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. 2018:1661:43-60.
doi: 10.1007/978-1-4939-7258-6_4.

Selenocysteine tRNA[Ser]Sec, the Central Component of Selenoprotein Biosynthesis: Isolation, Identification, Modification, and Sequencing

Bradley A Carlson  1 , Byeong Jae Lee  2 , Petra A Tsuji  3 , Paul R Copeland  4 , Ulrich Schweizer  5 , Vadim N Gladyshev  6 , Dolph L Hatfield  7
Affiliations

Selenocysteine tRNA[Ser]Sec, the Central Component of Selenoprotein Biosynthesis: Isolation, Identification, Modification, and Sequencing

Bradley A Carlson et al. Methods Mol Biol. 2018.

Abstract

The selenocysteine (Sec) tRNA[Ser]Sec 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[Ser]Sec population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec 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[Ser]Sec for identifying their occurrence in other organisms is also presented.

Keywords: Chromatography; Gel electrophoresis; Selenium; Selenocysteine tRNA; Selenocysteine tRNA detection; Selenocysteine tRNA gene modification.

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Figures

Fig 1
Fig 1
Selenocysteine tRNA[Ser]Sec (a) The primary structure of human tRNA[Ser]Sec is shown in a cloverleaf model. Mammalian tRNA[Ser]Sec is 90 bases in length. The acceptor stem constitutes the paired 5’ and 3’ terminal bases, the D stem and loop constitute the six paired and four unpaired bases of the left portion of the tRNA, the anticodon stem and loop, the six paired and seven unpaired bases of the lower portion of the tRNA, the variable stem and loop, the five paired and 4 unpaired bases, and the TψC stem and loop, the four paired and seven unpaired bases of the right portion of the tRNA. Mammalian tRNA[Ser]Sec contains modifications at positions 34 (mcm5U or mcm5Um), 37 (i6A), 55 (ψ) and 58 (m1A). (b) The two tRNA[Ser]Sec isoforms (containing either mcm5U or mcm5Um at position 34) differ from each other by a single methyl group on the 2’-O-ribosyl moiety. (c) Representative fractionation of [75Se]selenocysteyl-tRNA[Ser]Sec isoforms by RPC5 chromatography. [75Se]selenocysteyl-tRNA[Ser]Sec was extracted from HL-60 cells and run on an RPC5 column as described in Section 3.6. The initial eluting peak from the column is the mcm5U-containing isoform and the later eluting peak is the mcm5Um-containing isoform. The isoforms are pooled as shown by the hatched areas and collected as described in Section 3.6 for further study.
Fig 2
Fig 2
Schematic diagram demonstrating the procedure for sequencing uncharacterized tRNA[Ser]Sec from any organism as described in the text.
See this image and copyright information in PMC

References

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