NMR structure of the apoB mRNA stem–loop and its interaction with the C to U editing APOBEC1 complementary factor

Abstract

We have solved the NMR structure of the 31-nucleotide (nt) apoB mRNA stem-loop, a substrate of the cytidine deaminase APOBEC1. We found that the edited base located at the 5 end of the octa-loop is stacked between two adenosines in both the unedited (cytidine 6666) and the edited (uridine 6666) forms and that the rest of the loop is unstructured. The 11-nt "mooring" sequence essential for editing is partially flexible although it is mostly in the stem of the RNA. The octa-loop and the internal loop in the middle of the stem confer this flexibility. These findings shed light on why APOBEC1 alone cannot edit efficiently the cytidine 6666 under physiological conditions, the editing base being buried in the loop and not directly accessible. We also show that APOBEC1 does not specifically bind apoB mRNA and requires the auxiliary factor, APOBEC1 complementary factor (ACF), to edit specifically cytidine 6666. The binding of ACF to both the mooring sequence and APOBEC1 explains the specificity of the reaction. Our NMR study lead us to propose a mechanism in which ACF recognizes first the flexible nucleotides of the mooring sequence (the internal loop and the 3 end octa-loop) and subsequently melts the stem-loop, exposing the amino group of the cytidine 6666 to APOBEC1. Thus, the flexibility of the mooring sequence plays a central role in the RNA recognition by ACF.

Hypermutation induced by APOBEC-1 overexpression can be eliminated

“…These data suggest that site 6666 has greater accessibility/affinity to the editing complex compared to other sites. This is consistent with the mooring sequence being near site 6666 and the mooringlike sequence being near site 6802 and the proposal that canonical editing by the editing complex is associated with mooring motif recognition (Hersberger and Innerarity 1998;Maris et al 2005).…”

“…ACF and APOBEC-1 form a complex minimally required for in vitro apoB mRNA editing. It has been proposed that ACF first recognizes the flexible nucleotides of the apoB mRNA mooring sequence and subsequently melts the stem-loop, exposing the amino group of cytidine 6666 to APOBEC-1 (Maris et al 2005). ACF also lowers the temperature requirement to promote a conformational transition in the RNA substrate (Chester et al 2004).…”

Apobec-1 Complementation Factor Modulates Liver Regeneration by Post-transcriptional Regulation of Interleukin-6 mRNA Stability

“…ACF is the RNA binding component of the apoB RNA editing enzyme and functions both to bind an AU-rich region of the nuclear apoB transcript and also to anchor and position the dimeric RNA-specific cytidine deaminase Apobec-1 into an optimal configuration for C to U deamination of the targeted base at position 6666 (4,(23)(24)(25). Structural studies using nuclear magnetic resonance demonstrated that ACF exhibited greater binding affinity for apoB than did Apobec-1 and suggested that increasing concentrations of ACF restricts the RNA binding of Apobec-1 and promotes site-specific C to U editing (25).…”

Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells

“…This fragment has been considered the minimal stem-loop recognized by the editosome. 29,55 However, this construct resulted completely uneditable in our system (data not shown), possibly due to secondary/tertiary structures in the transcript that masked the editing site.…”

“…Thus APOBEC1 Complementation Factor (ACF) is required for the specificity of the targeting through its interaction with the mooring sequence. [24][25][26][27][28][29][30] Another molecule, RBM47, has been recently shown to be sufficient for the APOBEC1-mediated RNA editing. 31 Other molecules, GRY-RBP and hnRNP-C1 among them, have been characterized as negative regulators of the editing process.…”