Abstract
Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3′ untranslated region (3′UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3′UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3′UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the level of REQ mRNA via STAU1-mediated mRNA decay (SMD). 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.
Highlights
Requiem (REQ), known as DPF2, ubi-d4 or BAF45D, was initially identified as an essential gene for apoptosis following IL-3 deprivation in myeloid cells and was named for its role in cell death [1]
We show that specific RNA sequences residing in the 3 untranslated region (3 UTR) of REQ, which we cumulatively term the protein binding site (PBS) core, trigger REQ messenger ribonucleic acid (mRNA) decay
Clone G8, which encodes a region of the REQ 3 UTR, regulates REQ mRNA abundance
Summary
Requiem (REQ), known as DPF2, ubi-d4 or BAF45D, was initially identified as an essential gene for apoptosis following IL-3 deprivation in myeloid cells and was named for its role in cell death [1]. 7000 Nucleic Acids Research, 2014, Vol 42, No 11 termed G8, which encodes a portion of the 3 -untranslated region (3 UTR) of REQ, as a putative factor involved in ␥ to -globin switching. Since the overexpression of G8 appeared to mediate globin switching, we speculated that 3 UTR-mediated translational control could play a role in the regulation of REQ gene expression. The recognition and binding of proteins or protein complexes to defined sequences or structural RNA elements in the mRNA 3 UTR make up a common translational control mechanism [see [15] for review]. We show that specific RNA sequences residing in the 3 UTR of REQ, which we cumulatively term the protein binding site (PBS) core, trigger REQ mRNA decay. Our data indicate that REQ mRNA is an SMD target and suggest that the PBS core in the G8 region of the REQ 3 UTR is a key regulatory element in SMD
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