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. 2016 Dec 23:7:14021.
doi: 10.1038/ncomms14021.

ATP hydrolysis by UPF1 is required for efficient translation termination at premature stop codons

Lucas D Serdar  1 , DaJuan L Whiteside  1 , Kristian E Baker  1
Affiliations

ATP hydrolysis by UPF1 is required for efficient translation termination at premature stop codons

Lucas D Serdar et al. Nat Commun. .

Abstract

Nonsense-mediated mRNA decay (NMD) represents a eukaryotic quality control pathway that recognizes and rapidly degrades transcripts harbouring nonsense mutations to limit accumulation of non-functional and potentially toxic truncated polypeptides. A critical component of the NMD machinery is UPF1, an RNA helicase whose ATPase activity is essential for NMD, but for which the precise function and site of action remain unclear. We provide evidence that ATP hydrolysis by UPF1 is required for efficient translation termination and ribosome release at a premature termination codon. UPF1 ATPase mutants accumulate 3' RNA decay fragments harbouring a ribosome stalled during premature termination that impedes complete degradation of the mRNA. The ability of UPF1 to impinge on premature termination, moreover, requires ATP-binding, RNA-binding and NMD cofactors UPF2 and UPF3. Our results reveal that ATP hydrolysis by UPF1 modulates a functional interaction between the NMD machinery and terminating ribosomes necessary for targeting substrates to accelerated degradation.

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Figures

Figure 1
Figure 1. Accumulation of a 3′ RNA fragment from nonsense-containing mRNA in UPF1 ATP hydrolysis mutants.
(a) Schematic diagram of point mutations within the C-terminal helicase domain of UPF1 that impair ATP binding (K436E), ATP hydrolysis (DE572AA) or RNA binding (RR793AA). The cysteine/histidine-rich domain (CH) within the N-terminus of UPF1 is indicated. (b–d) Northern blot analysis of PGK1 reporter mRNA in upf1Δ cells (−) complemented with wild-type (WT) or mutant UPF1 using a probe complementary to the mRNA 3′ end. Reporter mRNA either harboured a PTC within its 416 codon open reading frame (b–d) or lacked a PTC (c; −). Full-length reporter mRNA (FL) and 3′ RNA fragments (arrow) are indicated. RNA levels were normalized to NMD-insensitive SCR1 RNA. Results are representative of three independent experiments. Nt, nucleotide.
Figure 2
Figure 2. 3′ RNA fragments are decay intermediates dependent on XRN1 activity and translation.
(a,b) Northern blot analysis of PTC-containing PGK1 reporter mRNA in upf1Δ cells expressing ATPase-deficient UPF1 in the presence (UPF1-DE) or absence of XRN1 (UPF1-DE/xrn1Δ). (b) Reporter mRNA inhibited for translation by a 5′ stemloop in upf1Δ cells expressing either wild-type (WT) or ATPase-deficient (DE) UPF1. Full-length reporter mRNA (FL) and 3′ RNA fragments (arrow) indicated. RNA levels were normalized to NMD-insensitive SCR1 RNA. Results are representative of three independent experiments. Nt, nucleotide.
Figure 3
Figure 3. 3′ RNA decay fragments are ribosome bound.
(a) Ultraviolet absorbance trace from polyribosome analysis of cells expressing ATPase-deficient UPF1 and one of three PTC-containing PGK1 reporter mRNAs; ribosome-free RNA (RNP), 40S, 60S and 80S ribosomal subunits, and polyribosomes are indicated. (b) Northern blot analysis of PTC-containing PGK1 reporter mRNA from each gradient fraction. Full-length reporter mRNA (FL) is indicated. Results are representative of three independent experiments. Nt, nucleotide.
Figure 4
Figure 4. UPF1 function in premature translation termination requires RNA binding and cofactors UPF2 and UPF3.
(a–c) Northern blot analysis of PTC-containing PGK1 reporter mRNA. (a) upf1Δ cells complemented with either ATPase-deficient UPF1 (UPF1-DE) or the same mutant also lacking RNA-binding activity (UPF1-DE/RR). (b) PGK1-PTC344 reporter mRNA in upf1Δ cells expressing wild-type (WT) or mutant UPF1 (DE) and also lacking UPF2 (upf2Δ), UPF3 (upf3Δ) or both (upf2Δupf3Δ). (c) PGK1-PTC344 reporter mRNA in upf1Δ cells expressing ATPase-deficient UPF1 (DE), mutant UPF1 relieved for allosteric inhibition of ATPase and helicase activities (F), or UPF1 harbouring both mutations (F/DE) in cells either with or without UPF2 (upf2Δ). Full-length reporter mRNA (FL) and 3′ RNA fragments (arrow) are indicated. RNA levels were normalized to NMD-insensitive SCR1 RNA. Results are representative of three independent experiments. Nt, nucleotide.
Figure 5
Figure 5. ATP hydrolysis by UPF1 promotes efficient translation termination on nonsense-containing mRNAs.
Premature translation termination allows UPF1 to remain associated with transcripts at sites downstream of the PTC and in a 3′ UTR length-dependent manner. ATP binding by UPF1, in conjunction with cofactors UPF2 and UPF3, promotes association of the NMD machinery with a terminating ribosome, mediated through interaction with translation release factors eRF1 and eRF3. For wild-type UPF1, ATP hydrolysis promotes rapid disassembly of the trimeric complex (ribosome, RNA and UPF proteins) leading to efficient translation termination and release of the ribosome and NMD factors from the RNA. How this event is communicated to the decay machinery to accelerate decapping and turnover of the mRNA remains unclear (bottom left; Fast). Failure to catalyze ATP hydrolysis leads to a defect in translation termination and a kinetic stall in ribosome release, presenting a block to XRN1-mediated 5′→3′ exonucleolytic decay and, critically, failure to trigger rapid turnover of the mRNA (bottom right; Slow).
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