A biochemical landscape of A-to-I RNA editing in the human brain transcriptome.

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called "inosine chemical erasing" (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions.

Inosine cyanoethylation identifies A-to-I RNA editing sites in the human transcriptome.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional processing event involved in diversifying the transcriptome responsible for various biological processes. Although bioinformatic approaches have predicted a number of A-to-I editing sites in cDNAs, the human transcriptome is thought to still harbor large numbers of as-yet-unknown editing sites. Exploring new editing sites requires a biochemical method to accurately identify inosines on RNA strands. We here describe a chemical method to identify inosines, called inosine chemical erasing (ICE), that is based on cyanoethylation combined with reverse transcription. We carried out a large-scale verification of the ICE method focusing on 642 regions in human cDNA and identified 5,072 editing sites, including 4,395 new sites. Functional study revealed that A-to-I intronic editing in the SARS gene mediated by ADAR1 is involved in preventing aberrant exonization of Alu sequence into mature mRNA.

Creation of a cellooligosaccharide-assimilating Escherichia coli strain by displaying active beta-glucosidase on the cell surface via a novel anchor protein.

We demonstrated direct assimilation of cellooligosaccharide using Escherichia coli displaying beta-glucosidase (BGL). BGL from Thermobifida fusca YX (Tfu0937) was displayed on the E. coli cell surface using a novel anchor protein named Blc. This strain was grown successfully on 0.2% cellobiose, and the optical density at 600 nm (OD(600)) was 1.05 after 20 h.

Creation of cellobiose and xylooligosaccharides-coutilizing Escherichia coli displaying both ß-glucosidase and ß-xylosidase on its cell surface.

We demonstrated direct utilization of xylooligosaccharides using ß-xylosidase-displaying Escherichia coli. After screening active ß-xylosidases, BSU17580 from Bacillus subtilis or Tfu1616 from Thermobifida fusca YX, were successfully displayed on the E. coli cell surface using Blc or HdeD as anchor proteins, and these transformants directly assimilated xylooligosaccharides as a carbon source. The final OD 600 in minimal medium containing 2% xylooligosaccharides was 1.09 (after 12 h of cultivation) and 1.30 (after 40 h of cultivation). We then constructed an E. coli strain displaying both ß-glucosidase and ß-xylosidase. ß-glucosidase- and ß-xylosidase-displaying E. coli was successfully grown on a 1% cellobiose and 1% xylooligosaccharides mixture, and the OD 600 was 1.76 after 10 h of cultivation, which was higher and reached faster than that grown on a glucose/xylose mixture (1.20 after 30 h of cultivation).