Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA.

Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m1A sites and revealed hundreds of m1A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m1A biology.

Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors.

Avermectins are a group of compounds isolated from a soil-dwelling bacterium. They have been widely used as parasiticides and insecticides, acting by relatively irreversible activation of invertebrate chloride channels. Emamectin is a soluble derivative of an avermectin. It is an insecticide, which persistently activates glutamate-gated chloride channels. However, its effects on mammalian ligand-gated ion channels are unknown. To this end, we tested the effect of emamectin on two cation selective nicotinic receptors and two GABA-gated chloride channels expressed in Xenopus oocytes using two-electrode voltage clamp. Our results demonstrate that emamectin could directly activate α7 nAChR, α4ß2 nAChR, α1ß2γ2 GABAA receptor and ρ1 GABAC receptor concentration dependently, with similar potencies for each channel. However, the potencies for it to activate these channels were at least two orders of magnitude lower than its potency of activating invertebrate glutamate-gated chloride channel. In contrast, ivermectin only activated the α1ß2γ2 GABAA receptor.