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

2019 ◽  
Author(s):  
Chhaminder Kaur ◽  
Mayank Kumar ◽  
Swati Patankar
Keyword(s):  
Plasmodium Falciparum ◽  
Open Reading Frames ◽  
Luciferase Reporter ◽  
Messenger Rnas ◽  
Leaky Scanning ◽  
Luciferase Reporter Gene ◽  
Genome Data ◽  
Large Numbers ◽  
Upstream Open Reading Frames ◽  
Specific Proteins

AbstractThe 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 inter-cistronic 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, parasite-specific proteins.

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

Parasitology ◽  
2020 ◽  
Vol 147 (10) ◽  
pp. 1100-1113
Author(s):  
Chhaminder Kaur ◽  
Mayank Kumar ◽  
Swati Patankar
Keyword(s):  
Plasmodium Falciparum ◽  
Open Reading Frames ◽  
Luciferase Reporter ◽  
Messenger Rnas ◽  
Leaky Scanning ◽  
Luciferase Reporter Gene ◽  
Reading Frame ◽  
Genome Data ◽  
Upstream Open Reading Frames ◽  
Reading Frames

AbstractThe 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.

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

Bioengineered ◽  
2014 ◽  
Vol 5 (3) ◽  
pp. 186-192 ◽  
Author(s):  
Joshua P Ferreira ◽  
William L Noderer ◽  
Alexander J Diaz de Arce ◽  
Clifford L Wang
Keyword(s):  
Protein Translation ◽  
Open Reading Frames ◽  
Leaky Scanning ◽  
Precise Control ◽  
Upstream Open Reading Frames ◽  
Reading Frames

scholarly journals Conserved Upstream Open Reading Frame Nascent Peptides That Control Translation

2020 ◽  
Vol 54 (1) ◽  
pp. 237-264
Author(s):  
Thomas E. Dever ◽  
Ivaylo P. Ivanov ◽  
Matthew S. Sachs
Keyword(s):  
Gene Expression ◽  
Open Reading Frames ◽  
Upstream Open Reading Frame ◽  
Messenger Rnas ◽  
Regulate Gene Expression ◽  
Reading Frame ◽  
Ribosome Stalling ◽  
Evolutionarily Conserved ◽  
Upstream Open Reading Frames ◽  
Control Translation

Cells utilize transcriptional and posttranscriptional mechanisms to alter gene expression in response to environmental cues. Gene-specific controls, including changing the translation of specific messenger RNAs (mRNAs), provide a rapid means to respond precisely to different conditions. Upstream open reading frames (uORFs) are known to control the translation of mRNAs. Recent studies in bacteria and eukaryotes have revealed the functions of evolutionarily conserved uORF-encoded peptides. Some of these uORF-encoded nascent peptides enable responses to specific metabolites to modulate the translation of their mRNAs by stalling ribosomes and through ribosome stalling may also modulate the level of their mRNAs. In this review, we highlight several examples of conserved uORF nascent peptides that stall ribosomes to regulate gene expression in response to specific metabolites in bacteria, fungi, mammals, and plants.

scholarly journals Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs

2021 ◽  
Vol 118 (40) ◽  
pp. e2018899118
Author(s):  
Divya Ram Jayaram ◽  
Sigal Frost ◽  
Chanan Argov ◽  
Vijayasteltar Belsamma Liju ◽  
Nikhil Ponnoor Anto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Kinase Inhibitor ◽  
Open Reading Frames ◽  
Biologically Active ◽  
Messenger Rnas ◽  
Active Peptides ◽  
Protein Levels ◽  
Lower Protein ◽  
Cancer Tumor ◽  
Upstream Open Reading Frames

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

scholarly journals Upstream Open Reading Frames (uORFs) in the Apicomplexan Parasites Plasmodium falciparum and Toxoplasma gondii: Small yet Powerful Regulators of Translation

2021 ◽  
Author(s):  
Chhaminder Kaur ◽  
Swati Patankar
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Toxoplasma Gondii ◽  
Translational Regulation ◽  
Life Cycles ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Apicomplexan Parasites ◽  
Upstream Open Reading Frames ◽  
Reading Frames

During their complex life cycles, the Apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii employ several genetic switches to regulate their gene expression. One such switch is mediated at the level of translation through upstream Open Reading Frames (uORFs). As uORFs are found in the upstream regions of a majority of genes in both the parasites, it is essential that their roles in translational regulation be appreciated to a greater extent. This review provides a comprehensive summary of studies that show uORF-mediated gene regulation in these parasites and highlights examples of clinically and physiologically relevant proteins that exhibit uORF-mediated regulation. In addition to these examples, several studies that use bioinformatics, transcriptomics, proteomics, and ribosome profiling also indicate the possibility of widespread translational regulation by uORFs. Further analysis of genome-wide datasets will reveal novel genes involved in key biological pathways such as cell-cycle progression, stress-response, and pathogenicity. The cumulative evidence from studies presented in this review suggests that uORFs will play crucial roles in regulating gene expression during clinical disease caused by these important human pathogens.

