An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Summary

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

Abstract

N⁶-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N⁶-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i⁶A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i⁶A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

Introduction

At least 163 distinct posttranscriptional RNA modifications have been identified, with these modifications conferring diverse and context-dependent functions to RNAs, directly influencing RNA structure, and affecting interactions of RNA with other molecules^1,2. As the appreciation for the number and variety of RNA modifications increases, it is critical to develop assays that can reliably interrogate both the RNA modifications and the enzymes that catalyze them.

One of the first RNA modifications to be identified occurs at base 37 in tRNAs, adjacent to the anti-codon on the 3' s....

Protocol

NOTE: The protocol was adapted from Ref. ²⁴.

1. Obtain RNA and Enzyme of Interest

1. Use in vitro transcribed RNAs internally labeled with ³²P, and recombinant His6-Mod5 expressed in and purified from E. coli, as previously described²⁴.
   1. Introduce in vitro transcribed RNAs (section 3) using T7 RNA polymerase in the presence of unlabeled ATP, UTP, CTP, GTP and 10 µCi of gel-purified, ethanol-precipitated α-ATP, resuspended in 1x TE (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA), and stored at -80 °C²⁴.
   ....

Results

Mod5 was incubated with a tyrosine tRNA or serine tRNA in the presence or absence of DMAPP. Following the modification reaction, products were RNase T1-digested, which cleaves the 3' end of all guanosines leaving a 3' guanosine monophosphates (GMP)²⁴ (Figure 1). Full digestion of the RNAs produces a predictable pattern of radiolabeled fragments (Figure 2A), which are then resolved on a 20%.......

Discussion

RNA modifications continue to be shown to play ever more important and diverse roles in cellular and organismal function. As such, the development of assays to interrogate RNA modifying enzymes is central to better understanding the fundamental aspects of biology. This protocol describes a high-resolution in vitro assay to characterize the tRNA modification activity of Mod5.

This protocol has the distinct advantage of providing a direct, and easily interpretable readout of isopentenyl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
UreaGel Concentrate	National Diagnostics	EC-833	As part of a kit
UreaGel Diluent	National Diagnostics	EC-833	As part of a kit
UreaGel Buffer	National Diagnostics	EC-833	As part of a kit
10x TBE	National Diagnostics	EC-833	As part of a kit
Ammonium persulfate (APS)	Sigma-Aldrich	7727-54-0
N,N,N′,N′-Tetramethylethylenediamine (TMED)	Sigma-Aldrich	T9281
Tris base	Sigma-Aldrich	T1503
Boric acid	Sigma-Aldrich	10043-35-3
EDTA	Sigma-Aldrich	60-00-4
ATP	Sigma-Aldrich	34369-07-8
MgCl2	Sigma-Aldrich	7786-30-3
DMAPP	Caymen Chemical	1186-30-7
Super RNaseIN	ThermoFisher Scientific	AM2694
2-mercaptoethanol	Sigma-Aldrich	60-24-2
Ethanol	Sigma-Aldrich	64-17-5
Sodiume acetate	Sigma-Aldrich	127-09-3
Rnase T1	ThermoFisher Scientific	EN0541
Glycerol	Sigma-Aldrich	56-81-5
Xylene cyanol	Sigma-Aldrich	2650-17-1
Equipment and Supplies
Short glass plates (20-40 cm W x 40 cm L)	The Gel Company
Long glass plates (20-40 cm W x 40 cm L)	The Gel Company
Vertical gel apparatus	The Gel Company	S2-3040
50 mL disposable syringe	Fisher Scientific	03-377-26
Stainless steel binder clips	Idea Scientific	1066
Phosphoscreen	Sigma-Aldrich	28-9564-74
Plastic wrap	(local grocery store)