Preston MA, Porter DF, Chen F, Buter N, Lapointe CP, Keles S, Kimble J, Wickens M.

Nat Methods. 2019 May;16(5):437-445. doi:10.1038/s41592-019-0370-6.

Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase.

TRAID-seq (tethered rNTase activity identified by high-throughput sequencing). “Enzymes were fused to MS2 coat protein (MS2) and coexpressed in yeast with a reporter RNA bearing high-affinity MS2-binding sites. Interaction of MS2 with its binding sites tethered the fusion protein to the RNA23 and circumvented the activity of proteins that might bring the rNTase to its endogenous substrates.” Final substrate: “ tRNASer(AGA), in which the 4-bp variable arm was replaced by a single MS2-binding site”. Problem with background polyadenlyation in previous attempt with RNase P–derived RNA. “CeMUT-2 added tails with a 1:1 ratio of uridines to guanosines.” These might form a hairpin to prime RdRP. “CeNPOL-1 added tails composed of random combinations of all four nucleotides.”