Analysis of tRNA Editing in Native and Synthetic Substrates

Abstract

The primary sequence of all nucleic acids in a cell contain 4 canonical nucleotides (G, A, T, and C for DNA and G, A, U, and C for RNA). However, post-transcriptionally, nucleic acids can undergo a number of chemical modifications, which may change their structure and function. tRNAs contain the most diverse array of post-transcriptionally added chemical groups that involve both editing and modification. Because editing and modification events can serve vital roles in cell function, it is important to develop techniques that allow for fast and accurate analysis of these events. This chapter describes the methods used to purify tRNAs from total native RNA pools and for subsequent analysis of their edited and modified states using reverse transcriptase-based approaches. These techniques, in combination with 2D-TLC, allow for the routine analysis and quantitation of edited and modified nucleotides in a fast, cost effective manner and without the need for special equipment such as HPLC or a mass spectrometer. Admittedly, the techniques described here are only applicable to a subset of post-transcriptional changes occurring in a tRNA such as C to U and A to I editing as well as modifications that prevent reverse transcriptase elongation; these have been highlighted throughout the chapter.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii

“…Qiagen-Tip 2500 was used to purify tRNAs (approximately 4 mg) from total RNA (86). A Qiagen Q-2500 column was equilibrated with 50 ml fresh equilibration buffer (0.05 M morpholinepropanesulfonic acid [MOPS], pH 7.0) two times.…”

Diversity of the biosynthesis pathway for threonylcarbamoyladenosine (t6A), a universal modification of tRNA

“…Indeed, as recently shown by the Alfonzo laboratory, reverse-transcriptase bypassed t 6 A to stop downstream at m 3 C 32 . 48 Analysis of bulk tRNA from a pcc1-4 allele by LC-MS/MS found t 6 A was reduced 30%. 42 HPLC and LC-MS/MS analysis of bulk tRNAs purified from whole gene deletions in S. cerevisiae found that the bud32D and gon7D strains were devoid of t 6 A, while t 6 A levels in the pcc1D and cgi121D strains were reduced 30% and 60%, respectively, versus wild-type (Thiaville and de Cr ecy-Lagard, unpublished data).…”

The absence of A-to-I editing in the anticodon of plant cytoplasmic tRNAArgACGdemands a relaxation of the wobble decoding rules

“…Whereas Mass spectrometry. The tRNA Arg ACG isoacceptor was isolated from wheat leaves by oligonucleotide capture, 27 subjected to RNase T1 digestion, and analyzed by LC-MS/MS. 28,29 Digestion products of the tRNA containing inosine were not detected (Fig.…”