Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation

Ferreira, Joshua P.; Noderer, William L; Arce, Alexander J Diaz de; Wang, Clifford L.

doi:10.4161/bioe.27607

Cited by 12 publications

(7 citation statements)

References 33 publications

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“…Alleviation of translational repression due to numerous uORFs can also be achieved through leaky scanning, possibly the most metabolically efficient way to handle multiple uORFs. A known contributor to leaky scanning is the strength of the Kozak sequence (Ferreira et al, 2013(Ferreira et al, , 2014Kozak, 1999). This report confirms published observations that the nucleotide following the start codon (+4 position) plays a significant role in determining the strength of the Kozak sequence in P. falciparum (Kumar et al, 2015), unlike other eukaryotes where nucleotides at -3 and +4 positions are both important (Pisarev et al, 2006).…”

Section: The Kozak Sequence Determines the Extent Of Leaky Scanningsupporting

confidence: 88%

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

Kaur

Kumar

Patankar

2019

Preprint

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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 10 uORFs in their leader sequences. Similar to other eukaryotes, uORFs repress the translation of the downstream gene (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 codon usage of the uORF, translation of the dORF is governed by the Kozak sequence and length of the uORF, and intercistronic distance between the uORF and dORF. Based on these features whole genome data was analyzed 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, parasitespecific proteins.

show abstract

Section: The Kozak Sequence Determines the Extent Of Leaky Scanningsupporting

confidence: 88%

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

Kaur

Kumar

Patankar

2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Alleviation of translational repression due to numerous uORFs can also be achieved through leaky scanning, possibly the most metabolically efficient way to handle multiple uORFs. A known contributor to leaky scanning is the strength of the Kozak sequence (Kozak, 1999; Ferreira et al ., 2013, 2014). This report confirms published observations that the nucleotide following the start codon (+4 position) plays a significant role in determining the strength of the Kozak sequence in P. falciparum (Kumar et al ., 2015), unlike other eukaryotes where nucleotides at −3 and +4 positions are both important (Pisarev et al ., 2006).…”

Section: Discussionmentioning

confidence: 99%

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

2020

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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.

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“…Weak initiation starts (e.g., ACG or CTG), if surrounded by appropriate nucleotide context, may initiate protein synthesis in a noncanonical manner. This mechanism is called leaky ribosomal scanning and is recently attracting more attention in engineering and manipulation of protein expression [23,24]. The strength of the leaky ribosomal scanning at VP1 will determine the extent of the ribosomal "leakage" to VP2 and VP3 and the subsequent strength of protein expression.…”

Section: Discussionmentioning

confidence: 99%

Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system

et al. 2018

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Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.

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Engineering ribosomal leaky scanning and upstream open reading frames for precise control of protein translation

Cited by 12 publications

References 33 publications

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

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

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

Optimization of viral protein ratios for production of rAAV serotype 5 in the baculovirus system

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