5'-End sequencing in Saccharomyces cerevisiae offers new insights into 5'-ends of tRNAH is and snoRNAs.

tRNAH is guanylyltransferase (Thg1) specifies eukaryotic tRNAH is identity by catalysing a 3'-5' non-Watson-Crick (WC) addition of guanosine to the 5'-end of tRNAH is . Thg1 family enzymes in Archaea and Bacteria, called Thg1-like proteins (TLPs), catalyse a similar but distinct 3'-5' addition in an exclusively WC-dependent manner. Here, a genetic system in Saccharomyces cerevisiae was employed to further assess the biochemical differences between Thg1 and TLPs. Utilizing a novel 5'-end sequencing pipeline, we find that a Bacillus thuringiensis TLP sustains the growth of a thg1Δ strain by maintaining a WC-dependent addition of U-1 across from A73 . Additionally, we observe 5'-end heterogeneity in S. cerevisiae small nucleolar RNAs (snoRNAs), an observation that may inform methods of annotation and mechanisms of snoRNA processing.