Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR.
Bottom Line: By screening a complementary deoxyribonucleic acid (cDNA) expression library with an RNA-ligand binding assay, we identified STAU1 as an interactor of the REQ mRNA 3'UTR.Specifically, we provide evidence that STAU1 binds to putative 30-nucleotide stem-loop-structured RNA sequences within the G8 region, which we term the protein binding site core; this binding triggers the degradation of REQ mRNA and thus regulates translation.Furthermore, we demonstrate that siRNA-mediated silencing of either STAU1 or UPF1 increases the abundance of cellular REQ mRNA and, consequently, the REQ protein, indicating that REQ mRNA is a target of SMD.
Affiliation: Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences.Show MeSH
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Mentions: If STAU1 binds to the REQ 3′UTR, it is plausible that endogenous REQ mRNA is degraded by SMD. To test this hypothesis, we employed HeLa cells, which exhibit higher transfection efficiency than either MEL or K562 cells and have been used in many studies of SMD. We transiently transfected HeLa cells with siRNA targeting either STAU1 and UPF1 (both essential factors in mammalian SMD) or a nonspecific control siRNA (Figure 5A and B). Western blot analysis revealed that siRNA-mediated silencing of UPF1 and STAU1 reduced the levels of these proteins to ∼20% and 10%, respectively, of the amounts found in control cells (the levels of UPF1 and STAU1 were also normalized to the level of PLCγ in order to control variations in protein loading) (Figure 5A). Moreover, silencing of UPF1 did not affect the level of STAU1, and silencing of STAU1 did not affect the level of UPF1 (Figure 5A). When UPF1 and STAU1 were individually depleted, the abundance of endogenous REQ was upregulated by ∼1.5- and 1.7-fold, respectively. These results suggest that SMD reduces REQ expression by degradation of REQ mRNA.
Affiliation: Department of Life Science and Research Institute for Natural Sciences, College of Natural Sciences.