Selenocysteine tRNA, the Central Component of Selenoprotein Biosynthesis: Isolation, Identification, Modification, and Sequencing.

The selenocysteine (Sec) tRNA population consists of two isoforms that differ from each other by a single 2'-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA for identifying their occurrence in other organisms is also presented.