Abstract
Recognition and rapid degradation of mRNA harboring premature translation termination codons (PTCs) serves to protect cells from accumulating non-functional and potentially toxic truncated polypeptides. Targeting of PTC-containing transcripts is mediated by the nonsense-mediated mRNA decay (NMD) pathway and requires a conserved set of proteins including UPF1, an RNA helicase whose ATPase activity is essential for NMD. Previously, we identified a functional interaction between the NMD machinery and terminating ribosomes based on 3' RNA decay fragments that accrue in UPF1 ATPase mutants. Herein, we show that those decay intermediates originate downstream of the PTC and harbor 80S ribosomes that migrate into the mRNA 3' UTR independent of canonical translation. Accumulation of 3' RNA decay fragments is determined by both RNA sequence downstream of the PTC and the inactivating mutation within the active site of UPF1. Our data reveal a failure in post-termination ribosome recycling in UPF1 ATPase mutants.
Highlights
Messenger RNA degradation provides a robust means to regulate gene expression and limit the quantity of protein produced from information transcribed from individual genes
Characterization of products derived from GFP reporter Messenger RNA (mRNA) harboring a premature translation termination codons (PTCs) at codon position 125 (i.e. GFPPTC125 mRNA) on high-resolution polyacrylamide gels revealed cDNA corresponding to fulllength mRNA in both wild type and UPF1 ATPase mutants (Figure 1A, lane 1 and 2, respectively; Full-length cDNAs (FL))
We have leveraged the 3’ RNA decay intermediates that accumulate from PTC-containing mRNA in the presence of ATPase-deficient UPF1 to enhance our understanding of the functional relationship between the translation and nonsense-mediated mRNA decay (NMD) machinery
Summary
Messenger RNA (mRNA) degradation provides a robust means to regulate gene expression and limit the quantity of protein produced from information transcribed from individual genes. The dependency on PTC position, translation of the mRNA, and for NMD cofactors UPF2 and UPF3 for fragment accumulation led us to propose that the block to XRN1 was a consequence of a stalled trimeric mRNP complex that forms between the ribosome, mRNA, and the NMD machinery, and that ATP hydrolysis by UPF1 is critical for efficient translation termination at a premature stop codon (Serdar et al, 2016) These data revealed an important functional interaction between the NMD and translation machinery and provided a direct role for ATP hydrolysis by UPF1 in events occurring during premature termination. Our data reveal that a failure to hydrolyze ATP by UPF1 results in a defect in posttermination ribosome recycling at PTCs and migration of ribosomes into the mRNA 3’ UTR, and provide novel evidence linking UPF1 catalytic activity with ribosome dynamics during premature termination
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