Poster abstracts
Poster number 85 submitted by Paul Russell
Investigating how upstream open reading frame length regulates re-initiation and mRNA stability
Paul J. Russell (Cellular, Molecular, and Biochemical Sciences Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Elaina P. Boyle (Cellular, Molecular, and Biochemical Sciences Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210), Michael G. Kearse (Cellular, Molecular, and Biochemical Sciences Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology, The Ohio State University, Columbus, OH 43210)
Abstract:
Approximately 50% of all mammalian transcripts contain at least one upstream open reading frames (uORFs). Since uORFs exist in the 5' untranslated region (UTR), they are classically thought to inhibit translation of the downstream primary open reading frame (ORF) as they directly compete for the scanning ribosome. However, it is well accepted that ribosomes can re-initiate after translating uORFs. Additionally, uORFs have been implicated in regulating the stability of the mRNA via nonsense-mediated mRNA decay (NMD) and re-initiation may be a method used to evade NMD. Here we use re-initiation specific luciferase reporters with custom uORF sequences of different lengths in rabbit reticulocyte lysate and in HeLa cells to assess uORF length dependency on re-initiation and mRNA stability. Our data shows our custom uORFs capture all detectable scanning ribosomes and that re-initiation is more efficient after translating short uORFs. Using class I-IV viral internal ribosome entry site (IRES) elements to drive initiation with varying smaller sets of initiation factors, our data also supports the model that stubbornly-bound initiation factors aid re-initiation. We are now primed to test if re-initiation is a robust method for mRNAs to use to evade NMD. Together, this study will expand our understanding of how uORFs influence translational control and mRNA stability in mammalian cells.
Keywords: translational control, eIF, ribosome