Editing does not exist for mammalian selenocysteine tRNAs

Abstract

It has been reported that selenocysteine tRNA from bovine liver is completely edited to two isoacceptor species, called tRNA([Ser]SecNCA) and tRNA([Ser]SecCmCa), which differ from the gene sequence. We used direct tRNA sequencing, mobility shift analyses, primer extension, restriction enzyme digestion and single strand conformational polymorphism (SSCP) analyses of products from reverse transcription coupled with polymerase chain reaction (RT/PCR), sequencing of RT/PCR products and HPLC-coupled mass spectrometry to reproduce this result and show here that editing of these tRNAs does not occur.

How Selenium Has Altered Our Understanding of the Genetic Code

“…For example, at 90 nucleotides in length, it is the longest eukaryotic tRNA sequenced to date (2,24,26,39). This is due to an atypically long variable arm and the presence of 13 nucleotides in the acceptor and T⌿C stem helices (where ⌿ indicates pseudouridine) instead of the 12 normally found in all other tRNAs.…”

“…2). One isoform contains methylcarboxymethyl-5Ј-uridine (mcm 5 U) at position 34, and the other contains methylcarboxymethyl-5Ј-uridine-2Ј-O-methylribose (2,26). mcm 5 U is the precursor of mcm 5 Um (17,50), and addition of this methyl group marks the final step in Sec tRNA [Ser] responsive to selenium status (15,26,40), and its presence confers dramatic changes in tertiary structure (26).…”

Selenium Metabolism in Drosophila

“…The Sec tRNA [Ser]Sec population was resolved into two major bands on polyacrylamide gels that appear to be the result of conformational and not structural changes. Sequence analysis of the two major bands shows that they have indistinguishable structures, which are 90 nucleotides in length, and that they contain only three modified nucleosides, mcm 5 U, i 6 A, and , at positions 34, 37, and 55, respectively. P]dCTP (specific activity Ͼ5000 Ci/mmol) were obtained from Amersham Pharmacia Biotech.…”

“…Sec tRNAs are initially aminoacylated with serine by seryl-tRNA synthetase and are therefore designated Sec tRNA [Ser]Sec . The tRNA [Ser]Sec population in higher vertebrates consists primarily of two isoacceptors that differ from each other by a single methyl group on the 2Ј-O-ribose of the nucleoside in the wobble position (4,5), resulting in either 5-methylcarboxymethyluridine (mcm 5 U) or 5-methylcarboxymethyl-2Ј-O-methyluridine (mcm 5 Um). Exogenous selenium alters the levels and distributions of these isoacceptors in mammalian cells grown in culture (6), in the tissues of rats maintained on a selenium-deficient diet (4), in the tissues of selenium-deficient rats supplemented intravenously with this element (7), in Xenopus oocytes maintained in culture (8), and in mouse embryonic stem cells harboring only one functional copy of the Sec tRNA [Ser]Sec gene (9).…”

“…They are colinear with the corresponding Sec tRNA genes from each animal (4,10) and contain only four modified nucleosides. In addition to mcm 5 U or mcm 5 Um at position 34, both tRNAs contain N 6 -isopentenyladenosine (i 6 A) at position 37, pseudouridine () at position 55, and 1-methyladenosine at position 58. A single copy gene for Sec tRNA has been shown to be present in the genomes of higher vertebrates as well as in other animals such as Drosophila and Caenorhabditis elegans (11).…”