Nucleotide sequence ofPhysarumU6 small RNA

Small RNA (sRNA) U6 was isolated from total cellular RNA of vegetative Physarum microplasmodia by preparative polyacrylamide electrophoresis. It was end-labeled in vitro to high-specific-activity using (32-P)-pCp and RNA-ligase; and sequenced by direct chemical methods. In this sequencing process, pseudouridine scores as a uridine, methylcytosine scores as a cytosine, and methyladenosine scores as an adenosine. Physarum U6 is 99 N long. Its primary sequence was compared to the published sequence of rat U6. One region, 93 N long, shows 65.6% homology (61/93) with nucleotides 10 to 108 of the rat U6 molecule. One 15 N subdomain, corresponding to nucleotides 18-32 of the rat molecule, is 100% conserved. Those functional rat U6 sequence domains proposed to base-pair with rat U4 RNA show 58.3% (21/36 N) homology with the rat sequence. Potential secondary structures of the Physarum U6 molecule were analyzed by computer programs. The most stable structure is shown below. Within the proposed stem regions, 86.7% (26/30) of the base changes preserved the hairpin structure. Interestingly, in those domains where primary structure shows poor homology, secondary structure is highly conserved.