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Abstract
Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.
Highlights
Ribosomal frameshifing is a recoding process of translation where a specific messenger RNA-mediated signal directs a ribosome to shift its reading frame and to continue in the new frame
To search for cellular proteins that directly interacted with infectious bronchitis virus (IBV) pseudoknot RNA, a RNA pull down assay was performed in the presence of cell extracts (Figure 2A)
Wild-type and mutant IBV pseudoknot RNAs were end-labeled with biotin to serve as bait for cellular proteins that might precipitate with streptavidin-coated beads
Summary
Ribosomal frameshifing is a recoding process of translation where a specific messenger RNA (mRNA)-mediated signal directs a ribosome to shift its reading frame and to continue in the new frame. Programmed-1 ribosomal frameshifting is the most widely used translational recoding mechanism of many viruses [1]. For most other viruses, including coronavirus avian infectious bronchitis virus (IBV), frameshifting generates RNA dependent RNA polymerases. Since translation is a complex process involving many regulatory factors in addition to the ribosome, binding of RNA binding protein (RBP) at a nearby RNA signal could affect RNA conformation and somehow redirect the translational machinery [5,6]. The identification of RBPs involved in frameshifting is important in elucidating the regulatory mechanism for RNA translation
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