Poster abstracts
Poster number 170 submitted by Sarah Nock
UPF1 binding to neo-UTRs marks aberrant transcripts for nonsense-mediated mRNA decay in yeast
Jenna E. Smith (Department of Genetics & Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH; School of Biological and Environmental Studies, Millikin University, Decatur, IL), Sarah L.H. Nock (Department of Genetics & Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH), DaJuan L. Whiteside (Department of Genetics & Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH), Whitney R. Smith-Kinnaman (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN), Amber L. Mosley (Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN), Kristian E. Baker (Department of Genetics & Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, OH)
Abstract:
In eukaryotic cells, mRNAs harboring premature translation termination codons are recognized by the nonsense-mediated RNA decay (NMD) pathway and targeted for rapid degradation. We have previously shown that, in yeast, targeting of transcripts to NMD is independent of a role for the mRNA poly(A) tail or its associated binding protein, Pab1. Current models, however, continue to invoke that NMD is triggered by Upf1 recruitment to prematurely terminating ribosomes as a consequence of the failure of Pab1 to promote efficient translation termination through its interaction with the ribosome release factor, eRF3. The mechanism by which the NMD machinery surveils transcripts and is recruited to mRNA thus remains unclear.
We demonstrate here that the region of RNA between the premature and natural stop codons – referred to here as the neo-UTR – is the critical determinant for targeting transcripts to NMD in yeast. The efficiency of targeting of a transcript to NMD is dependent upon neo-UTR length and, importantly, independent of the mRNA’s native 3’ UTR and poly(A) tail. RNA affinity purification of an NMD mRNP identified the core NMD factor, Upf1, as associated with the neo-UTR sequence, and that its copurification is proportional with neo-UTR length. To further support the importance of Upf1 recruitment to mRNA downstream of a translation termination event as the key first step in NMD, we show that tethering Upf1 downstream of a normal translation termination event is sufficient to trigger NMD, and that the efficiency of this process is dependent upon the level of Upf1 association with the mRNA. Our findings reveal that Upf1 binding to neo-UTRs marks transcripts for NMD and triggers its rapid decay, independent of a role for the mRNA poly(A) tail or Pab1.
References:
Baker, K.E. & Parker, R. (2006) RNA. 12(8):1441-1445.
Dower, K. et al. (2004) RNA. 10(12):1888-1899.
Meaux, S. et al. (2008) Mol Cell. 29(1):134-140.
Smith, J.E. & Baker, K.E. (2017) Methods Mol Biol. 1648:201-220.
Keywords: NMD, UPF1-RNA binding, RNA surveillance and degradation