Translation reinitiation after uORFs does not fully protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability in HeLa cells. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives in HeLa cells and mining available mRNA half-life datasets for cumulative predicted uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to prevent NMD. Together, these data suggests that the decision of whether NMD ensues after translating uORFs occurs before re-initiation in mammalian cells.

[1]  Guramrit Singh,et al.  Features and factors that dictate if terminating ribosomes cause or counteract nonsense-mediated mRNA decay , 2022, The Journal of biological chemistry.

[2]  Christopher P. Lapointe,et al.  eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining , 2022 .

[3]  Christopher P. Lapointe,et al.  eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining , 2022, Nature.

[4]  J. Puglisi,et al.  Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation , 2021, bioRxiv.

[5]  Guramrit Singh,et al.  The Branched Nature of the Nonsense-Mediated mRNA Decay Pathway. , 2020, Trends in genetics : TIG.

[6]  O. Mühlemann,et al.  Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay , 2020, Nucleic acids research.

[7]  V. Ramakrishnan,et al.  Structure of a human 48S translational initiation complex , 2020, Science.

[8]  A. Teleman,et al.  Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells. , 2020, Molecular cell.

[9]  A. Hinnebusch,et al.  Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes , 2019, bioRxiv.

[10]  R. Green,et al.  Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides , 2019, bioRxiv.

[11]  T. Preiss,et al.  Translation initiation by cap‐dependent ribosome recruitment: Recent insights and open questions , 2018, Wiley interdisciplinary reviews. RNA.

[12]  R. Wek.  Role of eIF2α Kinases in Translational Control and Adaptation to Cellular Stress. , 2018, Cold Spring Harbor perspectives in biology.

[13]  A. Hinnebusch,et al.  Please do not recycle! Translation reinitiation in microbes and higher eukaryotes , 2017, FEMS microbiology reviews.

[14]  A. Hinnebusch.  Structural Insights into the Mechanism of Scanning and Start Codon Recognition in Eukaryotic Translation Initiation. , 2017, Trends in biochemical sciences.

[15]  L. Qian,et al.  Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector , 2017, Scientific Reports.

[16]  Yutaka Suzuki,et al.  A GC-rich sequence feature in the 3′ UTR directs UPF1-dependent mRNA decay in mammalian cells , 2017, Genome research.

[17]  J. Kieft,et al.  Translation initiation by the hepatitis C virus IRES requires eIF1A and ribosomal complex remodeling , 2016, eLife.

[18]  R. Wek,et al.  Upstream Open Reading Frames Differentially Regulate Gene-specific Translation in the Integrated Stress Response* , 2016, The Journal of Biological Chemistry.

[19]  Caitlin M. Rodriguez,et al.  CGG Repeat-Associated Non-AUG Translation Utilizes a Cap-Dependent Scanning Mechanism of Initiation to Produce Toxic Proteins. , 2016, Molecular cell.

[20]  J. Puglisi,et al.  Multiple Parallel Pathways of Translation Initiation on the CrPV IRES. , 2016, Molecular cell.

[21]  R. Bradley,et al.  Translational plasticity facilitates the accumulation of nonsense genetic variants in the human population , 2016, bioRxiv.

[22]  T. Jensen,et al.  Nonsense-mediated mRNA decay: an intricate machinery that shapes transcriptomes , 2015, Nature Reviews Molecular Cell Biology.

[23]  Jian Kong,et al.  Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity , 2015, Nucleic acids research.

[24]  M. Wilkinson,et al.  The unfolded protein response is shaped by the NMD pathway , 2015, EMBO reports.

[25]  S. Sugano,et al.  Analysis of RNA decay factor mediated RNA stability contributions on RNA abundance , 2015, BMC Genomics.

[26]  S. Rivella,et al.  Identi fi cation and Characterization of Small Molecules That Inhibit Nonsense-Mediated RNA Decay and Suppress Nonsense p53 Mutations , 2022 .

[27]  F. Baas,et al.  Premature termination of SMARCB1 translation may be followed by reinitiation in schwannomatosis-associated schwannomas, but results in absence of SMARCB1 expression in rhabdoid tumors , 2014, Acta Neuropathologica.

[28]  Q. Gong,et al.  Early LQT2 nonsense mutation generates N-terminally truncated hERG channels with altered gating properties by the reinitiation of translation. , 2012, Journal of molecular and cellular cardiology.

[29]  D. Ron,et al.  New insights into translational regulation in the endoplasmic reticulum unfolded protein response. , 2012, Cold Spring Harbor perspectives in biology.

[30]  A. Wakamatsu,et al.  Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals , 2012, Genome research.

[31]  R. Jackson,et al.  The mechanism of eukaryotic translation initiation and principles of its regulation , 2010, Nature Reviews Molecular Cell Biology.

[32]  Tilman Schneider-Poetsch,et al.  Inhibition of Eukaryotic Translation Elongation by Cycloheximide and Lactimidomycin , 2010, Nature chemical biology.

[33]  V. Mootha,et al.  Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans , 2009, Proceedings of the National Academy of Sciences.

[34]  Lawrence B. Gardner.  Hypoxic Inhibition of Nonsense-Mediated RNA Decay Regulates Gene Expression and the Integrated Stress Response , 2008, Molecular and Cellular Biology.

[35]  Olga Anczuków,et al.  The 185delAG mutation (c.68_69delAG) in the BRCA1 gene triggers translation reinitiation at a downstream AUG codon , 2006, Human mutation.

[36]  J. Winther,et al.  Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites. , 2006, American journal of human genetics.

[37]  Andrea L. Szymczak,et al.  Development of 2A peptide-based strategies in the design of multicistronic vectors , 2005, Expert opinion on biological therapy.

[38]  D. Ron,et al.  Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response , 2004, The Journal of cell biology.

[39]  R. Wek,et al.  Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[40]  C. Hellen,et al.  Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. , 2003, Genes & development.

[41]  M. Kozak,et al.  Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6 , 1997, The EMBO journal.

[42]  M. Kozak.  Structural features in eukaryotic mRNAs that modulate the initiation of translation. , 1991, The Journal of biological chemistry.

[43]  M. Kozak,et al.  Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. , 1987, Molecular and cellular biology.

[44]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[45]  Michael A. Cortazar,et al.  How to get away with nonsense: Mechanisms and consequences of escape from nonsense‐mediated RNA decay , 2019, Wiley interdisciplinary reviews. RNA.