Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein

Abstract

We previously developed TRIBE, a method for the identification of cell-specific RNA-binding protein targets. TRIBE expresses an RBP of interest fused to the catalytic domain (cd) of the RNA-editing enzyme ADAR and performs adenosine-to-inosine editing on RNA targets of the RBP. However, target identification is limited by the low editing efficiency of the ADARcd. Here we describe HyperTRIBE, which carries a previously characterized hyperactive mutation (E488Q) of the ADARcd. HyperTRIBE identifies dramatically more editing sites, many of which are also edited by TRIBE but at a much lower editing frequency. HyperTRIBE therefore more faithfully recapitulates the known binding specificity of its RBP than TRIBE. In addition, separating RNA binding from the enhanced editing activity of the HyperTRIBE ADAR catalytic domain sheds light on the mechanism of ADARcd editing as well as the enhanced activity of the HyperADARcd.

TRIBE editing reveals specific mRNA targets of eIF4E-BP inDrosophilaand in mammals

“…TRIBE was first developed in Drosophila and worked well both in S2 tissue culture cells and in fly neurons (McMahon et al 2016;Luo et al 2018;Xu et al 2018). To adapt this method to mammalian systems, we first tried the improved hyperTRIBE version with its hyperactive form of the Drosophila ADAR catalytic domain.…”

“…Although 4E-BP-mediated editing is enriched in the 5'UTR, editing clearly occurs elsewhere within the transcript, in fly cells as well as in mammalian cells. This could reflect 4E-BP overexpression, but it is likely that the ADARcd can also "reach" and edit a susceptible sequence element a considerable distance away from the binding site of its protein complex (McMahon et al 2016;Xu et al 2018).…”

“…Transcripts hosting these A-to-G editing sites are considered binding targets of the RBP. The upgraded version of TRIBE, hyperTRIBE, contains the one amino acid substitution E488Q in the ADARcd region (hyper-ADARcd), which improves target identification efficiency by reducing bias originating from the intrinsic editing preference of the ADARcd (Kuttan and Bass 2012;Rahman et al 2018;Xu et al 2018).…”

Ecdysone signaling promotes expression of multifunctional RNA binding proteins essential for ovarian germline stem cell self-renewal inDrosophila

“…Our data suggest that Sqd, Heph, and Hrb27C post-transcriptionally promote the expression of key components of the BMP signaling pathway in GSCs. In support of this model, recent studies profiling RNA targets of Hrb27C in Drosophila neuronal cell lines identified several transcripts essential for BMP signaling (McMahon et al, 2016;Xu et al, 2018). Given the complexity of the sax, tkv, and put gene loci, we postulate that hnRNPs regulate the splicing or stability of these essential BMP signaling transcripts in GSCs.…”

The m6A reader Ythdf restricts axonal growth inDrosophilathrough target selection modulation of the Fragile X mental retardation protein

“…Peak fractions containing the recombinant proteins were pooled, aliquoted, and snap-frozen in liquid nitrogen. Frozen aliquots were stored at − 80 °C.TRIBETRIBE was performed as previously described(McMahon et al, 2016;Worpenberg et al, 2019;Xu et al, 2018). Briefly, for the identification of mRNA targets in S2R+ cells, Flagtagged Ythdf and Fmr1 versions fused to the catalytic domain of Adar (cdAdar) were ectopically expressed using a metal inducible expression system.…”