Hyperphosphorylation amplifies UPF1 activity to resolve stalls in nonsense-mediated mRNA decay

Introduction

The correct control of gene expression requires coordination of multiple transcriptional and post-transcriptional processes. Thousands of genes or gene products use a shared pool of core gene expression machineries to carry out each step of gene expression. This is orchestrated by regulatory DNA- and RNA-binding factors, many of which target subsets of genes or gene products for regulation of specific steps in gene expression. However, the mechanisms by which gene-specific factors ensure timely regulation of their target genes or gene products in the face of changing demands for the core gene expression machineries is poorly understood.

RNA quality-control pathways maintain fidelity in gene expression by targeting faulty RNAs for decay1. Nonsense-mediated decay (NMD) is a quality-control pathway that monitors the integrity of gene expression by degrading messenger RNAs (mRNAs) that have acquired premature termination codons (PTCs), for example, through mutations, or errors in transcription or mRNA processing2,3,4,5,6. Given the potential for mRNAs with PTCs to cause accumulation of detrimental truncated protein products, the ability of NMD to degrade these mRNAs likely needs to be continuously sustained to avoid deleterious consequences, no matter the current availability of RNA decay machinery. Moreover, a critical aspect of NMD is that non-target mRNAs must remain immune to the pathway.

The detection of mRNAs with PTCs occurs during translation termination and is directed by the superfamily 1 RNA helicase UPF1 and co-factors7,8,9,10,11. In metazoans, subsequent to PTC recognition, UPF1 is phosphorylated by the phosphatidylinositol-kinase related kinase (PIKK) SMG1 at [S/T]Q motifs12,13. This activates downstream steps in the pathway carried out by the endonuclease SMG6 as well as the adaptor proteins SMG5, SMG7 and PNRC2, which connect UPF1 to the general decapping, deadenylation and exonucleolytic decay machineries14,15,16,17,18,19,20,21,22,23. While UPF1 specifically targets NMD substrates for degradation, our recent evidence suggests that UPF1 transiently associates with all translated mRNAs, but a mechanism dependent on UPF1 ATPase activity prevents the stable assembly of UPF1 with non-targets24.

Intriguingly, an evolutionary conserved property of UPF1 is its ability to undergo hyperphosphorylation13,19,22,25,26,27,28, a feature that is shared with many prominent factors in gene expression, including RNA polymerase II and SR proteins for which the importance of phosphorylation in gene expression is well described29,30,31. Metazoan UPF1 proteins contain a multitude of [S/T]Q motifs in the N- and C-terminal regions, the majority of which are evolutionarily conserved (for example, 19 in humans; Supplementary Fig. 1a). Specific [S/T]Q motifs in human UPF1 have been characterized as phosphorylation-dependent binding sites for downstream factors in the NMD pathway10,17,32,33, but the functional role of other [S/T]Q motifs and the significance of UPF1 undergoing hyperphosphorylation has remained uncharacterized.

Previous studies conducted to understand principles of UPF1 phosphorylation observed that phosphorylation of UPF1 increases on depletion of SMG5, SMG6 or SMG7 in Caenorhabditis elegans and human cells10,22,25,28. Those observations, together with an observed association of phosphatase 2A with SMG5-7 (refs 22, 25, 34), led to the conclusion that SMG5-7 promote UPF1 dephosphorylation. Here given the more recently demonstrated role of SMG5-7 in linking UPF1 to mRNA decay14,16,17,18,19,21,23, we considered the alternative but not necessarily mutually exclusive possibility that the increase in UPF1 phosphorylation on SMG5-7-depletion is caused by continuous phosphorylation of UPF1 as a consequence of a stall in the NMD pathway. Indeed, we find that multiple interventions that impair the NMD pathway downstream of UPF1 mRNA assembly, including mutation of the UPF1 ATPase and depletion of NMD-specific and general mRNA decay factors, all result in increased UPF1 phosphorylation. Moreover, UPF1 undergoes increased phosphorylation upon stimulation of the AU-rich element (ARE)-mediated mRNA decay pathway, which uses shared mRNA decay machinery. Mutational analyses demonstrate that no single phosphorylation site in UPF1 is essential for NMD, but multiple phosphorylation sites contribute to NMD efficiency with some sites being more important than others. Depletion of downstream NMD factors, SMG5 or SMG7, causes increased dependence of NMD on UPF1 hyperphosphorylation. Taken together, our observations suggest that UPF1 hyperphosphorylation serves as a feedback mechanism to ensure efficient degradation of mRNAs that stably assemble with UPF1. This could serve to ensure that NMD non-targets evade NMD despite their transient association with UPF1 (ref. 24), and to resolve stalls in the NMD pathway ensuring that mRNAs targeted for NMD are efficiently degraded even under conditions where downstream NMD-specific or general mRNA decay factors are limiting.