Involvement of upstream open reading frames in regulation of rat V1b vasopressin receptor expression

2001 ◽  
Vol 280 (5) ◽  
pp. E780-E787 ◽  
Author(s):  
Atsushi Nomura ◽  
Yasumasa Iwasaki ◽  
Masayuki Saito ◽  
Yoshiaki Aoki ◽  
Etsuko Yamamori ◽  
...  
Keyword(s):  
Receptor Gene ◽  
Suppressive Effect ◽  
Open Reading Frames ◽  
Receptor Expression ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Vasopressin Receptor ◽  
Start Site ◽  
Upstream Open Reading Frames ◽  
Reading Frames

The V1b vasopressin receptor, expressed mainly in the corticotroph of the anterior pituitary, mediates the stimulatory effect of vasopressin on ACTH release. To clarify the regulation of receptor expression, we cloned, sequenced (up to ∼5 kb from the translation start site), and characterized the 5′-flanking region of the rat V1b receptor gene. We identified the transcription start site by amplification of cDNA ends and found a new intron within the 5′-untranslated region (5′-UTR) by comparing the sequence with that of cDNA. We then confirmed that the obtained promoter indeed has transcriptional activity by use of the luciferase reporter in AtT-20 mouse corticotroph cells. Interestingly, there were five short upstream open reading frames (uORFs) located within the 5′-UTR that were found to suppress V1b expression. Subsequent mutational analyses showed that the two downstream uORFs have an inhibitory effect on expression in both homologous and heterologous contexts. Furthermore, the inhibition did not accompany a parallel decrease in mRNA, suggesting that the suppressive effect occurs at a level downstream of transcription. Taken together, our data strongly suggest that the expression of the V1b receptor is regulated at the posttranscriptional as well as transcriptional level through uORFs within the 5′-UTR region of the mRNA. Whether the uORF-mediated regulation of V1b expression is functionally linked to any intracellular and/or extracellular factor(s) awaits further research.

scholarly journals Chi-miR-130b-3p regulates Inner Mongolia cashmere goat skin hair follicles in fetuses by targeting Wnt family member 10A

2020 ◽  
Vol 11 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Zhihong Wu ◽  
Erhan Hai ◽  
Zhengyang Di ◽  
Rong Ma ◽  
Fangzheng Shang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Family Member ◽  
Dermal Fibroblast ◽  
Hair Follicles ◽  
Dermal Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Messenger Rnas ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Abstract The development of hair follicles (HFs) is dependent on interactions between epithelial cells and dermal fibroblasts, which may play an important role in maintaining the structure of HFs during their development and maturation. Wnt family member 10 (WNT10A) is a hub gene during HF development and maturation that may regulate the proliferation of dermal fibroblasts and epithelial cells through microRNAs (miRNAs) and messenger RNAs (mRNAs) to maintain the structural stability of HFs. In the present study, we confirmed that WNT10A is the target gene of chi-miR-130b-3p by real-time quantitative PCR, western blotting, and a dual-luciferase reporter gene assay. We successfully cultured fetal epithelial cells and dermal fibroblasts using the tissue block attachment method, and Cell Counting Kit-8 (CCK8) results showed that chi-miR-130b-3p regulates epithelial cell and dermal fibroblast proliferation by targeting WNT10A.

scholarly journals DAP5 enables translation re-initiation on structured messenger RNAs

2021 ◽  
Author(s):  
Ramona Weber ◽  
Leon Kleemann ◽  
Insa Hirschberg ◽  
Min-Yi Chung ◽  
Eugene Valkov ◽  
...  
Keyword(s):  
Cell Fate ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Messenger Rnas ◽  
Upstream Open Reading Frames ◽  
Reading Frames

SummaryHalf of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is selectively required for re-initiation at the main CDS following uORF translation. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated re-initiation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates re-initiation by unrecycled post-termination 40S subunits. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

Planta Medica ◽  
2009 ◽  
Vol 75 (09) ◽  
Author(s):  
S Vogl ◽  
P Picker ◽  
N Fakhrudin ◽  
A Atanasov ◽  
E Heiß ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Plant Extracts ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Reporter Gene Assays
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