. 5
Fig. 5
Firefly luciferase readings of the constructs Pf86 and Pf86-60var containing different point mutations. Firefly luciferase readings of the constructs Pf86, Pf86-60var and Pf86-60var(mut 1 to 6), Pf86-60var(mut 2), Pf86-60var(mut 2 to 6), Pf86-60var(mut 1, 3, 4, 5, 6), and Pf86-60var(mut 1 to 5) obtained after transiently transfecting them in parasite. Construct Pf86-stop where the second codon of the luciferase gene in the vector Pf86 was converted to a stop codon was used as a negative control. Firefly luminescence units were normalized against those of Renilla for each construct. Normalized Firefly readings of each construct have been expressed with respect to normalized Firefly readings of Pf86. Firefly luciferase readings and standard deviations have been calculated from five replicates. Normalized firefly readings were in the range of 1000–7000 RLUs, measured in a luminometer
Fig. 6
Fig. 6
Luciferase assay of the constructs containing upstream region from a gene with unknown function and upstream region with mutated ATGs. Firefly luciferase readings of the constructs Pf86, Pf86-60_0517100 and Pf86-60_0517100(mut 1 to 6) obtained after transiently transfecting them in the parasite. Construct Pf86-stop was used as a negative control where the second codon of the luciferase CDS in the vector Pf86 was converted to a stop codon. Firefly luminescence units were normalized against those of Renilla for each construct. Normalized Firefly readings of each constructs have been expressed with respect to normalized Firefly readings of Pf86. Firefly luciferase readings and standard deviations have been calculated from four replicates. Normalized firefly readings of Pf86 were in the range of 2300–4000 RLUs, measured in a luminometer
Fig. 7
Fig. 7
Firefly luciferase readings of the plasmid Pf86 and its derivatives under different Kozak sequences with their frequencies. Firefly luciferase readings of the plasmid Pf86 and its derivatives carrying luciferase start codon under different Kozak sequences have been shown (bar graph). Frequency of occurrence of different Kozak sequences in the genome tested for their strength has been shown on secondary Y-axis (line). Firefly luminescence units were normalized against those of Renilla for each constructs. Normalized Firefly readings of each constructs have been expressed with respect to normalized Firefly readings of Pf86. Luciferase readings have been measured in a scintillation counter. Firefly luciferase readings and standard deviations have been calculated from four replicates. Normalized Firefly readings were in the range of ~800,000 to ~28,00,000 CPM, measured using a scintillation counter. Constructs containing different Kozak sequences were grouped into three categories based on the +4 position: those containing ‘G’ at +4 position (a), ‘C’ at +4 position (b) and ‘A’ at +4 position (c)
Fig. 8
Fig. 8
Folding energy of regions surrounding AUGs. The cumulative frequency graph of minimum folding energy, ΔG (Kcal/mol) of regions around AUGs (−15 bases to +20 bases). AUGs were characterized based on their positions in the mRNA: upstream, downstream and main AUGs

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