Abstract
Eukaryotic upstream Open Reading Frames (uORFs) are short translated regions found in many transcript leaders (Barbosa et al. PLoS Genet 9:e1003529, 2013; Zhang et al. Trends Biochem Sci 44:782–794, 2019). Modern transcript annotations and ribosome profiling studies have found thousands of AUG-initiated uORFs, and many more uORFs initiated by near-cognate codons (CUG, GUG, UUG, etc.). Their translation generally decreases the expression of the main encoded protein by preventing ribosomes from reaching the main ORF of each gene, and by inducing nonsense mediated decay (NMD) through premature termination. Under many cellular stresses, uORF containing transcripts are de-repressed due to decreased translation initiation (Young et al. J Biol Chem 291:16927–16935, 2016). Traditional experimental evaluation of uORFs involves comparing expression from matched uORF-containing and start-codon mutated transcript leader reporter plasmids. This tedious process has precluded analysis of large numbers of uORFs. We recently used FACS-uORF to simultaneously assay thousands of yeast uORFs in order to evaluate the impact of codon usage on their functions (Lin et al. Nucleic Acids Res 2:1–10, 2019). Here, we provide a step-by-step protocol for this assay.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Barbosa C, Peixeiro I, Romão L (2013) Gene expression regulation by upstream open reading frames and human disease. PLoS Genet 9:e1003529
Zhang H, Wang Y, Lu J (2019) Function and evolution of upstream ORFs in eukaryotes. Trends Biochem Sci 44:782–794
Young SK, Wek RC (2016) Upstream open Reading frames differentially regulate gene-specific translation in the integrated stress response. J Biol Chem 291:16927–16935
Lin Y, May GE, Kready H et al (2019) Impacts of uORF codon identity and position on translation regulation. Nucleic Acids Res 2:1–10
Hinnebusch AG, Ivanov IP, Sonenberg N (2016) Translational control by 5′-untranslated regions of eukaryotic mRNAs. Science 352:1413–1416
McGillivray P, Ault R, Pawashe M et al (2018) A comprehensive catalog of predicted functional upstream open reading frames in humans. Nucleic Acids Res 46:3326–3338
Melnikov A, Murugan A, Zhang X et al (2012) Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat Biotechnol 30:271–277
Sharon E, Kalma Y, Sharp A et al (2012) Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters. Nat Biotechnol 30:521–530
Soemedi R, Cygan KJ, Rhine CL et al (2017) Pathogenic variants that alter protein code often disrupt splicing. Nat Genet 49:848–855
Wong MS, Kinney JB, Krainer AR (2018) Quantitative activity profile and context dependence of all human 5′ splice sites. Mol Cell 71:1012–1026.e3
Adamson SI, Zhan L, Graveley BR (2018) Vex-seq: high-throughput identification of the impact of genetic variation on pre-mRNA splicing efficiency. Genome Biol 19:1–12
Dvir S, Velten L, Sharon E et al (2013) Deciphering the rules by which 5′-UTR sequences affect protein expression in yeast. Proc Natl Acad Sci U S A 110:E2792–E2801
Weingarten-Gabbay S, Elias-Kirma S, Nir R et al (2016) Comparative genetics: systematic discovery of cap-independent translation sequences in human and viral genomes. Science 351:aad4939
Cuperus JT, Groves B, Kuchina A et al (2017) Deep learning of the regulatory grammar of yeast 5′ untranslated regions from 500,000 random sequences. Genome Res 27:2015–2024
Sample PJ, Wang B, Reid DW et al (2019) Human 5′ UTR design and variant effect prediction from a massively parallel translation assay. Nat Biotechnol 37:803–809
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
May, G.E., McManus, C.J. (2022). High-Throughput Quantitation of Yeast uORF Regulatory Impacts Using FACS-uORF. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 2404. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1851-6_18
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1851-6_18
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1850-9
Online ISBN: 978-1-0716-1851-6
eBook Packages: Springer Protocols