INVESTIGATING THE DYNAMICS OF TRANSLATION READTHROUGH IN LIVE CELLS Kelsey Bettridge, Agnes Karasik, and Nicholas R. Guydosh | Laboratory of Biochemistry and Genetics, NIDDK, NIH, Bethesda MD The possible mechanisms by which translational readthrough occur References and Acknowledgements 1. Young, David J., et al. "Rli1/ABCE1 recycles terminating ribosomes and controls translation reinitiation in 3′ UTRs in vivo." Cell 162.4 (2015): 872-884. 2. Loughran, Gary, et al. "Evidence of efficient stop codon readthrough in four mammalian genes." Nucleic acids research 42.14 (2014): 8928- 8938. 3. Bidou, Laure, et al. "Sense from nonsense: therapies for premature stop codon diseases." Trends in molecular medicine 18.11 (2012): 679- 688. 4. Wilschanski, Michael, et al. "Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations." New England Journal of Medicine 349.15 (2003): 1433-1441. 5. Wu, Bin, et al. "Translation dynamics of single mRNAs in live cells and neurons." Science 352.6292 (2016): 1430-1435. 6. Yan, Xiaowei, et al. "Dynamics of translation of single mRNA molecules in vivo." Cell 165.4 (2016): 976-989. 7. Boersma, Sanne, et al. "Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding." Cell(2019). 8. Wangen, Jamie R., and Rachel Green. "Stop codon context influences genome-wide stimulation of termination codon readthrough by aminoglycosides." Elife 9 (2020). We would like to thank Dr. Marvin Tanenbaum for the gift of Moontag-containing cell strains. Additionally, thanks to Agnes Karasik for development of reporters used in this poster, and initial data collection and data collection optimization. Controls to verify translation status of signals induction of reporter puromycin treatment translation inhibition removes ribosomes from mRNA inducing expression of reporter shows gradual increase in translation events Moontag/Suntag5-7 are repeat epitopes recognized by fluorescent nanobodies. RNA is visualized using PP7 stem loops and fluorescently-labelled coat proteins. UGAC stop codon no stop codon moontag suntag RNA mRNA first ORF second ORF (A)n x12 DNA (A)n x12 Why use single molecule reporters? Can directly correlate multiple simultaneous signals over time. Single molecule reporters allow us to directly visualize readthrough in real time Comparing real data to simulations suggests “burst-like” readthrough Modeling different readthrough mechanisms via stochastic simulations Different stop codons have unique readthrough propensities8. Can we detect this using our single molecule reporters? : readthrough ratio: (∆t readthrough) (total traj len) on events threshold Future experiments no stop UGAC 3’UTR translation frequency 100% 75% 50% 25% We can detect 3’UTR translation events and see differences between stop codon contexts. Stochastic readthrough model shows a single, Gaussian like distribution 1 Investigate more stop codon contexts, including programmed stop codon sequences and premature stop codon contexts 2 Knockdown recycling factors to determine each factor’s contribution to readthrough dynamics Termination factor depletion can lead to ribosomes in the 3’UTR 1 stop codon 60s subunit termination factor normal ribosome recycling AAAAAA main ORF 3’UTR Ribosomes are recycled at stop codons with the help of termination factors. When those are depleted, ribosomes undergo readthrough1. loss of recycling factors readthrough into 3’UTR AAAAAA main ORF Some genes have programmed readthrough signals 2 specialized readthrough sequence Some genes encode readthrough sequences to make C- terminal extensions2. Treatment with aminoglycosides can induce readthrough of both normal and premature stop codons 3 C-terminal extension AAAAAA main ORF readthrough into 3’UTR/ORF AAAAAA normal stop codon OR premature stop codon 3’UTR OR remainder of main ORF Many human genetic diseases are caused by premature stop codons within the coding region3. Treatment of cells with am