Results

Inhibiting late NMD steps increases UPF1 phosphorylation

To determine whether increased phosphorylation of UPF1 is a general response to stalls in downstream steps of the NMD pathway, we inhibited various steps of the NMD pathway and performed anti-UPF1 immunoprecipitation (IP) followed by western blotting using a general phospho-[S/T]Q antibody to monitor relative levels of [S/T]Q-phosphorylated UPF1. Consistent with the previous reports10,22,25,28,35, depletion of NMD factors SMG7, SMG6 or SMG5 resulted in increased UPF1 phosphorylation (Fig. 1a; depletion efficiencies shown inSupplementary Fig. 1b) and mutant UPF1 proteins with deficiencies in ATP hydrolysis (DE636/637AA) or ATP binding (K498A, G495R and G497E), known to become trapped on mRNAs and stalling NMD24,36,37, all accumulate with higher levels of phosphorylation than wild-type UPF1 (Fig. 1b). In addition, depletion of PNRC2 resulted in increased UPF1 phosphorylation (Fig. 1a, depletion efficiencies shown inSupplementary Fig. 1b,e). We also tested UPF1 phosphorylation levels following interventions that impair general mRNA decay factors. As seen in Fig. 1c,d, depletion of the 5′-to-3′ exonuclease XRN1 and the decapping activator HEDLS, as well as exogenous expression of a dominant negative form of the decapping enzyme DCP2 (DCP2-E148Q) augmented UPF1 phosphorylation (Fig 1c,d; see Supplementary Fig. 1c,dfor depletion and expression levels), whereas exogenous expression of a dominant negative form of the deadenylase CAF1B (CAF1B-DDAA) showed inconsistent effects on UPF1 phosphorylation (Fig. 1d). We conclude that interventions that impair steps in the NMD pathway downstream of UPF1 assembly with mRNA substrates, including manipulations of NMD-specific factors as well as general mRNA decay factors, lead to increased UPF1 phosphorylation. We note that a correlation appears to exist between the severity of the NMD defect and the extent of UPF1 phosphorylation, as those of the tested conditions known to most severely inhibit NMD—that is, depletion of SMG6 and mutations of the UPF1 ATPase10,16,35,36,38,39—also cause the largest increase in phosphorylation (Fig. 1a–d).

Figure 1: Inhibition of UPF1 ATPase and disruption of decay activities increase UPF1 phosphorylation.

(a) Western blots monitoring phosphorylation levels of endogenous UPF1 in HeLa tet-off cells transfected with indicated siRNAs followed by IP for UPF1 and anti-phospho-[S/T]Q (P-UPF1) and anti-UPF1 (UPF1) western blotting. Indicated phosphorylation levels were calculated from at least three independent experiments by dividing the P-UPF1 signal with that from anti-UPF1 (UPF1) and are shown normalized to control (LUC) conditions±s.e.m. Pvalues are indicated below panels and are relative to control conditions (paired two-tailed Student’s t-test). (b) Same as in a in HeLa tet-off cells transfected with myc-tagged UPF1 wild-type (‘WT’), ATP-hydrolysis mutant (DE636/637AA) or ATP-binding mutants (K498A, G495R and G497E). Indicated phosphorylation levels were calculated as in a and are shown normalized to control (‘WT’) conditions±s.e.m. (c) Same as in a in HeLa tet-off cells transfected with indicated siRNAs. UPF1 phosphorylation levels were quantified as in a. (d) Same as in a in HeLa tet-off cells transfected with plasmids coding for myc-tagged UPF1 together with an empty vector (none) or vectors expressing CAF1B DDAA or DCP2 E148Q. UPF1 phosphorylation levels were quantified as in a and are shown normalized to control (‘none’) conditions±s.e.m.