Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance

Upf1, Upf2, and Upf3 are the central regulators of nonsense-mediated mRNA decay (NMD), the eukaryotic mRNA quality control pathway generally triggered when a premature termination codon is recognized by the ribosome. The NMD-related functions of the Upf proteins likely commence while these factors are ribosome-associated, but little is known of the timing of their ribosome binding, their specificity for ribosomes translating NMD substrates, or the nature and role of any ribosome:Upf complexes. Here, we have elucidated details of the ribosome-associated steps of NMD. By combining yeast genetics with selective ribosome profiling and co-sedimentation analyses of polysomes with wild-type and mutant Upf proteins, our approaches have identified distinct states of ribosome:Upf association. All three Upf factors manifest progressive polysome association as mRNA translation proceeds, but these events appear to be preceded by formation of a Upf1:80S complex as mRNAs initiate translation. This complex is likely executing an early mRNA surveillance function. Overall design: We have employed selective profiling of ribosomes bound by FLAG-tagged NMD factors Upf1, Upf2, or Upf3, and of ribosomes from strains harboring NMD-altering mutations. Ribosome profiling libraries were prepared from both total and immunopurified ribosomes in duplicate or triplicate. RNAseq libraries were prepared from cell lysates of the same strains. High throughput sequencing was performed using Illumina NextSeq500 or HiSeq4000. The size and transcript location of ribosome protected fragments with respect to transcript coding regions, 5' and 3'UTRs, and start or stop codons from both NMD and non-NMD substrates was determined.