Identification of exceptionally potent adenosine deaminases RNA editors from high body temperature organisms

Abstract

The most abundant form of RNA editing in metazoa is the deamination of adenosines into inosines (A-to-I), catalyzed by ADAR enzymes. Inosines are read as guanosines by the translation machinery, and thus A-to-I may lead to protein recoding. The ability of ADARs to recode at the mRNA level makes them attractive therapeutic tools. Several approaches for Site-Directed RNA Editing (SDRE) are currently under development. A major challenge in this field is achieving high on-target editing efficiency, and thus it is of much interest to identify highly potent ADARs. To address this, we used the baker yeast Saccharomyces cerevisiae as an editing-naïve system. We exogenously expressed a range of heterologous ADARs and identified the hummingbird and primarily mallard-duck ADARs, which evolved at 40–42°C, as two exceptionally potent editors. ADARs bind to double-stranded RNA structures (dsRNAs), which in turn are temperature sensitive. Our results indicate that species evolved to live with higher core body temperatures have developed ADAR enzymes that target weaker dsRNA structures and would therefore be more effective than other ADARs. Further studies may use this approach to isolate additional ADARs with an editing profile of choice to meet specific requirements, thus broadening the applicability of SDRE.

New Protein Variants Resulting from RNA Editing May Lead to Proteotoxic Stress

“…Ssa1 and Ssa2 are HSPs that belong to the Hsp70 family 49 and are 98% identical. We deleted both SSA1 and SSA2 simultaneously 50 in WT cells and in cells expressing the hbADAR2 (that has a milder effect on yeast viability but is still responsible for considerable editing 41 ), and found that the expression of hbADAR2 in Δssa1Δssa2 had a similar synthetic growth defect as that detected for rpt6-1 mutant expressing the highly active mdADAR1 (Fig. 3C).…”

“…This makes them suitable for in vitro studies on ADAR site targeting preferences [37][38][39][40][41][42] . Recently we expressed exogenously a range of heterologous ADARs in Saccharomyces cerevisiae (bakers' yeast) and identi ed the mallard-duck ADAR1 (mdADAR1) as an exceptionally potent editor 41 . Expression of this editor in the editing-naïve bakers' yeast resulted in severe growth impairment associated with ADAR catalytic activity.…”

A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases

“…These studies have focused on hADARs and identified a novel set of editing substrates. 33 , 34 Furthermore, the co-expression of hADARs with selected substrates has allowed monitoring the effects of mutations within the ADAR domains or the target dsRNA structure on the editing levels. 35 , 36 These studies nicely demonstrated that ADAR enzymes can be expressed in yeast and carry out their biological function.…”

Detection of Developmental Asexual Stage-Specific RNA Editing Events in Plasmodium falciparum 3D7 Malaria Parasite

“…In this study, we observed that RNA editing occurred within a relatively short period of 8 h. In our previous study in HEK-293 cells, we observed that a considerable level of RNA editing occurs in engineered human deaminase enzymes within a short period [ 34 , 35 , 36 , 37 , 38 ]. Recently, it was reported that adenosine deaminase acting on RNA (ADAR) enzymes in birds evolved at 40 °C and targeted different groups of temperature-sensitive RNAs [ 39 ]. Plasmodium RNA recognition by ADAR complementary factors may be altered during the early ring and late schizont stages, when the organism faces higher core body temperature in its host.…”

“…Ducks’ and hummingbirds’ elevated core body temperature reprograms their deaminase systems. Then, the deaminase system recognizes the elevated temperature-sensitive RNA and thus they adapt to their environment [ 39 ]. Recently, it was reported that temperature influences RNA editing in the octopus neural proteome and affects the protein function to a considerable degree [ 40 ].…”