Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity
ABSTRACT Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3'-untranslated regions (3' UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ∼10-fold more UPF1 and ∼two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1-STAU1, STAU2-STAU2, and STAU1-STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3' UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by "greasing the wheels" of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed.
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ABSTRACT: Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.Genes & Development 09/2014; 28(17):1900-16. DOI:10.1101/gad.245506.114 · 12.64 Impact Factor
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ABSTRACT: Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions.Trends in Neurosciences 09/2014; 37(9). DOI:10.1016/j.tins.2014.05.009 · 12.90 Impact Factor